Enzymatic catalysis of hydrogen oxidation and production

Abstract

Enzymatic catalysis of hydrogen oxidation and production The present invention relates to a means and methods for catalyzing the redox reaction of hydrogen, in particular oxidation of molecular hydrogen and reduction of protons. Specifically, it pertains to a composition for catalyzing reduction of protons and / or oxidation of hydrogen which comprises hydrogenase and a redox-active matrix having a reduction potential identical to or lower than that of the hydrogenase, thereby protecting the hydrogenase from oxygen, oxidative stress and oxidative potentials. The present invention also relates to specific hydrogenases that can be used with specific redox-active matrices. The invention also relates to methods and uses for the reduction of protons and / or oxidation of hydrogen making use of the composition of the invention, as well as devices, for producing molecular hydrogen or electricity with the composition of the present invention and the corresponding methods. Furthermore, the invention relates to novel redox-active matrices and methods and uses thereof to protect hydrogenases from oxygen, oxidative stress and / or oxidative potentials.

Description

[0001] New PCT Application based on EP 25 305 533.9

[0002] Technische Universität München, in Vertretung des Freistaates Bayern; Centre National de la Recherche Scientifique (CNRS); Aix-Marseille Université

[0003] Vossius Ref.: AJ2983 PCT S3

[0004] Enzymatic catalysis of hydrogen oxidation and production

[0005] The present invention relates to a means and methods for catalyzing the redox reaction of hydrogen, in particular oxidation of molecular hydrogen and reduction of protons. Specifically, it pertains to a composition for catalyzing reduction of protons and / or oxidation of hydrogen which comprises a hydrogenase and a redox-active matrix having a reduction potential identical to or lower than the inactivation potential of the hydrogenase, thereby protecting the hydrogenase from oxygen, oxidative stress and oxidative potentials. The present invention also relates to specific hydrogenases that can be used with specific redox-active matrices. The invention also relates to methods and uses for the reduction of protons and / or oxidation of hydrogen making use of the composition of the invention, as well as devices, for producing molecular hydrogen or electricity with the composition of the present invention and the corresponding methods. Furthermore, the invention relates to novel redox-active matrices and methods and uses thereof to protect hydrogenases from oxygen, oxidative stress and / or oxidative potentials.

[0006] In light of the increasingly evident consequences of decades of global reliance on fossil energy sources, a radical shift in our energy economy towards renewable and sustainably usable energy carriers is essential. Molecular hydrogen, due to its chemical and physical properties, is considered to be one of the most promising energy carriers in a so-called “green system”. Hydrogen can be used as an efficient storage medium for electrical energy. An increase in the use of hydrogen produced sustainably, for instance, through the electrolysis of water, will inevitably lead to a growing demand for systems dedicated to the production and conversion of hydrogen into electricity.

[0007] Today's fuel cell and electrolyzer technologies still predominantly rely on platinum as a catalyst, a precious metal with extremely limited availability on Earth. The extraction of platinum is costly, associated with environmental damage, and limited to a few regions worldwide. According to the “Global Hydrogen Review 2022” by the IEA, in 2022, approximately 80% of the world's platinum was mined in South Africa (70%) and Russia (10%). To reduce the dependency of the global energy supply on limited resources and specific raw material source countries, it is necessary to develop alternative methods to carry out these reactions using different catalysts.

[0008] In the case of hydrogen production and electricity generation, nature offers a fully regenerative alternative in the form of biocatalysts (enzymes), a particular subgroup of which can catalyze these reactions with efficiency comparable to that of platinum. These enzymes, known as hy-drogenases, are metalloproteins that can oxidize hydrogen and reduce protons to molecular hydrogen independently of rare elements like platinum metals.

[0009] Based on the metal composition of their active centers, the enzyme group of hydrogenases is divided into three families: [Fe]-, [NiFe]-, and [FeFe]-hy drogenases. The latter two hydrogenase families catalyze the conversion of hydrogen bidirectionally with high efficiency through the formation of an intermediate hydride. The [FeFe] -hydrogenase group is particularly noteworthy due to its high catalytic activity, which is comparable to that of platinum-based catalysts. Additionally, the cofactor of this hydrogenase family can be chemically synthesized and integrated in vitro into the relatively easily expressible protein scaffold via a self-assembly process, enabling large-scale enzyme production. These characteristics make [FeFe]-hydrogenases particularly interesting for scalable applications of hydrogen production and electricity generation.

[0010] The use of [FeFe] -hydrogenases as fully regenerative and indefinitely available substitute catalysts for the rare precious metal platinum has so far been hindered by two intrinsic properties of this enzyme group: (1) their irreversible inactivation by molecular oxygen, which, even at very low concentrations, irreversibly damages the enzyme's active site, and (2) their sensitivity to high voltages, which makes their application in electrically operated devices, such as fuel cells and electrolyzers, practically impossible.

[0011] Thus, there is a need to develop a cost-effective regenerative system for reversible hydrogen conversion that does not rely on platinum, in which a hydrogenase is protected both from high potentials and from oxygen (to prevent irreversible activity loss).

[0012] This protection is required not only during operational phases but also during storage and intermittent idle periods. Both of these challenges arise in systems for hydrogen production and electricity generation and avoiding them has proven to be extremely difficult or costly. While the limitation caused by inactivation at oxidative potentials has been tackled in recent years by the development of electrically conductive hydrogels for enzyme embedding, the issue of oxygen inactivation has not been resolved.

[0013] Although it has been possible to protect the enzymes from oxygen inactivation through the use of electrically conductive and oxygen-reducing hydrogels, this protection requires the presence of hydrogen and is effective only during catalytic activity. In practical terms, this means that the protection is limited to the operational phase and does not offer any safeguarding of the biocatalyst during production, storage, or operational downtimes (as reported in the literature, references 1-6).

[0014] A direct protection mechanism for an enzyme has been described for a [FeFe]-hydrogenase, through the reversible binding of sulfide at the active site, leading to an inactive but oxygen-protected state. This protective state allows the enzyme to be handled in an oxygen-containing atmosphere; however, the initial transition to the catalytically active state is irreversible (as discussed in the literature, references 7 & 8). This protective effect, therefore, extends onlythrough the production and storage of the bio-electrodes up until the first operational use; it does not provide reversible protection that can be reactivated during operational pauses, such as shutdowns, malfunctions, maintenance work, etc. Thus, there is a need to provide a composition for catalyzing reduction of protons and / or oxidation of hydrogen capable of protecting a hydrogenase from oxygen during production, operational phases, storage phases and intermittent idle periods.

[0015] The embedding of enzymes in an electrically conductive redox hydrogel can protect against voltage spikes and molecular oxygen under certain conditions. However, systems so far provided only incomplete protection or significantly restricted system flexibility because the chemical composition of the gel only allowed for catalytic hydrogen activation (H2oxidation / electricity generation) by the enzymes, and oxygen protection was only possible in the presence of hydrogen itself (Plumeré et al. 2014; Oughli et al. 2015; Fourmond et al. 2015; Ruff et al. 2017; Oughli et al. 2018; Li et al. 2019). More recent studies have demonstrated that modifications to the chemical composition of the gel's redox groups can facilitate both catalytic activation and hydrogen production (Hardt et al. 2021).

[0016] A further limitation of the prior art system is that it only protects the enzyme during operational phases and not during system shutdowns or storage of the biocatalyst, as the protective effect is tied to the catalytic hydrogen oxidation by the embedded enzyme. With the addition of the reversible inhibitor sulfide (e.g., Na2S), it is possible to protect the inactive enzyme from oxygen outside of operational conditions. This allows, for instance, the functionalization of electrodes with the combination of hydrogel and enzyme under aerobic conditions. Sulfide induces an inactive but oxygen-protected enzyme state (Hinact). However, this protection mechanism can only be effectively applied to one of the established [FeFe] -hydrogenases (DdHydAB) and does not allow for reversible and instantaneous protection (Rodríguez-Maciá et al. 2020). The transient protection effect mediated by sulfide is irreversibly lost once the enzyme is activated in the system for the first time. Since flexible electrical systems cannot be continuously maintained in the same operational mode, and transient shutdowns, e.g., for maintenance purposes, must be possible without requiring the entire enzyme stock to be replaced, reversible protection against oxygen during such idle phases is essential.

[0017] Thus, there is a need to provide an improved composition for catalyzing reduction of protons and / or oxidation of hydrogen which is capable of reversibly protecting a hydrogenase from oxygen.

[0018] In summary, while the current state of the art provides some means of protecting enzymes from high potentials, the protection offered by sulfide as an external inhibitor is limited to the production phase of the catalysts and requires a cumbersome procedure, whereas the oxygen protection afforded by the hydrogel is restricted to the catalytic phase. Reversible protection of the enzyme during operational pauses for maintenance, repairs, etc., cannot be achieved with these methods.Despite the substantial advances in the field of hydrogenase-based catalysis, there remains a need for improved systems and compositions that can enhance the stability and activity of hydrogenases under oxidative conditions. The problem to be solved by the present invention is therefore the improvement of the catalyzation of the reduction of (hydrogen) protons and / or the catalyzation of the oxidation of (molecular) hydrogen.

[0019] The above-mentioned problem is solved by the embodiments provided herein and the claims. In particular, the problem is solved by the provision of a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix (RAM) having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0020] The exemplary [FeFe] -hydrogenase CbA5H of Clostridium beijerinckii has been embedded into a RAM whose reduction potential is finely tuned, the experienced potential is thereby buffered in a sharp potential range that is compatible with H2 oxidation, without compromising resistance of this system towards O2. This redox buffering matrix enables to gain high H2-oxidation activity with an O2-resistant [FeFe]-hydrogenase even at high potentials. The protection from oxygen and therefore the catalytic activity of CbA5H of Clostridium beijerinckii is maintained throughout proton reduction and oxidation of hydrogen. Furthermore, the protection from oxygen can be achieved as long as the reduction potential of the RAM is identical to or lower than the inactivation potential of CbA5H. The inactivation potential of CbA5H of Clostridium beijerinckii is about +0.170 V vs. RHE (Winkler et al. 2021). The inactivation potential was estimated from the enzyme’s calculated rate constants for the inactivation and reactivation, which were reported by Winkler et al. (2021). Protection from oxidative potentials as well as protection in the presence and absence of H2 has been demonstrated. Taken together, the Examples and in particular Example 1 demonstrated that the full protection of the hydrogenase from oxygen is achieved not only during H2-oxidation / reduction catalysis (aerobic or anaerobic) but also during intermediate resting stages or storage. The composition of claim 1 consequently solves the above-mentioned problem since it effectively protects the hydrogenase via the RAM from oxygen or oxidative potentials, thereby allowing the use of the composition for catalyzing reduction of protons and / or oxidation of hydrogen. This in turn leads to the advantage that the composition can be consistently used instead of platinum in a fuel cell or an electrolyzer for catalyzing H2 evolution or oxidation.

[0021] Furthermore, the problem of the present invention is solved by the provision of a method for reduction of protons comprising catalyzing the reduction with the composition of the present invention and a method for oxidation of hydrogen comprising catalyzing the oxidation with the composition of the present invention.

[0022] The advantages discussed above in context of the composition of the present invention apply mutatis mutandis. In particular, the methods take advantage of the capability of the composition to catalyze the reduction of protons and the oxidation of hydrogen. This leads to an improve-ment over the prior art since prior art methods either lack because of hydrogenases being inactivated by oxygen stress or oxidative potentials or rely on platinum as a catalyzer. Both prejudices are overcome by the methods of the present invention.

[0023] Furthermore, the problem of the present invention is solved by the provision of a use of the composition of the present invention in the oxidation of hydrogen, particularly in a fuel cell and by the provision of the corresponding use of the composition of the present application in the reduction of protons, particularly for the electrolysis of water, more particularly to produce hydrogen. The above-mentioned advantages apply mutatis mutandis. In particular, the prejudice in the prior art uses - necessitating platinum as an expensive catalyst for the oxidation of hydrogen in a fuel cell or the evolution of hydrogen in an electrolyzer - is overcome.

[0024] Furthermore, the problem of the present invention is solved by the provision of a redox-active matrix comprising a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase (RLM). This redox-active matrix allows efficient protection of hydrogenases thereby leading to the above-mentioned advantages, namely the full protection from oxygen or oxidative potentials as well as the advantage that platinum is not necessary when the hydrogenase can be protected efficiently.

[0025] Furthermore, the problem of the present invention is solved by the provision of a method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with a redox-active matrix of the present invention and the corresponding use of a redox-active matrix of the present invention for protecting a hydrogenase from oxygen. This leads to the advantage that efficient protection as described above is achieved when a composition according to the present invention is formulated with the redoxactive matrices of the present invention.

[0026] Furthermore, the problem of the present invention is solved by the provision of a fuel cell comprising an anode, a cathode, an electrolyte and the composition of the present invention as well as the corresponding method for producing electricity comprising catalyzing the oxidation of hydrogen in a fuel cell. When the composition of the present invention is used in a fuel cell, the hydrogenase protected by the redox-active matrix can be used to produce electricity. This is advantageous over the prior art which either has to rely on platinum as a catalyzer since prior art hydrogenases have not been able to be stable enough for application in a fuel cell, where oxidative stress and oxidative potentials are occurring.

[0027] Furthermore, the problem of the present invention is solved by the provision of a device, such as an electrolyzer, for the production of hydrogen, e.g. molecular hydrogen H2, comprising an anode, a cathode, an electrolyte and the composition of the present invention, as well as thecorresponding method for producing hydrogen, e.g. molecular hydrogen H2, comprising catalyzing the reduction of protons with the device of the present invention. The same advantages apply as discussed above in the context of the fuel cell, since the oxidation of hydrogen and the reduction of protons is a reversible redox reaction.

[0028] Preferred embodiments of the present invention are defined in the dependent claims.

[0029] Although the above paragraph refers to the Examples of the invention, it is noted that the invention is not limited to the Examples, e.g. limited to CbA5H of Clostridium beijerinckii. Example 1 has been performed with CbA5H of Clostridium beijerinckii as an exemplary hydrogenase for illustrating the successful protection from oxygen and the successful catalysis of the hydrogen redox reaction. However, these results can be extrapolated to any hydrogenase, since any hydrogenase will be protected from oxygen and oxidative potential as long as the reduction potential of the redox-active matrix is lower, identical or slightly more positive than the inactivation potential of the hydrogenase, preferably as long as the reduction potential of the redoxactive matrix is lower or identical compared to the inactivation potential of the hydrogenase, most preferably as long as the reduction potential of the redox-active matrix is lower than the inactivation potential of the hydrogenase. Accordingly, the general principle is applicable to any oxygen sensitive enzyme. This is demonstrated in Example 4, which explains that the structure of the active site of the hydrogenase, rather than the specific hydrogenase per se is relevant for a successful protection from oxygen by a redox-active matrix. Accordingly, the principle of the present invention which has been demonstrated for CbA5H of Clostridium beijerinckii as an exemplary hydrogenase applies at least to all hydrogenases making use of the same protection mechanism that is based on the presence of a H-cluster comprising a [4Fe4S]-cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe- sub cluster (2FeH) as the active site. The presence of such an active site can for example be confirmed by Fourier Transform Infrared (FTIR) spectroscopy, in which it results in a signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1in the presence of oxygen, oxidative stress and / or oxidative potentials. The general applicability of the present invention has been demonstrated for three different hydrogenases, namely CbA5H of Clostridium beijerinckii, Hnd of Desulfovibrio fructosovorans and CpIII of Clostridium pasteurianum. Accordingly, it has been made plausible that protection from oxygen or oxidative potentials can be achieved by the composition of the present invention for any hydrogenase as long as the protection mechanism corresponds to that of the hydrogenases mentioned above and in the examples.

[0030] The invention, inter alia, provides the following items:

[0031] 1. A composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.2. The composition of item 1, wherein the hydrogenase is a hydrogenase sensitive to oxidative stress induced by oxygen and / or oxidative potentials.

[0032] 3. The composition of item 1 or 2, wherein the hydrogenase has a side chain capable of protecting its active site from oxygen, oxidative stress and / or oxidative potentials.

[0033] 4. The composition of item 3, wherein the side chain is selected from a cysteine side chain, an aspartic acid side chain, a glutamic acid side chain or a serine side chain, preferably a cysteine side chain.

[0034] 5. The composition of any one of items 1 to 4, wherein the hydrogenase is a hydrogenase having a cysteine side chain capable of coordinating the substrate binding site of its active site, thereby protecting its active site from oxygen, optionally wherein the protection is independent of diffusion channels for gas transport within the enzyme.

[0035] 6. The composition of any one of items 1 to 5, wherein the hydrogenase is a hydrogenase having a cysteine side chain capable of reversibly coordinating its substrate binding diiron active site.

[0036] 7. The composition of any one of items 1 to 6, wherein the hydrogenase is a [FeFe]-hydro- genase.

[0037] 8. The composition of any one of items 1 to 7, wherein the active site of the hydrogenase is a H-cluster comprising a [4Fe4S]-cluster (4FeH) coupled to a uniquely ligated dinu- clear FeFe-subcluster (2FeH).

[0038] 9. The composition of any one of items 1 to 8, wherein the active site of the hydrogenase exhibits a Fourier Transform Infrared (FTIR) spectroscopy signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1in the presence of oxygen, oxidative stress and / or oxidative potentials.

[0039] 10. The composition of any one of items 1 to 9, wherein the hydrogenase is selected from the group of CbA5H of Clostridium beijerinckii. Hnd of Desulfovibrio fructosovorans and CpIII of Clostridium pasteurianum or variants thereof having a sequence identity of at least about 90%.

[0040] 11. The composition of any one of items 1 to 10, wherein the hydrogenase is a hydrogenase having an amino acid sequence set forth in any one of SEQ ID NOs: 1-3, or variants thereof having a sequence identity of at least about 90%.12. The composition of any one of items 1 to 11, wherein the RAM has a redox potential of about 0.13 to about -1.86 V vs. RHE, preferably at a pH of about 7.0.

[0041] 13. The composition of any one of items 1 to 12, wherein the RAM comprises a redox- polymer, a redox-dendrimer and / or a redox-active lipidic mesophase.

[0042] 14. The composition of item 13, wherein the redox-polymer comprises polyvinyl alcohol (PVA), polyvinyl imidazole (PVI), polyvinyl imine (PEI) and / or polyvinyl pyridine (PVP).

[0043] 15. The composition of item 13, wherein the redox-dendrimer comprises a symmetrical or asymmetrical dendrimer with 8, 16 or 32 linking positions, such as a poly(amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a ester functionalized dendrimer, a polypropylene imine (PPI) dendrimer, a poly(aryl ether) dendrimers, a poly(benzyl ether) dendrimer and / or a peptide dendrimer.

[0044] 16. The composition of item 13, wherein the redox-active lipidic mesophase comprises a carboxylic acid and a hydrophobic hydrocarbon.

[0045] 17. The composition of any one of items 13 to 16, wherein the redox-polymer, the redoxdendrimer and / or the redox-active lipidic mesophase further comprises a viologen moiety.

[0046] 18. The composition of item 17, wherein the viologen moiety is linked directly or indirectly, e.g. via an additional linking group, to the polymer, the dendrimer and / or the lipidic mesophase.

[0047] 19. The composition of item 17 or 18, wherein the viologen moiety is selected from

[0048]

[0049] wherein R is selected from CH3, (CH2)nR2, or R2,

[0050] wherein R2 is selected from H, OH, COOH, SOAc or CN, and

[0051] wherein n can be any number, preferably n is between 1 and 25, and / or

[0052]

[0053] wherein R is selected from H, CH3, (CH2)nR2, or R2,

[0054] wherein R2 is selected from OH, COOH, SOAc or CN, and

[0055] wherein n can be any number, preferably n is between 1 and 25.

[0056] 20. The composition of any one of items 17 to 19, wherein the viologen moiety is selected from Table 1.

[0057] 21. The composition of any one of items 1 to 20, wherein the RAM is selected from Table 2.

[0058] 22. The composition of any one of items 1 to 21, wherein the RAM comprises a 2,2’-vio- logen moiety

[0059]

[0060] linked to a polyvinyl alcohol (PVA), preferably n is between 1 and 25.

[0061] 23. The composition of any one of items 1 to 21, wherein the RAM comprises a poly(ami- doamine) (PAMAM) dendrimer core

[0062]

[0063] wherein R is a 2,2’ -viologen moiety (DV2),

[0064]

[0065] The composition of any one of items 1 to 21, wherein the RAM comprises a redoxactive lipidic mesophase (RLM) with the formula

[0066]

[0067] The composition of any one of item 22 to 24, wherein:

[0068] (i) DV2 has a reduction potential of about -0.40 V vs. SHE and / or about 0.014 V vs. RHE,(ii) PV2 has a reduction potential of about -0.43V vs. SHE and / or about -0.016 V vs. RHE, and / or

[0069] (iii) RLM has a reduction potential of about -0.285 V vs. SHE and / or about 0.13 V vs. RHE,

[0070] preferably at a pH of about 7.0.

[0071] 26. The composition of any one of items 1 to 25, wherein the hydrogenase is embedded in the redox-active matrix.

[0072] 27. The composition of any one of items 1 to 26, wherein the reduction potential relative to a reversible hydrogen electrode (V vs. RHE) is the reduction potential relative to a reversible hydrogen electrode (V vs. RHE) at a pH of about 7.0.

[0073] 28. A method for reduction of protons comprising catalyzing the reduction with the composition of any one of items 1 to 27.

[0074] 29. A method for oxidation of hydrogen comprising catalyzing the oxidation with the composition of any one of items 1 to 27.

[0075] 30. Use of the composition of any one of items 1 to 27 in the oxidation of hydrogen, particularly in a fuel cell.

[0076] 31. Use of the composition of any one of items 1 to 27 in the reduction of protons, particularly for the electrolysis of water, more particularly to produce hydrogen.

[0077] 32. A RAM comprising a redox-active lipidic mesophase (RLM) with the formula

[0078]

[0079] 33. A method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with the redox-active matrix of item 32.

[0080] 34. The method of item 33, wherein the hydrogenase is a hydrogenase sensitive to oxidative stress induced by oxygen and / or oxidative potentials.35. The method of item 33 or 34, wherein the hydrogenase has a side chain capable of protecting its active site from oxidative stress induced by oxygen and / or oxidative potentials.

[0081] 36. The method of item 35, wherein the side chain is selected from a cysteine side chain, an aspartic acid side chain, a glutamic acid side chain or a serine side chain.

[0082] 37. The method of any one of items 33 to 36, wherein the hydrogenase is a hydrogenase having a cysteine side chain capable of coordinating the substrate binding site of its active site, thereby protecting its active site from oxygen.

[0083] 38. The method of any one of items 33 to 37, wherein the hydrogenase is a hydrogenase having a cysteine side chain capable of reversibly coordinating its substrate binding diiron active site.

[0084] 39. The method of any one of items 33 to 38, wherein the hydrogenase is a [FeFe]-hydro- genase.

[0085] 40. The method of any one of items 33 to 39, wherein the active site of the hydrogenase is a H-cluster comprising a [4Fe4S] -cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe- sub cluster (2FeH).

[0086] 41. The method of any one of items 33 to 40, wherein the active site of the hydrogenase exhibits a Fourier Transform Infrared (FTIR) spectroscopy signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1in the presence of oxygen, oxidative stress and / or oxidative potentials.

[0087] 42. The method of any one of items 33 to 41, wherein the hydrogenase is selected from the group of CbA5H of Clostridium beijerinckii. Hnd of Desulfovibrio fructosovorans and CpIII of Clostridium pasteurianum or variants thereof having a sequence identity of at least about 90%.

[0088] 43. The method of any one of items 33 to 42, wherein the hydrogenase is a hydrogenase having an amino acid sequence set forth in any one of SEQ ID NOs: 1-3, or variants thereof having a sequence identity of at least about 90%.

[0089] 44. The method of any one of items 33 to 43, comprising protecting the hydrogenase from oxygen during storage of the hydrogenase and / or resting stages during the reduction of protons or oxidation of hydrogen.45. Use of the RAM of item 32 for protecting a hydrogenase from oxygen.

[0090] 46. A fuel cell comprising an anode, a cathode, an electrolyte and the composition of any one of items 1 to 27.

[0091] 47. The fuel cell of item 46, wherein the anode is coated with the composition of any one of items 1 to 27.

[0092] 48. A method for producing electricity comprising catalyzing the oxidation of hydrogen in the fuel cell of item 46 or 47.

[0093] 49. A device (electrolyzer) for the production of hydrogen comprising an anode, a cathode, an electrolyte and the composition of any one of items 1 to 27.

[0094] 50. The device of item 49, wherein the cathode is coated with the composition of any one of items 1 to 27.

[0095] 51. A method for producing hydrogen comprising catalyzing the reduction of protons with the device of item 49 or 50.

[0096] Composition

[0097] Accordingly, the present invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix, wherein the reduction potential of the RAM is identical to or lower than the inactivation potential of the hydrogenase.

[0098] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0099] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0100] The subject matter described is a composition designed to facilitate the reduction of protons and / or the oxidation of hydrogen. This composition includes two primary components: a hydrogenase and a redox-active matrix. Hydrogenase is an enzyme that catalyzes the reversible oxidation of molecular hydrogen. RAM is a material capable of undergoing redox reactions, which means it can either gain or lose electrons. The matrix in the composition can have a specific reduction potential, which is a measure of its tendency to acquire electrons and thereby be reduced. However, the oxygen protection of the hydrogenase can be achieved as long as thereduction potential of the redox-active matrix is identical to or lower than the inactivation potential of the hydrogenase.

[0101] As used herein “catalyze” refers to the enzyme' s / hydrogenase’s ability to accelerate redox reactions involving hydrogen. As used herein “reduction of protons” refers to hydrogen evolution reactions, wherein single protons forming the core of the hydrogen atom are reduced to molecular hydrogen H2. In particular, the following reactions are envisioned:

[0102] As used herein “oxidation of hydrogen” generally refers to the following reaction:

[0103] H2→ 2 H++ 2 e−

[0104] As used herein “production / evolution of hydrogen” generally refers to the following reaction:

[0105] 2 H++ 2 e−→ H2

[0106] Hydrogenase

[0107] Hydrogenases are enzymes that catalyze the reversible oxidation of molecular hydrogen H2. As discussed above, it previously has not been possible to efficiently use hydrogenases in hydrogen evolution or oxidation. This is because hydrogenases are sensitive to oxygen and oxidative potentials. The present invention overcomes this prejudice. A hydrogenase in the sense of the present invention is preferably a hydrogenase sensitive to oxidative stress induced by oxygen and / or oxidative potentials. Such hydrogenases can efficiently be protected in the composition of the present invention via the co-formulation with a redox-active matrix. Furthermore, the hydrogenase may have a side chain capable of protecting its active site from oxygen, oxidative stress and / or oxidative potentials. However, this intrinsic protection mechanism against oxygen leads to some irreversible enzyme inactivation leading to a further decrease of the catalytic activity when repeatedly exposed to oxygen, e.g. air. This is solved by the RAM which protects from oxygen and therefore takes advantage of the intrinsic protection mechanism. The side chain capable of protecting the hydrogenase active site from oxygen, oxidative stress and / or oxidative potentials is part of this intrinsic protection mechanism. This side chain is a cysteine side chain in the hydrogenase CbA5H of Clostridium beijerinckii. however, also other side chains may contribute to the intrinsic protection mechanism in other hydrogenases; these other side chains may be an aspartic acid side chain, a glutamic acid side chain or a serine side chain. This side chain can coordinate the substrate binding site of the hydrogenase active site thereby leading to the intrinsic protection from oxygen. In general, the hydrogenase is preferably a hydrogenase having such an intrinsic protection mechanism as described above. This enables the RAM to take advantage of the intrinsic protection and further protect the hydrogenase from oxygen, oxidative stress and / or oxidative potentials when the hydrogenase is formulated with the redox-active matrix thereby avoiding irreversible enzyme inactivation and decreased catalytic activity after repeated exposure to oxygen.

[0108] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less. The hydrogenasemay be a hydrogenase sensitive to oxidative stress induced by oxygen and / or oxidative potentials. The hydrogenase may have a side chain capable of protecting its active site from oxygen, oxidative stress and / or oxidative potentials. Preferably, the side chain is selected from a cysteine side chain, an aspartic acid side chain, a glutamic acid side chain or a serine side chain. Most preferably, the side chain is a cysteine side chain. The hydrogenase may be a hydrogenase having a cysteine side chain capable of coordinating the substrate binding site of its active site, thereby protecting its active site from oxygen. The hydrogenase may advantageously be a hydrogenase having a cysteine side chain capable of reversibly coordinating its substrate binding di -iron active site.

[0109] An exemplary class of hydrogenases in the sense of the present invention are [FeFe]-hydrogen-ases. The [FeFe]-hydrogenases are known for their high efficiency in catalyzing these reactions due to their unique active sites, which consist of iron-sulfur clusters that facilitate rapid electron transfer. The RAM, with a reduction potential of about 0.13 V relative to a reversible hydrogen electrode or less, provides an optimal environment for these electron transfer processes. [FeFe]-hydrogenases are known for their higher catalytic activity compared to other types of hydrogenases, such as [NiFe]-hydrogenases. This increased activity can lead to more efficient catalysis of proton reduction and hydrogen oxidation, thereby enhancing the overall performance of the composition. [FeFe]-hydrogenases have a mechanism of action distinct from other hydrogenases involving the formation of hydride intermediates, which can facilitate faster and more efficient electron transfer processes. These hydrogenases comprise a di-iron center with a bridging dithiolate cofactor. The active site of [FeFe]-hydrogenases can comprise a so-called H-cluster comprising a [4Fe4S]-cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe-sub-cluster (2FeH). The [4Fe4S]-cluster serves as an electron relay, facilitating the transfer of electrons within the hydrogenase. This cluster is involved in the hydrogenase's ability to catalyze the reduction of protons and / or the oxidation of hydrogen. The uniquely ligated dinuclear FeFe-subcluster (2FeH) is involved in the binding and activation of hydrogen molecules. The coupling of these two clusters within the H-cluster allows for efficient electron transfer and proton-coupled electron transfer processes, which are important for the catalytic activity of the hydrogenase. The specific mechanism of communication between the [4Fe4S]-cluster and the FeFe-subcluster involves the relay of electrons through the iron-sulfur bonds, which is facilitated by the unique ligation of the FeFe-subcluster.

[0110] This active site configuration can be identified with Fourier Transform Infrared (FTIR) spectroscopy and results in a specific signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1in the presence of oxygen, oxidative stress and / or oxidative potentials. The FTIR spectroscopy signature serves as a distinct identifier for hydrogenases comprising the corresponding active site. The presence of these specific FTIR bands indicates the structural and electronic environment of the active site, which can be correlated with its catalytic activity and stability. Hydrogenases falling within this definition can successfully be used in the composition of the present invention.

[0111] Accordingly, the invention provides a composition for catalyzing reduction of protons and / oroxidation of hydrogen comprising a [FeFe] -hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0112] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase, e.g. a [FeFe] -hydrogenase, and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the active site of the hydrogenase is a H-cluster comprising a [4Fe4S]-cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe-subcluster (2FeH).

[0113] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase, e.g. a [FeFe] -hydrogenase, and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the active site of the hydrogenase exhibits a Fourier Transform Infrared (FTIR) spectroscopy signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm-1 in the presence of oxygen, oxidative stress and / or oxidative potentials.

[0114] Exemplary [FeFe]-hydrogenases in the sense of the invention are HydA1 and HydA2 from Chlamydomonas reinhardtii, CpI and CpIII from Clostridium pasteurianum, DdH from Desulfovibrio desulfuricans, CaHydA from Clostridium acetobutylicum, CbA5H from Clostridium beijerinckii, HydA from Shewanella oneidensis, HydS from Thermotoga maritima, and Hnd from Desulfovibrio fructosovorans. Preferred [FeFe]-hydrogenases of the present invention are CbA5H from Clostridium beijerinckii, Hnd from Desulfovibrio fructosovorans and CpIII from Clostridium pasteurianum.

[0115] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the hydrogenase is selected from HydA1 and HydA2 from Chlamydomonas reinhardtii, CpI and CpIII from Clostridium pasteurianum, DdH from Desulfovibrio desulfuricans, CaHydA from Clostridium acetobutylicum, CbA5H from Clostridium beijerinckii, HydA from Shewanella oneidensis, HydS from Thermotoga maritima, and Hnd from Desulfovibrio fructosovorans, or variants thereof having a sequence identity of at least about 90%.

[0116] Also envisioned herein are variants of the mentioned hydrogenases that share a substantial part of the sequence identity. Thus, the hydrogenase may be selected from HydA1 and HydA2 from Chlamydomonas reinhardtii, CpI and CpIII from Clostridium pasteurianum, DdH from Desulfovibrio desulfuricans, CaHydA from Clostridium acetobutylicum, CbA5H from Clostridium beijerinckii, HydA from Shewanella oneidensis, HydS from Thermotoga maritima, and Hnd from Desulfovibrio fructosovorans or variants of thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99% sequence identity. The hydrogenase may be a hydrogenase having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at leastabout 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99% to a hydrogenase selected from HydA1 and HydA2 from Chlamydomonas reinhardtii, CpI and CpIII from Clostridium pasteurianum, DdH from Desulfovibrio desulfuricans, CaHydA from Clostridium acetobutylicum, CbA5H from Clostridium beijerinckii, HydA from Shewanella oneidensis, HydS from Thermotoga maritima, and Hnd from Desulfovibrio fructosovorans.

[0117] An exemplary amino acid sequence of CbA5H from Clostridium beijerinckii is set forth in SEQ ID NO: 1. An exemplary amino acid sequence of Hnd of Desulfovibrio fructosovorans is set forth in SEQ ID NO: 2. An exemplary amino acid sequence of CpIII from Clostridium pasteur-ianum is set forth in SEQ ID NO: 3.

[0118] Also envisioned herein are variants of the preferred hydrogenases defined in any one of SEQ ID NOs: 1-3. Accordingly, the hydrogenase may be a hydrogenase having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99% to any one of SEQ ID NOs: 1-3. Preferably, the hydrogenase is a hydrogenase having a sequence identity of at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% to SEQ ID NO: 1.

[0119] Example 3 has demonstrated that also mutated versions of hydrogenases can be functional. In Example 3 the M382E mutation, i.e. an amino acid exchange of the wild type methionine to glutamic acid has been performed, is described resulting in increased oxygen resistance (Rutz et al. 2022). Accordingly, it is plausible that also hydrogenases whose sequence is adapted work in the sense of the present invention, as long as the catalytic activity is still maintained at some level. Accordingly, the hydrogenase preferably is a hydrogenase defined in SEQ ID NO: 1 having a M382E mutation.

[0120] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii or a variant thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0121] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii as defined in SEQ ID NO: 1 or a variant thereof having a sequence identity of at least about 90%, preferably atleast about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0122] Further, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a Hnd hydrogenase of Desulfovibrio fructosovorans or a variant thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0123] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a Hnd hydrogenase of Desulfovibrio fructosovorans as defined in SEQ ID NO: 2 or a variant thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0124] Furthermore, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CpIII hydrogenase of Clostridium pasteurianum or a variant thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0125] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CpIII hydrogenase of Clostridium pasteurianum as defined in SEQ ID NO: 3 or a variant thereof having a sequence identity of at least about 90%, preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, or most preferably at least about 99%, and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less.

[0126] As used herein “a variant thereof having a sequence identity of at least about 90%” refers to a variant of the hydrogenase or the specific sequence mentioned having a sequence identity of at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%,more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, or most preferably at least 99% to the mentioned hydrogenase or specific sequence. Thus, variants with slight modifications or naturally occurring variations of the hydrogenases are envisioned herein, provided they retain the essential functional characteristics, namely catalyzing the reduction of protons and / or the oxidation of hydrogen.

[0127] The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.

[0128] 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpetc / a / ., Nucleic Acids’ Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

[0129] Sequence identity between nucleic acid sequences, or between amino acid sequences, can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same nucleotide, or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

[0130] Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29;), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410;), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: thetheory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley). All programs may be run using default parameters.

[0131] For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.

[0132] Redox-active matrix

[0133] The redox-active matrix (RAM) refers to a material capable of undergoing redox reactions, which means it can either gain or lose electrons. The redox-active matrix facilitates electron transfer to and from the hydrogenase, providing a suitable electrochemical environment for the hydrogenase, offering protection against oxidative stress by maintaining a reducing environment. The redox-active matrix does not only facilitate electron transfer but also modulates the local redox conditions in favor of the stability and activity of the hydrogenase by preventing inactivation via oxygen exposure. This is beneficial for applications e.g. in a fuel cell or eletro-lyzer where the presence of oxygen or oxidative potentials is unavoidable.

[0134] The specific mechanisms of communication between the hydrogenase and the redox-active matrix involve electron transfer processes, where the matrix acts as an electron donor or acceptor, facilitating the catalytic cycle of the hydrogenase, and facilitating electron transfer between the hydrogenase, the matrix and an anode or cathode.

[0135] The potential of the RAM can be finely tuned to allow protection of the hydrogenase from oxygen and oxidative potentials under anaerobic / operating, aerobic / operating and aerobic / non-operating conditions. The RAM has a reduction potential of about 0.13 V vs. RHE or less. Preferably, the RAM has a reduction potential of about 0.13 to about -1.86 V vs. RHE, more preferably at a pH of about 7.0. Advantageously, the redox-active matrix has a reduction potential of about 0.13 to about -1.86 V vs. RHE or less, more preferably at a pH of about 7.0. Accordingly, exemplarily RAM may have a reduction potential of about 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0, -0.01, -0.02, -0.03, -0.04, -0.05, -0.06, -0.07, -0.08, -0.09, -0.1, -0.11, -0.12, -0.13, -0.14, -0.15, -0.16, -0.17, -0.18, -0.19, -0.2, -0.21, -0.22, -0.23, -0.24, -0.25, -0.26, -0.27, -0.28, -0.29, -0.3, -0.31, -0.32, -0.33, -0.34, -0.35, -0.36, -0.37, -0.38, -0.39, -0.4, -0.41, -0.42, -0.43, -0.44, -0.45, -0.46, -0.47, -0.48, -0.49, -0.5, -0.51, -0.52, -0.53, -0.54, -0.55, -0.56, -0.57, -0.58, -0.59, -0.6, -0.61, -0.62, -0.63, -0.64, -0.65, -0.66, -0.67, -0.68, -0.69, -0.7, -0.71, -0.72, -0.73, -0.74, -0.75, -0.76, -0.77, -0.78, -0.79, -0.8, -0.81, -0.82, -0.83, -0.84, -0.85, -0.86, -0.87, -0.88, -0.89, -0.9, -0.91, -0.92, -0.93, -0.94, -0.95, -0.96, -0.97, -0.98, -0.99, -1, -1.01, -1.02, -1.03, -1.04, -1.05, -1.06, -1.07, -1.08, -1.09, -1.1, -1.11, -1.12, -1.13, -1.14, -1.15, -1.16, -1.17, -1.18, -1.19, -1.2, -1.21, -1.22, -1.23, -1.24, -1.25, -1.26, -1.27, -1.28, -1.29, -1.3, -1.31, -1.32, -1.33, -1.34, -1.35, -1.36, -1.37, -1.38, -1.39, -1.4, -1.41, -1.42, -1.43, -1.44, -1.45, -1.46, -1.47, -1.48, -1.49, -1.5, -1.51, -1.52, -1.53, -1.54, -1.55, -1.56, -1.57, -1.58, -1.59, -1.6, -1.61, -1.62, -1.63, -1.64, -1.65, -1.66, -1.67, -1.68, -1.69, -1.7, -1.71,-1.72, -1.73, -1.74, -1.75, -1.76, -1.77, -1.78, -1.79, -1.8, -1.81, -1.82, -1.83, -1.84, -1.85, or -1.86 V vs. RHE or less, preferably at a pH of about 7.0, wherein reduction potentials of about 0.13, 0.014 and -0,016 V vs. RHE or less are preferred herein. Preferably, the RAM may have a reduction potential of about 0.194 V vs. RHE or less, preferably at a pH of about 7.0. Advantageously, the RAM may have a reduction potential of about 0.014 V vs. RHE or less, preferably at a pH of about 7.0. Preferably, the RAM may have a reduction potential of about -0.016 V vs. RHE or less, preferably at a pH of about 7.0. Advantageously, the RAM may have a reduction potential of about 0.13 V vs. RHE or less, preferably at a pH of about 7.0.

[0136] Preferably, the RAM may have a reduction potential of about -0.22 V vs. SHE or less. Advantageously, the RAM may have a reduction potential of about -0.40 V vs. SHE or less. Preferably, the RAM may have a reduction potential of about -0.43 V vs. SHE or less. Advantageously, the RAM may have a reduction potential of about -0.285 V vs. SHE or less. Preferably, the RAM may have a reduction potential of about 0.170 V vs. RHE or less.

[0137] In general, the potential of the RAM can be expressed as V vs. RHE or V vs. SHE. V vs. SHE refers to potentials measured relative to the Standard Hydrogen Electrode (SHE), which is defined as 0 V under standard conditions (25°C, 1 atm H2, 1 M H+activity, pH 0). SHE is pH-independent. A potential reported vs. SHE remains constant regardless of the solution's pH. V vs. RHE (Reversible Hydrogen Electrode) refers to potentials measured relative to a hydrogen electrode reversible in the same solution, accounting for its pH. RHE shifts with pH according to the Nernst equation at 25°C: ERHE= ESHE- 0.059 × pH. Accordingly, V vs. SHE provides a universal reference (pH 0), while V vs. RHE adjusts for solution pH, enabling direct comparison of electrochemical behavior across different environments. Both values are envisioned herein. Accordingly, whenever a potential is mentioned as V vs. RHE also the corresponding V vs. SHE value is disclosed herein. A V vs. RHE value can be converted to V vs. SHE value by the following formula at a given pH: ESHE= ERHE+ 0.059 × pH. The pH dependency of hydrogenases and redox-active matrices of the present invention is demonstrated in Example 5.

[0138] In general, the RAM can comprise any components that allow the RAM to acquire a specific and stable potential. This potential should be lower that the inactivation potential of the hydrogenase. In the example of the CbA5H hydrogenase of Clostridium beijerinckii the inactivation potential is at about 0.170 V vs. RHE, a corresponding RAM for use in a composition with CbA5H should thus have a reduction potential of about 0.170 V vs. RHE or less.

[0139] The RAM comprises redox-moieties, e.g. viologens, and redox-silent compounds to fixate the redox-moieties. In general, the RAM can comprise redox-polymers, redox-dendrimers and / or redox active lipidic mesophases.

[0140] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase.

[0141] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii, such as defined inSEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90%, and a redox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a redox -polymer, a redox-dendrimer and / or a redox-active lipidic mesophase.

[0142] As used herein “polymer” is a large molecule composed of repeating structural units (monomers) connected by covalent bonds. Polymers can be natural (e.g., DNA, proteins) or synthetic (e.g., plastics, rubber). The redox-polymer, being a polymeric material with redox-active sites, facilitates electron transfer through its conjugated or non-conjugated backbone, enhancing the efficiency of electron relay between the hydrogenase and an electrode or other components. This polymeric structure can offer flexibility, processability, and the potential for forming thin films or coatings, which can be advantageous in various applications. A polymer preferably forms a hydrogel. Exemplary polymers in the sense of the present invention comprise polyvinyl alcohol (PVA), polyvinyl imidazole (PVI), polyvinyl imine (PEI) and / or polyvinyl pyridine (PVP). In general, any polymer backbone can be used in the sense of the present invention. The main criterion for the redox-polymer is to have a functional group that allows for linking of a viologen moiety, such as alcohols, amines, imidazoles. In general, the chain length of the polymer does not influence the redox-potential of the redox-active matrix, but can be used to finetune the stability of the same.

[0143] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a redox-polymer, comprising polyvinyl alcohol (PVA), polyvinyl imidazole (PVI), polyvinyl imine (PEI) and / or polyvinyl pyridine (PVP).

[0144] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90%, and a redox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, comprising polyvinyl alcohol (PVA), polyvinyl imidazole (PVI), polyvinyl imine (PEI) and / or polyvinyl pyridine (PVP).

[0145] As used herein “dendrimer” refers to a highly branched, symmetric or asymmetric macromolecule with a tree-like structure. It comprises a central core, branching units, and terminal functional groups. The redox-dendrimer, characterized by its highly branched, tree-like structure with multiple redox-active end groups, provides a high density of redox sites in a confined space. This unique architecture allows for efficient electron transfer due to the proximity of the redox-active sites to the hydrogenase. Additionally, the dendrimer's structure can be tailored to optimize the interaction with the hydrogenase and the overall stability of the composition. This can be done by fine tuning its redox-potential via the redox-active end groups, e.g. viologens.Exemplary redox-dendrimers in the sense of the present invention can be symmetrical or asymmetrical, this does not result in a change of the electrochemical property of the redox-dendrimer. Dendrimers can be divided into different generations, wherein generation 1 has 8 linking positions, generation 2 has 16 linking positions and generation 3 which has 32 linking positions. Dendrimers of all three generations can be used to achieve a redox-potential suitable for catalytic H2and H+conversion with hydrogenases. The type of the dendrimer does not influence its suitability as a redox-dendrimer in the sense of the present invention, therefore any generation with any number of linking positions and type of dendrimer can be used to synthesize a redoxdendrimer as a redox-active matrix in the composition of the present invention. As long as the redox-dendrimer has a reduction potential that is identical to or lower than the inactivation potential of the hydrogenase it will work in the sense of the invention. Exemplary dendrimers that may be used in the present invention are a poly(amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a ester functionalized dendrimer, a polypropylene imine (PPI) dendrimer, a poly(aryl ether) dendrimers, a poly(benzyl ether) dendrimer and / or a peptide dendrimer. A dendrimer in the sense of the present invention may also comprise poly(ethylenimine).

[0146] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly (amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a ester functionalized dendrimer, a polypropylene imine (PPI) dendrimer, a poly(aryl ether) dendrimers, a poly(benzyl ether) dendrimer and / or a peptide dendrimer.

[0147] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90%, and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly(amidoamine) (PAMAM) dendrimer, a polyester dendrimer, a ester functionalized dendrimer, a polypropylene imine (PPI) dendrimer, a poly(aryl ether) dendrimers, a poly(benzyl ether) dendrimer and / or a peptide dendrimer.

[0148] As used herein “lipidic mesophase” refers to a self-assembled, ordered structure formed by lipids in water, exhibiting properties between a liquid and a solid. The redox-active lipidic (RLM) mesophase comprises lipid molecules organized in a mesophase structure, offering a distinct mechanism of electron transfer through its ordered yet dynamic arrangement. This mesophase can provide a biocompatible environment that mimics natural biological membranes. In general, the lipidic mesophase comprises a carboxylic acid and a hydrophobic hydrocarbon. The redox-potential of the RLM is defined when it is equipped with a viologen. The viologen moiety of the lipidic mesophase can be modified, e.g. can be exchanged for fine-tuning the redox potential of the RLM.The above mentioned redox-active matrices can further comprise a viologen moiety. A viologen is a class of organic compounds characterized by their structure, which consists of two nitrogencontaining heterocycles (typically pyridinium rings) connected by a bridge, often a methylene group (-CH2-). Viologens are dicationic (carry a +2 charge) in their oxidized state and can be reduced to form radical cations or neutral species.

[0149] The viologen moiety acts as an electron mediator, facilitating efficient electron transfer between the hydrogenase and the RAM. The viologen moiety is known for its excellent redox properties, including a stable and reversible redox couple, which makes it an ideal candidate for facilitating electron transfer processes. By incorporating the viologen moiety, the redox-potential of the RAM can be finely tuned resulting in a potential range where the hydrogenase can successfully catalyze the reduction of protons and / or oxidation of hydrogen. Additionally, the presence of the viologen moiety contributes to the stabilization of the potential of the RAM, thereby ensuring that the hydrogenase maintains its catalytic activity over a prolonged period. This stabilization is beneficial for practical applications in a fuel cell or electrolyzer where the composition is exposed to oxygen and oxidative potentials. Furthermore, the inclusion of a viologen moiety can also enhance the solubility and dispersibility of the redox-active components within the composition, promoting a more homogeneous distribution and interaction with the hydrogenase enzyme. This homogeneity can contribute to the catalytic efficiency.

[0150] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and wherein the redox-polymer, the redox-dendrimer and / or the redox-active lipidic mesophase further comprises a viologen moiety.

[0151] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and wherein the redox-polymer, the redox-dendrimer and / or the redox-active lipidic mesophase further comprises a viologen moiety.

[0152] The viologen moiety can be directly or indirectly linked to the polymer, dendrimer or lipidic mesophase. For example, a viologen moiety may be directly linked to a polymer, dendrimer or lipidic mesophase backbone or may be linked to the polymer, dendrimer or lipidic mesophase backbone via an additional linking or functional group. Exemplary linking or functional groups in the sense of the present invention are CH3, (CH2)nR2, or R2, wherein R2 is selected from H, OH, COOH, SOAc or CN. n can be any number, preferably n is between 1 and 25.Furthermore, linking or functional groups may be selected from the following:

[0153]

[0154] wherein R is CH3, (CH2)nR2, or R2, wherein R2 is selected from H, OH, COOH, SOAc or CN, or wherein R is the polymer backbone, dendrimer backbone or lipidic mesophase backbone, n can be any number, preferably n is between 1 and 25.

[0155] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase further comprises a viologen moiety, preferably wherein the viologen moiety is linked directly or indirectly, e.g. via an additional linking group, to the polymer, the dendrimer and / or the lipidic mesophase. The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase further comprises a viologen moiety, preferably wherein the viologen moiety is linked directly or indirectly, e.g. via an additional linking group, to the polymer, the dendrimer and / or the lipidic mesophase.

[0156] In general, any viologen can be used to fine tune the redox-potential of the RAM. However, exemplary viologens that can be used in the sense of the present invention are

[0157]

[0158] wherein R is selected from CH3, (CH2)nR2, or R2,

[0159] wherein R2 is selected from H, OH, COOH, SOAc or CN, orwherein R or R2 is selected from any of the following functional groups

[0160]

[0161] and wherein n can be any number. In principle, n can be up to several hundreds or thousands, e.g. n is between 1 and 5000, between 1 and 1000, between 1 and 100, between 1 and 25, between 1 and 16. Typically, n will be between 1 and 25.

[0162] Further exemplary viologens that can be used in the sense of the present invention are

[0163]

[0164] wherein R is selected from H, CH3, (CH2)nR2, or R2,

[0165] wherein R2 is selected from OH, COOH, SOAc or CN, or

[0166] wherein R or R2 is selected from any of the following functional groups

[0167]

[0168] and wherein n can be any number. Again, in principle, n can be up to several hundreds or thousands, e.g. n is between 1 and 5000, between 1 and 1000, between 1 and 100, between 1 and 25, between 1 and 16. Typically, n will be between 1 and 25.Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a

[0169]

[0170] reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and a viologen moiety, wherein the viologen moiety is selected from

[0171] wherein R is selected from CH3, (CH2)nR2, or R2,

[0172] wherein R2 is selected from H, OH, COOH, SOAc or CN, or

[0173] wherein R or R2 is selected from any of the following functional groups

[0174]

[0175] and

[0176] wherein n can be any number, preferably n is between 1 and 25, and / or

[0177]

[0178] wherein R is selected from H, CH3, (CH2)nR2, or R2,

[0179] wherein R2 is selected from OH, COOH, SOAc or CN, orwherein R or R2 is selected from any of the following functional groups

[0180]

[0181] and

[0182] wherein n can be any number, preferably n is between 1 and 25.

[0183] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a redox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and a viologen moiety, wherein the viologen moiety is selected from

[0184]

[0185] wherein R is selected from CH3, (CH2)nR2, or R2,

[0186] wherein R2 is selected from H, OH, COOH, SOAc or CN, or

[0187] wherein R or R2 is selected from any of the following functional groups

[0188]

[0189] and

[0190] wherein n can be any number, preferably n is between 1 and 25, and / or

[0191]

[0192] wherein R is selected from H, CH3, (CH2)nR2, or R2,

[0193] wherein R2 is selected from OH, COOH, SOAc or CN, or

[0194] wherein R or R2 is selected from any of the following functional groups

[0195]

[0196] and

[0197] wherein n can be any number, preferably n is between 1 and 25.

[0198] Exemplary viologens that can be used in the sense of the present invention are provided in Table 1Table 1: Viologen moi eties of the present invention

[0199]

[0200]

[0201]

[0202]

[0203] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and a viologen moiety, wherein the viologen moiety is selected from Table 1 or Figure 8. The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer, a redox-dendrimer and / or a redox-active lipidic mesophase and a viologen moiety, wherein the viologen moiety is selected from Table 1 or Figure 8.Exemplary redox-active matrices are provided in Table 2.

[0204] Table 2: Exemplary redox-active matrices based on polymers or dendrimers linked to viologen moieties. Abbreviations: PVI = polyvinyl imidazole; PVP = polyvinyl pyridine; PVA = polyvinyl alcohol; PAMAM = poly(amidoamine) dendrimer.

[0205]

[0206]

[0207]

[0208] Accordingly, the invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a redox-polymer or a redox-dendrimer selected from Table 2 or Figure 9.

[0209] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a redox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-polymer or a redoxdendrimer selected from Table 2 or Figure 9.

[0210] Preferably, the viologen may be a 2,2’ -viologen moiety with the following structure

[0211]

[0212] wherein n can be any number, preferably n is between 1 and 25.

[0213] This viologen moiety can preferably be linked to a polyvinyl alcohol resulting in the redoxactive matrix termed “PV2” herein. PV2 has been used in Example 1 where it has been demonstrated that the redox-active matrix protects the Cb A5H hydrogenase of Clostridium beijerinckii oxygen and oxidative potentials in the presence of H₂. Full protection of the hydrogenase was achieved not only during H₂-oxidation catalysis (aerobic or anaerobic) but also during intermediate resting stages or storage.

[0214] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a 2,2’ -viologen moiety

[0215]

[0216] linked to a polyvinyl alcohol (PV2), wherein n can be any number, preferably n is between 1 and 25.

[0217] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii, such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and aredox-active matrix having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the redox-active matrix comprises a 2,2’ -viologen moiety

[0218]

[0219] linked to a polyvinyl alcohol (PV2), wherein n can be any number, preferably n is between 1 and 25.

[0220] Preferably, the viologen may be a 4,4’ -viologen moiety with the following structure

[0221]

[0222] This viologen moiety can be linked to a poly(amidoamine) (PAMAM) dendrimer core.

[0223] Preferably, the viologen may be a 2,2’ -viologen moiety with the following structure

[0224]

[0225] These viologen moi eties can be linked to a poly(amidoamine) (PAMAM) dendrimer core. ThePAMAM dendrimer may have the following structure

[0226]

[0227] wherein R represents the linked viologen moieties.

[0228] The 4,4’-viologen moieties linked to PAMAM results in the redox-active matrix termed “DVT” herein. DV1 has been shown in Example 1 and 5 as well as Figure 6 to promote EE oxidation via CbA5H under acidic conditions.

[0229] The 2,2’ -viologen moieties linked to PAMAM result in the redox-active matrix termed “DV2” herein. DV2 has been used in Example 1, where it has been demonstrated that the redox-active matrix fully protects the CbA5H hydrogenase of Clostridium beijerinckii under anaerobic / op-erating, aerobic / operating and aerobic / non-operating conditions.

[0230] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly(amidoamine) (PAMAM) dendrimer core

[0231]

[0232] wherein R is a 4,4’ -viologen moiety (DV1)

[0233]

[0234] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly(amidoamine) (PAMAM) dendrimer core

[0235]

[0236] wherein R is a 4,4’ -viologen moiety (DV1)

[0237]

[0238] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly(amidoamine) (PAMAM) dendrimer core

[0239]

[0240] wherein R is a 2,2’ -viologen moiety (DV2),

[0241]

[0242] The invention also provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a poly(amidoamine) (PAMAM) dendrimer core

[0243]

[0244] wherein R is a 2,2’ -viologen moiety (DV2),

[0245]

[0246] Preferably, the RAM comprises a redox active lipidic mesophase (RLM) with the following structure

[0247]

[0248] . This redox-active matrix has been used in Example 6 where it has been demonstrated that the RLM successfully protects the CbA5H hydrogenase of Clostridium beijerinckii from oxygen and high oxidative potentials. This is also reflected in Figures 7 and 10. Particularly panel e) of Figure 7 shows that catalytic activity is maintained throughout reduction of protons as wells as oxidation of molecular hydrogen. Thus, this RAM is especially beneficial to protect a hydrogenase from oxygen and oxidative potentials thereby avoiding inactivation and ensuring efficient catalysis of the reduction of protons and / or the oxidation of hydrogen.

[0249] Accordingly, the invention provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a RAM having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-active lipidic mesophase (RLM) with the formula

[0250]

[0251] The invention further provides a composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a CbA5H hydrogenase of Clostridium beijerinckii. such as defined in SEQ ID NO: 1, or a variant thereof having a sequence identity of at least about 90% and a RAM having a reduction potential of about 0.170 V relative to a reversible hydrogen electrode (V vs. RHE) or less, wherein the RAM comprises a redox-active lipidic mesophase (RLM) with the formula

[0252]

[0253] As mentioned before, the reduction potential of the redox-active matrix is important to ensure protection of the hydrogenase. In general, any reduction potential that is identical to or lower than the inactivation potential of the hydrogenase can work in the sense of the present invention. This has been demonstrated in the Examples for DV2, PV2 and RLM, which have a lower reduction potential than the inactivation potential CbA5H hydrogenase of Clostridium bei-jerinckii.

[0254] The reduction potentials of the redox-active matrices are defined by the viologen moieties. Accordingly, the redox-active matrices of the present invention have different reduction potentials, which however, all work in the sense of the present invention. As mentioned, the reduction potential can be expressed as V vs. SHE or V vs. RHE. DV1 has a reduction potential of about -0.22 V vs. SHE and / or about 0.194 V vs. RHE, DV2 has a reduction potential of about -0.40 V vs. SHE and / or about 0.014 V vs. RHE, PV2 has a reduction potential of about -0.43V vs. SHE and / or about -0.016 V vs. RHE, and RLM has a reduction potential of about -0.285 V vs. SHE and / or about 0.13 V vs. RHE. The reduction potential V vs. RHE is preferably the reduction potential at a pH of about 7.0.To achieve the beneficial effects of the present invention, the hydrogenase is formulated into the composition of the present invention together with the redox-active matrix. This can be realized by embedding the hydrogenase in the RAM, thereby facilitating mediated electron transfer. The RAM of the present invention can be in form of a hydrogel.

[0255] The invention also provides a RAM comprising a redox-active lipidic mesophase (RLM) with the formula

[0256]

[0257] . This RAM is particularly beneficial for protecting hydrogenases from oxygen and oxidative potentials. In particular, Example 6 has demonstrated that the RLM successfully protects the CbA5H hydrogenase of Clostridium beijerinckii from oxygen and high oxidative potentials. This shown in Figures 7 and 10. Particularly panel e) of Figure 7 shows that catalytic activity is maintained throughout reduction of protons as wells as oxidation of molecular hydrogen.

[0258] The invention further provides a redox-active matrix comprising any one of the polymers or dendrimers of Table 2.

[0259] Methods and uses

[0260] The composition of the present invention is particularly useful for reduction of protons and oxidation of hydrogen since the protected hydrogenase catalyzes both reactions. This allows for production of hydrogen and energy, e.g. electricity via the methods and uses of the present invention.

[0261] Accordingly, the invention provides a method for reduction of protons comprising catalyzing the reduction with the composition of the present invention. The hydrogen evolution reaction is described above.

[0262] The invention also provides a use of the composition of the present invention in the reduction of protons, particularly for the electrolysis of water, more particularly to produce hydrogen. The most common devices to produce hydrogen are electrolyzers. Exemplary electrolyzers are: (i) Proton Exchange Membrane (PEM) Electrolyzers: These use a solid polymer electrolyte and operate at relatively low temperatures. They are efficient and can produce high-purity hydrogen;

[0263] (ii) Alkaline Electrolyzers: These use an alkaline solution (usually potassium hydroxide) as the electrolyte and are known for their durability and lower cost; and

[0264] (iii) Solid Oxide Electrolyzers: These operate at high temperatures and use a ceramic electrolyte. They are highly efficient and can utilize waste heat from other processes.

[0265] The invention further provides a method for oxidation of hydrogen comprising catalyzing the oxidation with the composition of the present invention. The oxidation of molecular hydrogenis explained above.

[0266] Further, the invention provides a use of the composition of the present invention in the oxidation of hydrogen, particularly in a fuel cell.

[0267] The method and use for oxidating molecular hydrogen are particularly useful since the composition of the present invention catalyzes the underlying chemical reaction.

[0268] The invention also provides a method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with a redox-active matrix of the present invention. The features disclosed herein above in the sections “Composition”, “Hydrogenase” and “Redox-active matrix” (RAM) apply mutatis mutandis to the method for protecting a hydrogenase of the present invention. In particular, all features in context of the hydrogenase defined above in the section “Hydrogenase” apply to the hydrogenase of the method for protecting a hydrogenase mutatis mutandis. Furthermore, all features in context of the RAM defined above in the section “Redox-active matrix” apply to the RAM of the method for protecting a hydrogenase mutatis mutandis. It is explicitly disclosed herein that said features may be combined with the methods and uses herein.

[0269] Accordingly, the invention provides a method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with PV2, DV1, DV2 and / or RLM. The specific structures of PV2, DV1, DV2 and RLM are provided herein above.

[0270] The method for protecting a hydrogenase of the present invention is particularly useful since the redox-active matrices of the present invention allow for protection of the hydrogenase from oxygen and oxidative potentials under anaerobic / operating, aerobic / operating and aerobic / non-operating conditions. This provides a complete protection in all relevant situations, i.e. not only during the catalysis of the reduction of protons and / or the oxidation of hydrogen, but also when the hydrogenase is to be stored or in resting phases between operating phases. As used herein “operating phase” or “operating condition” refers to the phase or condition during which reduction of protons and / or oxidation of hydrogen takes place, i.e. a phase or condition where the composition of the present invention actively catalyzes the redox reaction of hydrogen. As used herein “non-operating phase” or “non-operating condition” refers to a phase or condition in which the reduction of protons and / or oxidation of hydrogen does not take place, i.e. a phase or condition where the composition of the present invention does not actively catalyze the redox reaction of hydrogen. Such conditions can for example be resting conditions during operating conditions, e.g. when catalysis is turned off for a certain amount of time, or storage conditions for storing the hydrogenase or preparation conditions e.g. when embedding a hydrogenase into a redox-active matrix thereby forming an enzymatic film.

[0271] Accordingly, the invention provides a method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with PV2, DV1, DV2 and / or RLM, comprising protecting the hydrogenase from oxygen during storage of the hydrogenase and / or resting stages during the reduction of protons or oxidation ofhydrogen.

[0272] The invention also provides a method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with PV2, DV1, DV2 and / or RLM, comprising protecting the hydrogenase from oxygen during anaero-bic / operating, aerobic / operating and aerobic / non-operating conditions.

[0273] Furthermore, the invention provides a use of a redox-active matrix of the present invention for protecting a hydrogenase from oxygen, oxidative stress induced by oxygen and / or oxidative potentials. Preferably, the invention provides a use of PV2, DV1, DV2 and / or RLM for protecting a hydrogenase from oxygen, oxidative stress induced by oxygen and / or oxidative potentials. The advantages and features described above in context of the method for protecting a hydrogenase of the present invention apply mutatis mutandis to the use for protecting a hydrogenase. In the sense of the present invention the methods and uses provided herein include the composition of the present invention as described in detail herein above and in particular in the sections “Composition”, “Hydrogenase” and “Redox-active matrix” (RAM). Accordingly, the features described in relation to the composition, hydrogenases and redox-active matrices in the sections “Composition”, “Hydrogenase” and “Redox-active matrix” above, apply mutatis mutandis to the methods and uses of the present invention. In particular, the methods and uses may comprise the composition of the present invention, the hydrogenases and / or the redox-active matrices described herein.

[0274] Devices

[0275] The invention also provides a fuel cell for oxidizing molecular hydrogen as well as a device, e.g. electrolyzer for producing molecular hydrogen.

[0276] A fuel cell is an electrochemical device that converts the chemical energy of hydrogen and an oxidizing agent, usually oxygen, into electricity through a pair of redox reactions. A fuel cell usually comprises at least three main components:

[0277] (i) an anode, which is the electrode where hydrogen is oxidized;

[0278] (ii) a cathode, which is the electrode where the oxygen is reduced; and

[0279] (iii) an electrolyte, which is a medium that allows ions to move between the anode and cathode while blocking electrons, forcing them to travel through an external circuit, thereby generating electricity. In the sense of the present invention a fuel cell further comprises the composition of the present invention. The following reactions take place within the fuel cell.

[0280] At the anode hydrogen gas is split into protons and electrons (H2 —> 2H++ 2e"). The protons pass through the electrolyte, while the electrons travel through an external circuit, creating an electric current. At the cathode oxygen gas or air reacts with the protons and electrons to form water (O2 + 4H++ 4e-—> 2H2O). This is the reduction reaction, and it produces water as the only byproduct. Exemplary types of fuel cells are: Proton Exchange Membrane Fuel Cell (PEMFC) with a Solid polymer membrane as an electrolyte (e.g., Nafion), Alkaline Fuel Cell (AFC) with aqueous alkaline solution as an electrolyte (e.g., potassium hydroxide), Phosphoric Acid Fuel Cell (PAFC) with liquid phosphoric acid as an electrolyte, Solid Oxide Fuel Cell(SOFC) with ceramic material as an electrolyte (e.g., yttria-stabilized zirconia), Molten Carbonate Fuel Cell (MCFC), with molten carbonate salts as an electrolyte (e.g., lithium potassium carbonate) or Direct Methanol Fuel Cell (DMFC) with a polymer membrane as an electrolyte. The fuel cell of the present invention comprises the composition of the present invention. The composition of the present invention is preferably coated onto the anode of the fuel cell. This allows the composition to catalyze the oxidation at the anode as described above.

[0281] Accordingly, the invention provides a fuel cell comprising an anode, a cathode, an electrolyte and the composition of the present invention.

[0282] The invention also provides a fuel cell comprising an anode, a cathode, an electrolyte and the composition of the present invention, wherein the anode is coated with the composition.

[0283] The fuel cell of the present invention is particularly advantageous, since it comprises the composition of the present invention thereby allowing the fuel cell to operate without expensive catalysts like platinum.

[0284] The invention also provides the corresponding method for producing electricity comprising catalyzing the oxidation of hydrogen in the fuel cell of the present invention.

[0285] The advantages and features in context of the fuel cell of the invention apply mutatis mutandis to the method for producing electricity.

[0286] Furthermore, the invention provides a device for the production of molecular hydrogen. Molecular hydrogen can for example be produced using an electrolyzer which is a device that uses electricity to split into hydrogen and oxygen through a process called electrolysis. At the anode water molecules are oxidized, releasing oxygen gas, protons, and electrons (2H2O —> O2 + 4H++ 4e-). At the cathode protons and electrons form hydrogen gas H2 (4H++ 4e-—> 2H2. Exemplary electrolyzers are Alkaline Electrolyzer (AEL) with aqueous alkaline solution (e.g., potassium hydroxide, KOH) as an electrolyte, Proton Exchange Membrane Electrolyzer (PEMEL) with a solid polymer membrane (e.g., Nafion) as an electrolyte, Solid Oxide Electrolyzer (SOEL) with ceramic material (e.g., yttria-stabilized zirconia) as an electrolyte, or Anion Exchange Membrane Electrolyzer (AEMEL) with a solid polymer membrane that conducts hydroxide ions (OH) as an electrolyte.

[0287] The device of the present invention comprises the composition of the present invention. The composition of the present invention is preferably coated onto the cathode of the device, e.g. electrolyzer. This allows the composition to catalyze the reduction at the cathode as described above.

[0288] Accordingly, the invention also provides a device, e.g. electrolyzer, for the production of hydrogen, e.g. molecular hydrogen H2, comprising an anode, a cathode, an electrolyte and the composition of the present invention.

[0289] The invention also provides a device, e.g. electrolyzer, for the production of hydrogen, e.g. molecular hydrogen H2, comprising an anode, a cathode, an electrolyte and the composition of the present invention, wherein the cathode is coated with the composition.The device, e.g. electrolyzer, of the present invention is particularly advantageous, since it comprises the composition of the present invention thereby allowing the device to operate without expensive catalysts like platinum.

[0290] The invention also provides the corresponding method for producing hydrogen comprising catalyzing the reduction of protons with the device of the present invention.

[0291] The advantages and features in context of the device of the invention apply mutatis mutandis to the method for producing hydrogen.

[0292] In the sense of the present invention the fuel cell and device provided herein, as well as the corresponding methods, include the composition of the present invention as described in detail herein above and in particular in the sections “Composition”, “Hydrogenase” and “Redox-active matrix”. Accordingly, the features described in relation to the composition, hydrogenases and redox-active matrices in the sections “Composition”, “Hydrogenase” and “Redox-active matrix” above, apply mutatis mutandis to the fuel cell, device and the corresponding methods of the present invention. In particular, the fuel cell, device and corresponding methods may comprise or make use of the composition of the present invention, the hydrogenases and / or the redox-active matrices described herein.

[0293] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art.

[0294] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0295] As used herein, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0296] As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise. As used herein, the term “about” indicates and encompasses an indicated value and a range above and below that value. As used herein, the term “about” indicates the designated value ± 10%, preferably ± 5%, or most preferably ± 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).

[0297] The disclosures in context of the methods described herein are disclosed as corresponding usesmutatis mutandis. The disclosures in context of the uses described herein are disclosed as corresponding methods mutatis mutandis.

[0298] Sequence listing:

[0299] SEQ ID NO: 1 CbA5H from Clostridium beijerinckii.

[0300] MGDNKKSFIQSALGSVFSVFSEEELKELSNGRKIAICGKVNNPGIIEVPEGATLNEIIQL CGGLINKSNFKAAQIGLPFGGFLTEDSLDKEFDFGIFYENIARTIIVLSQEDCIIQFEKF YIEYLLAKIKDGSYKNYEWKEDITEMFNILNRISKGVSNMREIYLLRNLAVTVKSKMNQ KHNIMEEIIDKFYEEIEEHIEEKKCYTSQCNHLVKLTITKKCIGCGACKRACPVDCINGE LKKKHEIDYNRCTHCGACVSACPVDAI SAGDNTMLFLRDLATPNKWITQMAPAVRVAIG EAFGFEPGENVEKKIAAGLRKLGVDYVFDTSWGADLTIMEEAAELQERLERHLAGDESVK LPILTSCCPSWIKFIEQNYGDMLDVPSSAKSPMEMFAIVAKEIWAKEKGLSRDEVTSVAI MPCIAKKYEASRAEFSVDMNYDVDYVITTRELIKIFENSGINLKEIEDEEIDTVMGEYTG AGIIFGRTGGVIEAATRTALEKMTGERFDNIEFEGLRGWDGFRVCELEAGDIKLRIGVAH GLREAAKMLDKIRSGEEFFHAIEIMACVGGCIGGGGQPKTKGNKQAALQKRAEGLNNIDR SKTLRRSNENPEVLAIYEKYLDHPLSNKAHELLHTVYFPRVKKD

[0301] SEQ ID NO: 2 Hnd of Desulfovibrio fructosovorans.

[0302] MSMLTITIDGKTTSVPEGSTILDAAKTLDIDIPTLCYLNLEALSINNKAASCRVCWEVE GRRNLAPSCATPVTDNMWKTNSLRVLNARRTVLELLLSDHPKDCLVCAKSGECELQTLA ERFGIRESPYDGGEMSHYRKDISASIIRDMDKCIMCRRCETMCNTVQTCGVLSGVNRGFT AWAPAFEMNLADTVCTNCGQCVAVCPTGALVEHEYIWEWEALANPDKWIVQTAPAVR AALGEDLGVAPGTSVTGKMAAALRRLGFDHVFDTDFAADLTIMEEGSEFLDRLGKHLAGD TNVKLPILTSCCPGWVKFFEHQFPDMLDVPSTAKSPQQMFGAIAKTYYADLLGIPREKLV WSVMPCLAKKYECARPEFSVNGNPDVDIVITTRELAKLVKRMNIDFAGLPDEDFDAPLG ASTGAAPIFGVTGGVIEAALRTAYELATGETLKKVDFEDVRGMDGVKKAKVKVGDNELVI GVAHGLGNARELLKPCGAGETFHAIEVMACPGGCIGGGGQPYHHGDVELLKKRTQVLYAE DAGKPLRKSHENPYIIELYEKFLGKPLSERSHQLLHTHYFKRQRL

[0303] SEQ ID NO: 3 CpIII from Clostridium pasteurianum.

[0304] MKSEYNDLFKSLIDAYYKDDFDEFIKKALSDSTVNKEELSNIISSFCGVELKYTDKDTYI KDLKNAIKNYNSDHKIVTKIRDCSVDCADENGKTSCQKSCPFDAILIDEANKTSYIDKDL CTDCGFCVEGCPNGSILDKVEFIPLANLLKEKQPVIAAVAPAITGQFGDDVTIDQLRTAF KKVGFADMIEVAFFADMLTLKEACEFNAHVKSKDDLMITSCCCPMWVGMLKRVYKDMVKY VSPSVSPMIAAGRVIKTLNSNCKWFVGPCIAKKAESKNKDIEGDIDFVLTFEEVKNIFE SLNINPSELPEDPSTDYASREGRLYARTGGVSISVSEAVAKLFPEKKDLFKSVQANGVIE CKKILEKAQNGEVAANFIEGMGCVGGCVGGPKALIPKEKGREKVNEFAENSNVKISLESD QMKKILNMLNITSAKDFMDEEKIKIFEREF

[0305] References:

[0306] 1. A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage. Plumere, N., Rudiger, O., Oughli, A. A., Williams, R., Vivekananthan, J., Poller, S., Schuhmann, W. & Lubitz, W. Nature Chemistry 6, 822-827; (2014).

[0307] 2. A redox hydrogel protects the O2-sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage. Oughli, A. A., Conzuelo, F., Winkler, M., Happe, T., Lubitz, W., Schuhmann, W., Rudiger, O. & Plumere, N. Angewandte Chemie International Edition 54, 12329-12333; (2015)

[0308] 3. Mechanism of Protection of Catalysts Supported in Redox Hydrogel Films.

[0309] Fourmond, V., Stapf, S., Li, H., Buesen, D., Birrell, J., Rudiger, O., Lubitz, W., Schuhmann, W., Plumere, N. & Leger, C. Journal of the American Chemical Society 137, 5494-5505; (2015).4. Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hil- denborough under Oxidative Conditions. Ruff, A., Szczesny, J., Zacarias, S., Pereira, I. A. C., Plumere, N. & Schuhmann, W. ACS Energy Letters 2, 964-968; (2017).

[0310] 5. Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential. Oughli, A. A., Velez, M., Birrell, J. A., Schuhmann, W., Lubitz, W., Plumere, N. & Rudiger, O. Dalton Transactions 47, 10685-10691.

[0311] 6. Complete Protection of O2-Sensitive Catalysts in Thin Films. Li, H., Buesen, D., Dementin, S., Leger, C., Fourmond, V. & Plumere, N. Journal of the American Chemical Society 141, 16734-16742; (2019).

[0312] 7. Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel.

[0313] Oughli, A. A., Hardt, S., Rudiger, O., Birrell, J. A. & Plumere, N. Chemical Communications 56, 9958-9961; (2020).

[0314] 8. Sulfide Protects [FeFe] Hydrogenases From O2. Rodriguez-Macia P, Reijerse EJ, van Gas- tel M, DeBeer S, Lubitz W, Rudiger O, Birrell JA. J Am Chem Soc. 2018 Aug 1;140(30):9346-9350; (2020),

[0315] 9. Reversible H2 Oxidation and Evolution by Hydrogenase Embedded in a Redox Polymer Film. Hardt, S., Stapf, S., Filmon, D. T., Birrell, J. A., Rudiger, O., Fourmond, V., Leger, C. & Plumere, N. Nature Catalysis 4, 251-258; (2021).

[0316] 10. Plumere, Nicolas, Olaf Rudiger, Alaa Alsheikh Oughli, Rhodri Williams, Jeevanthi Vive- kananthan, Sascha Poller, Wolfgang Schuhmann, and Wolfgang Lubitz. 2014. “A Redox Hydrogel Protects Hydrogenase from High-Potential Deactivation and Oxygen Damage.” Nature Chemistry 6 (9): 822-27.

[0317] 11. Oughli, Alaa A., et al. " Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential." Dalton Transactions 47.31 (2018): 10685-10691

[0318] 12. Oughli, Alaa Alsheikh, Felipe Conzuelo, Martin Winkler, Thomas Happe, Wolfgang Lubitz, Wolfgang Schuhmann, Olaf Rudiger, and Nicolas Plumere. 2015. “A Redox Hydrogel Protects the O2 -Sensitive [FeFe] -Hydrogenase from Chlamydomonas Reinhardtii from Oxidative Damage.” Angewandte Chemie 54 (42): 12329-33.

[0319] 13. Winkler, Martin, et al. " A safety cap protects hydrogenase from oxygen attack." Nature Communications 12.1 (2021): 756.

[0320] 14. Rutz, Andreas, et al. “Increasing the 02 resistance of the [FeFe] -hydrogenase CbA5H through enhanced protein flexibility.” ACS catalysis 13.2 (2022): 856-865.

[0321] Brief description of the Figures:

[0322] Figure 1: Potential window for H2-evolution, H2-oxidation and inactivation of the [FeFe]-hydrogenase of Clostridium bejerinckii (CbA5H) under DET (direct electron transfer) conditions (bottom). Redox-active dendrimers (DV1 and DV2) used for embedding the hydrogenase in a polymer matrix on the electrode surface (MET). DV1 fails to promote H2 oxidation since its reduction potential lies in the high potential inaction window of the hydrogenase. However, DV1 can promote H2 oxidation via CbA5H under more acidic conditions (not shown in the present study). In contrast, the reduction potential of DV2 lies in the narrow potential window for H2 oxidation of the hydrogenase (top). The redox buffering effect of DV2 maintains the [FeFe] -hydrogenase in its active state regardless of the applied potential at the electrode, which extends its catalytic activity for H2 oxidation far beyond the switch potential (Eswitch).Figure 2: Detailed Analysis of the Redox Hydrogel Systems DV1 and DV2 and Their Impact on CbA5H Catalytic Activity, (a) Non-Enzymatic Cyclic Voltammetry of DV1 and DV2. This panel compares the cyclic voltammetry curves of the redox hydrogels DV1 and DV2, demonstrating a potential difference of 0.18 V between them. Key Insight: The potential difference indicates distinct redox properties, with DV2 being more suitable for supporting the desired catalytic reactions under varying potential conditions, (b) Chemical structures of the two hydrogels. Structural differences are highlighted to explain the varying redox potentials and their implications for enzyme activity, (c) Cyclic voltammetry compares the catalytic hydrogen oxidation activities of CbA5H when embedded in DV2 (direct electron transfer, DET) versus without any hydrogel (mediated electron transfer, MET). Without the redox hydrogel, CbA5H's catalytic activity drops significantly in the potential range between -0.3 V and 0.2 V. With DV2, however, the potential is buffered, maintaining maximum catalytic current above -0.3 V, ensuring effective hydrogen oxidation activity, (d) Chronoamperometric response of CbA5H during potential cycling between -0.6 V and 0.2 V. CbA5H maintains consistent catalytic activity at higher potentials when protected by DV2, whereas activity declines sharply without the hydrogel. (e) Comparison of Cyclic Voltammograms for CbA5H Embedded in DV1 vs. DV2 at pH 7. DV1, with its relatively high redox potential (Eo = -0.22 V), provides limited catalytic activity in both directions (hydrogen oxidation and production), whereas DV2, with a lower redox potential (Eo = -0.4 V), supports both catalytic directions effectively and prevents the enzyme from transitioning into the inactive Hinact state even at higher potentials, (f) Comparison of Residual Activities of DdHydAB and CbA5H Embedded in DV2 After O2 Exposure During a Transient Operational Pause of 3 hours. While DdHydAB loses nearly all its catalytic activity after oxygen exposure, Cb A5H retains around 90% of its activity and maintains approximately 75% even after 18 hours of exposure, highlighting its superior resilience, (g) Chronoamperometric Measurement of Hydrogen oxidation activity of Cb A5H in DV2 under operational conditions, with periodic 5% oxygen addition to the gas supply. The slight decrease in current density during oxygen introduction phases is attributed to the electron demand for oxygen reduction within the redox hydrogel layers, demonstrating the oxygen protection provided by DV2 during operation, (h) Residual current % obtained from CVs after repetitive exposure of the same enzymatic hydrogel to O2. The enzymatic films were stored under ambient air at 5°C for each 1 hour.

[0323] Figure 3: The concentration profiles in MV0X(short-dashed gray line), MVred (long-dashed gray line), H2 (dotted-black line), O2 (gray line), and inactive enzyme (black line), and the viologen Nernst potential versus the H+ / H2Nernst potential (long-dashed black line) (related by the Nernst equation to the ratio MVox / MVred), under anaerobic (left) and aerobic (right) conditions, when E0MV= 60, 120 or 240 mV vs. SHE (from top to bottom).

[0324] Figure 4: Venn diagram of the already published protection mechanisms under operationalconditions, operational pauses and storage / production. The contribution of the present invention over the prior art is marked with the circled 4 and covers all three areas, while the protection mechanisms of the prior art each only provide protection under one condition (1: A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage. (Plumere, Nicolas, et al. " A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage." Nature chemistry 6.9 (2014): 822-827.) and 3: Sulfide Protects [FeFe] Hydrogenases From 02 (Oughli, Alaa A., et al. " Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpo¬ tential." Dalton Transactions 47.31 (2018): 10685-10691.)) or two conditions (2: Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel. (Oughli, Alaa A., et al. " Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel." Chemical Communications 56.69 (2020): 9958-9961.)).

[0325] Figure 5: Schematic overview of the Triple Protection Concept for [FeFe]-Hydrogenase CbA5H to prevent activity loss in Hydrogen production and conversion systems. (1) Under oxidative conditions, CbA5H adopts the inactive state (Hinact), with the intrinsic thiol group (from a cysteine residue) bound to the active site, providing protection against oxygen exposure. The arrow to demonstrates the reversible and potential dependent transitions between the inactive, protected state and the active, but O2 sensitive state. The enzyme is embedded in a redox hydrogel matrix (DV2), tailored specifically to the redox potential of CbA5H. DV2 provides protection against O2 (2) and high voltages (3) during operation, stabilizing the enzyme's activity by acting as a redox buffer.

[0326] Figure 6: pH-dependence of enzymatic inactivation and of redox-active matrices, (a) If the potentials are plotted against RHE, the potential of the redox-active matrices changes with the pH, but the inactivation potential of the hydrogenase remains relatively constant. This plot shows best how any redox-active matrix can be used for the hydrogenase protection at a specific pH. For instance, DV1 would protect the hydrogenase at acidic conditions, while at basic pHs, the enzyme is not protected, (b) If the potentials are plotted against SHE, the potential of the redox-active matrices remains constant and the inactivation changes with the pH.

[0327] Figure 7: a) Chemical structure of RLM. The RLM films were prepared with the addition of phytantriol as the structure-helper lipid, b) CVs of RLM recorded without enzyme under nitrogen atmosphere at different pH values, c) CVs of RLM with apo-CbA5H (catalytically inactive variant) also under nitrogen atmosphere at different pH values. The CVs without and with apoenzyme show a pH-independent redox potential of RLM. d) CVs of RLM with CbA5H recorded under hydrogen atmosphere at different pH values, e) Catalytic bias of RLM with CbA5H at different pH. The catalytic H2 conversion changes from catalytic hydrogen production to hydrogen oxidation with pH without deactivation of CbA5H by the high potentials.Figure 8: Exemplary viologen moieties of the present invention and their corresponding potential.

[0328] Figure 9: Exemplary redox-active matrices of the present invention and their corresponding potential.

[0329] Figure 10: A: CVs of CbA5H in RLM dropcasted on GCE under 100% N2, 5% O2 - 95% H2 and 5% N2 - 95% H2 at pH 8. The difference between the CVs in catalytic current density and CVs shape is minor, thus confirming that the protection from O2 is achieved by the catalysis.

[0330] B: CA of CbA5H in RLM dropcasted on GCE under 100% H2 for the first 10 minutes and 90% for the remaining 80 minutes showing stable catalytic H2 oxidation. O2 was added to the gas mixture at 2, 5 and 7% (gray shaded area) leading to slight drop of the catalytic current. This is due to electrons from the CbA5H catalyzed H2 oxidation are sacrificed to reduce the incoming O2. Both experiments confirm the O2 protection capability of the enzymatic RLM under turnover conditions.

[0331] Figure 11: A: CVs CbA5H (0.16nm) in DV2 2.8ug) at pH 8.8, 7.0 and 6.0 on GC-RDE (A = 0.07 cm2) in Tris-HCl / Citrate (50 mM) with KC1 (100 mM) at -0.2 V vs. SHE. The gas feed mixture of N2: H2 was varied as indicated. B: Plot of catalytic current density vs. the N2: H2 mixture measured with three individual experiments (n = 3). AT high concentrations of H2, the catalytic current is independent of H2 concentration. This excludes mass transport limitation inside the enzymatic hydrogel.

[0332] Figure 12: A: CVs CbA5H (0.16nm) in DV2 (2.8pg) at pH 8.8, 7.0 and 6.0 under 100% H2 on GC-RDE (A = 0.07 cm2) in Tris-HCl / Citrate (50 mM) with KC1 (100 mM) at u = 10 mV s'1. The rotation rate of the electrode was varied as indicated in the key. B: Plot of catalytic current density vs. rotation rate. The catalytic current vs. rotation rate was measured with three individual experiments (n = 3). At rotation rates higher than 2000 rpm no increase in catalytic current density can be observed excluding fast depletion of H2 in the RAM.

[0333] Figure 13: CVs of DdHydAB in DV2 before and after 3h exposure to air at pH 7.0. The shape of the CV changes from a sigmoidal shape to a diffusion-limited CV confirming the loss of catalytic activity due to 02-based enzyme inactivation.

[0334] Figure 14: CVs of CbA5H (0.16nm) in PV2 (1.13 pg) at pH 7.4 under 100% H2 on GC-RDE (A = 0.07 cm2, co = 2000 rpm) in Tris-citrate / HCl buffer (50 mM, pH = 7.0) with KC1 (100 mM) at u = 10 mV s'1. The CVs demonstrate the protection of CbA5H from oxidative potentials in the redox-polymer based RAM. The RAM (PV2) has a redox-potential similar to DV2 (see Fig.

[0335] 6).The invention is illustrated in the following examples.

[0336] Example 1: Turning the FeFe Hydrogenase from Clostridium beijerinckii into an Efficient H2 Oxidation Catalyst Using a Redox-active Matrix

[0337] Abstract

[0338] Hydrogenases are nature's sustainable catalysts for H2 / H+conversion. Among the three different subtypes, [FeFe]-hydrogenases show the highest efficiencies and lowest overpotentials in respect to bidirectional catalysis. The obstacles that prevent their utilization as H2 oxidation catalysts in fuel cells are being removed one by one, thanks to the continuous discovery of novel hydrogenase types and isoforms with unexpected catalytic properties, the development of new methods to produce these biocatalysts in large quantities and embed them in matrices that prevent their inactivation. Obtaining an efficient biohybrid electrode that is scalable and robust under a large range of experimental conditions remains nevertheless challenging. For H2 evolution the [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii is a highly active catalyst that can be handled in presence of oxygen and its production can be scaled up. However, under conditions of direct or wired electron exchange with an electrode, CbA5H cannot be used for H2 oxidation, as at high potentials the catalytic cofactor adopts an inactive but Ch-stable state. Here we show that when the enzyme is embedded into a redox-active hydrogel matrix whose reduction potential is finely tuned, the experienced potential is buffered in a sharp potential range that is compatible with H2 oxidation, without compromising resistance of this system towards O2. This redox buffering matrix enables to gain high H2-oxidation activity with an Ch-resistant [FeFe]-hydrogenase even at high potentials.

[0339] Introduction

[0340] The use of hydrogenases as efficient biological catalysts of H2 oxidation and production as potentially scalable alternatives of precious metal catalyst (Vincent, Parkin, and Armstrong 2007) in fuel cells has been an active field of research (Cracknell, Vincent, and Armstrong 2008; Qiao 2024; Le and Bren 2019; Karyakin et al. 2005; Vincent, Cracknell, Parkin, et al.

[0341] 2005). The main challenge in using hydrogenases comes from their sensitivity to oxidative (aerobic or anaerobic) conditions. Most hydrogenases especially those that exhibit high H2 oxidation rates under oxidative conditions are inhibited by oxygen to some extent. Under anaerobic oxidizing conditions, some hydrogenases reversibly inactivate, limiting their use as ^-oxidation catalysts even in the absence of Ch.

[0342] There are various ways to circumvent these drawbacks. One is to embed this fragile enzyme in a protective redox hydrogel matrix (Alaa A. Oughli et al. 2020; Alaa Alsheikh Oughli et al.

[0343] 2015; Plumeré et al. 2014) to mitigate the effect of O2 and high potentials at the electrode. Theother is to explore biodiversity to identify, produce, characterize and eventually use enzymes that are particularly robust (Fasano, Fourmond, and Leger 2024). Two main families of hydrogenases have been considered, [NiFe]- and [FeFe]-hydrogenases referring to the metal content of their active site. Some hydrogenases are described as " Ch-sensitive" because they quickly react with O2 to produce inactive states. This reaction may be irreversible leading to the irreversible decomposition of the active site, as known for most [FeFe] -hydrogenases or reversible. If most of the activity is recovered upon reduction, the enzyme is referred to as " Ch-resistant"; which is the case for all [NiFe]-hydrogenases and the [FeFe]- hydrogenase from Clostridium beijerinckii. If enzyme reactivation is very fast even above the Fh / Ffc equilibrium potential, the enzyme can oxidize H2 in the presence of Ch and is defined as " Ch-tolerant". This is the case for [NiFe]-hydrogenases of phylogenetic subgroup Id (Goldet et al., 2008; Fritsch et al., 2011). Some hydrogenases (including the [FeFe]- hydrogenase from Clostridium beijerinckii) also reversibly inactivate under anaerobic oxidizing conditions, which prevents their use as Fh-oxida-tion catalysts even in the absence of Ch.

[0344] The need of Ch in H2 / O2 fuel cells initially led to the preferential use of the oxygen-tolerant group Id [NiFe]-hydrogenases (Luo et al. 2009; Vincent, Cracknell, Lenz, et al. 2005; de Poulpiquet et al. 2014) since all other Ch-resistant [NiFe]-hydrogenases have very low activity (Fasano, Fourmond, and Leger 2024)). The subclass of [NiFeSe]-hydrogenases has also been considered (Ruff et al. 2020; Ruff et al. 2017) because these Ch-stable enzymes reactivate relatively quickly upon reduction. However, the generation and insertion (maturation) of the [NiFe] -cofactor requires a heavy biosynthesis system, and despite recent advances regarding their biosynthesis (Caserta et al. 2023), [NiFe] -hydrogenases cannot remain challenging to be produced in large quantities, as this significantly limits their application potential as a scalable alternative to precious metal catalysts.

[0345] In contrast, previous progress in the artificial maturation of [FeFe]-hydrogenases (Esselborn et al. 2013) whose active site (H-cluster) is defined by [4Fe4S]-cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe-subcluster (2FeH) (Fig 1 A), now enables the high-yield production of active [FeFe] -hydrogenase by the heterologous expression of the apo-hydrogenase (devoid of dinuclear cluster) and subsequent in vitro activation via incorporation of a synthetic mimic of the di-iron complex. All steps are potentially compatible with a large scalable production of the enzyme. However, [FeFe]-hydrogenases are deemed to be highly oxygen-sensitive (Kubas et al. 2017; Goldet et al. 2009). None of the so far characterized [FeFe]-hydrogen-ases can oxidize H2 in the presence of O2 under conditions of direct electron transfer (Rodriguez-Macia et al. 2017). The interaction of O2 with the vacant substrate binding site of the 2FeH subcluster initiates an irreversible degradation of both subclusters in a multistep process (Esselborn et al. 2019; Swanson et al. 2015). Embedding the [FeFe] -hydrogenase in redoxactive matrices protects [FeFe]-hydrogenases against incoming Ch-molecules but likewise against high potential inactivation thus enabling their employment for H2 oxidation even in thepresence of 5% O2 (Alaa Alsheikh Oughli et al. 2015). In presence of H2, the O2 reducing properties and the Nernst buffering effect of the matrix create a local anaerobic and redox-controlled environment for the hydrogenase (Plumeré et al. 2014; Li et al. 2019). However, this protection from O2 is strictly dependent on the presence of H2, and thus it is effective only under operational conditions, but not during shutdown or upon storage.

[0346] Some [FeFe] -hydrogenases such as DdHydAB of Desulfovibrio desulfuricans can be protected by adding exogenous sulfide (Rodriguez-Macia et al. 2018; Felbek, Arrigoni, et al. 2021) which reversibly inactivates the enzyme by occupying the substrate coordinaton site, thus preventing O2 binding and the initiation of the H-cluster degradation process (Fig lB)(Rodriguez-Macia et al. 2018). But this is only useful during electrode preparation and handling prior to initiating enzymatic catalysis. The redox-driven release of the sulfide ligand activates the enzyme but likewise renders it once more severely Ch-sensitive (Alaa A. Oughli et al. 2020; Felbek, Hardt, et al. 2021)). When catalysis is turned off, the hydrogenase does not return to the sulfide-inhib-ited state, and thus does not regain protection from O2 induced damage.

[0347] The recently discovered [FeFe]-hydrogenase of Clostridium bejerinckii (CbA5H) is unique among the FeFe-hydrogenases characterized so far: its H-cluster is stable in the presence of O2. This unique O2 resistance is due to a similar effect as known for sulfide protection, in this case however enabled by a nearby located Cys residue of the proton transfer pathway situated on a loop which is unusually flexible in case of CbA5H. The increased loop flexibility allows the thiol group of this Cys to come close enough to the substrate binding site of 2FeH to interact and block this location against incoming O2 molecules (Morra et al. 2016; Winkler et al. 2021) (Figure 1). As stable interaction with 2FeH requires oxidative conditions this O2 protection effect only occurs at sufficiently high potentials. However, oxidation (upon exposure to air or mildly oxidative potentials in DET experiments) and Cys binding transiently inactivates the enzyme during the high potential period. This significantly narrows the potential window for enzyme-catalyzed H2 oxidation and turns this enzyme into a very poor H2-oxidation catalyst (Morra et al. 2016; Winkler et al. 2021; Rutz et al. 2023; Corrigan, Tirsch, and Silakov 2020).

[0348] Here we demonstrate that [FeFe]-hydrogenase CbA5H can sustain a significant H2-oxidation capacity even under oxidizing conditions if it is embedded into a suitable polymer with redox active groups of fine-tuned electrochemical properties. In addition to that the polymer matrix renders the system Ch-resistant under turnover conditions. We could therefore design for the first time a hybrid H2-oxidizing electrode that is based on a scalable [FeFe]-hydrogenase which resists oxidative inactivation under all conditions, both under aerobic and anaerobic conditions and in the presence and absence of H2.

[0349] Results

[0350] Protection from oxidative potentials (anaerobic inactivation)Fig. 2C and 7D compare cyclic voltammograms (CVs) of [FeFe]-hydrogenase CbA5H recorded either under conditions of direct electron transfer (DET, red) by immobilizing the enzyme directly on the electrode surface or with the enzyme embedded in a film of 2,2’ -methylviologen modified redox dendrimer (DV2), undergoing mediated electron transfer (MET, green) with the electrode. Both CVs were recorded under anaerobic conditions with the electrode rotating in a solution saturated with EE. In both cases, EE evolution is observed as a negative current below Eeq = -0.4V vs. SHE, while H2 oxidation occurs above that equilibrium potential and is characterized by positive current values. However, the voltammetric response of DET shows that the enzyme inactivates (H2 oxidation current drops) atE > -0.3 V. The reductive current is recovered at low potential showing that this inactivation is reversible. It results from shifting to the O2 protected but inactive Hinact state due to cysteine binding to the active site (Fig. 1) under mildly oxidizing conditions, as investigated in reference (Winkler et al. 2021). Under conditions of MET the H2 oxidation current is stable in the high potential range (dashed line in Fig. 2C and 2D) contrasting the results gained for DET. The same difference is observed in chronoamperometry experiments where the potential is stepped up and down between -0.6 and +0.2V: H2 oxidation is only observed under conditions of MET (dashed line in Fig. 2C and 2D).

[0351] The reason why the enzyme film is insensitive to high electrode potentials is explained by the results in Fig. 3 (left col), showing the simulated concentrations of H2 (dotted-black line), of oxidized (short-dashed gray line) and reduced MV (long-dashed gray line) as a function of the normalized distance to the electrode (from ξ=0 at the electrode surface to ξ=1 at the film / solution interface), calculated under conditions of H2 oxidation (high electrode potential) and in the absence of O2. Unlike in our previous work (Fourmond et al. 2015; Li et al. 2019; Fourmond and Leger 2021) the calculations for CbA5H assume a bidirectional H+ / H2conversion. Panels A, C and E have been calculated with three different Eos for MV (60, 120 mV or 240 mV vs. RHE, respectively), all other parameters being equal. All panels show a small depletion of H2 inside the film (H2 concentration in the film is lower than in the bulk of the solution), and the accumulation of oxidized MV near the electrode surface. The steady-state concentration profile results from the compensation between "electron diffusion" (by hopping) and reduction of viologen groups by catalytic H2-oxidation.

[0352] The long-dashed black line and the right Y-axis also show the Nernst potential of the viologen, calculated in each case from the standard potential and the ratio of oxidized vs. reduced MV. The results of the simulation show that most of the enzyme is not exposed to the high electrode potential that has been applied. Near the electrode (0<^<0.2), where the concentration of oxidized viologen is high enough that H2 oxidation occurs, the Nernst potential of the MV is not very different from E0MV. Further away from the electrode (0.2<^<l), the potential equilibrates with H+ / H2, irrespective of the viologen standard potential. This last result is the consequence of the calculation assuming bidirectional catalysis, in contrast to previous work of ours: if catalysis were assumed to be unidirectional, the MV0Xconcentration would tend to zero as S, increases, and the calculated Nernst potential would unrealistically tend to minus infinity.The immediate implication of the above conclusion is that anaerobic inactivation is prevented when the enzyme is in the film, but only under the condition that the standard potential of the viologen is low enough. The study of the kinetics of reversible (in) activation of CbA5H hydrogenase in reference (Winkler et al. 2021) shows that the transition between an active and inactive enzyme occurs at E ~ -280 mV (at pH 8, 5°C). This observation is consistent with the fact that inactivation does not occur when the enzyme is embedded in DV2 (E = -400 mV vs. SHE, Fig. 2A and 2B). We however expect that inactivation would occur when using a dendrimer with redox active groups of a sufficiently high standard potential. We repeated the experiment with the 4,4’-methylviologen modified redox dendrimer (DV1) whose structure is shown in Fig- 2B, and whose redox potential is 180mV higher than the one of DV2 (Eo = -220 mV vs. SHE, Fig. 2A and 2B). Figure 2E compare CVs recorded with the same enzyme embedded into films of DV2 and DV1. As predicted, the anaerobic inactivation is seen only when the standard potential of the viologen derivative used in the redox hydrogel is higher than the peak potential for H2 oxidation in DET (>-0.3 V). The CVs of CbA5H in DV2 at different pHs show that the ratio of oxidative vs. reductive current changes with pH as described previously (Hardt et al. 2021). On the other hand, CVs with DV1 recorded for different pHs show a more pronounced inactivation at a higher pH, this is simply explained by the Eo of the viologen being pH independent, and the kinetics of inactivation being pH-dependent (Winkler et al. 2021). At neutral and slightly acidic pHs, the inactivation is less pronounced i.e. that DV1 in fact can promote H2 oxidation via CbA5H under more acidic conditions (results not shown). However, the catalytic bias is shifted in favor of H2 uptake due to the mismatch of the mediator potential and catalysis potential (Hardt et al. 2021). Limitations of H2 mass transport were excluded via control experiments showing that the catalytic current is independent of the H2 concentration at high H2 concentrations (Fig. 11) and of the rotation rate at high rates (Fig. 12).

[0353] Having demonstrated that CbA5H can be protected from anaerobic oxidative inactivation under MET conditions, we now turn to the protection against aerobic inactivation.

[0354] Protection from O2 in the absence of Hg

[0355] Figure 2F and 2G shows CVs of DV2 / CbA5H before (black) and after (grey) a 3h period of air exposure for with negligible change in the current density demonstrating effective protection against O2 via reversible enzyme inactivation to Hnact (Winkler et al. 2021; Morra et al. 2016). A plot of the residual current vs. exposure time to ambient air reveals how the catalytic current is retained with up to 74 ± 15% even after 18 hours (Fig. 2F). In contrast, [FeFe]-hydrogenase DdHydAB of D. desulfuricans is rapidly deactivated by O2 with its catalytic current dropping to less than 11 % after merely Ih air exposure and to zero after a 3h exposure period (Fig. 13).

[0356] After the first activation step, DdHydAB no longer harbors the protecting sulfide ligand and lacks the intrinsic O2 protection mechanism of CbA5H.Figure 2H shows the relative efficiency of the intrinsic protection mechanism of CbA5H embedded in redox hydrogel in the course of repeated 1 h-periods of air exposure, under conditions of open circuit. The decrease in Fh-oxidation activity is about 10% per exposure, resulting in an overall loss of activity of about 65% after 4 cycles for both H2 oxidation and H2 production. A similar behavior is observed for H2 production activity. This overall trend is consistent with the data published by Rutz et al. (Rutz et al. 2022), who reported a 75% loss in catalytic activity of CbA5H WT after two exposures to air.

[0357] Protection from O2 in the presence of Hg

[0358] We examined the protection provided by the hydrogel during aerobic catalysis, where O2 is reduced at the outer layer of the redox-active film, with CbA5H incorporated into a 2,2'-methyl viologen-modified polyvinyl alcohol hydrogel (PV2) with a redox potential of -0.431 ± 0.7 V vs. SHE, instead of the dendrimer DV2, which is based on a generation 3 poly(amidoamine) dendrimer (MW 6908.86 Da) functionalized with 322,2'-methyl viologen moieties. PV2, with a molecular weight exceeding 200 kDa, is significantly larger than DV2. Besides the synthetic advantages that polymers have over dendrimers, this larger weight also enhances long-term stability during catalysis. Cyclic voltammetry demonstrates that CbA5H is protected from high-potential inactivation when embedded in PV2 (Fig. 14), similar to the results gained with DV2 (Fig 7E). The chronoamperometry experiments with CbA5H in PV2 under a mixed EE and O2 gas feed show that the catalytic current for EE oxidation is largely unaffected by O2 (Fig. 2G).

[0359] As observed before with the Ch-sensitive [FeFe]-hydrogenase from D. desulfuricans, the decrease in current that results from an exposure to O2 is small and reversible (Alaa Alsheikh Oughli et al. 2015). The reason becomes obvious when examining the concentration profiles in the right column of Fig 3. The penetration of O2 throughout the film (gray line) inactivates the enzyme molecules present in the outer layers of the film (black concentration profile), and oxidizes the viologen (short-dashed gray line). By receiving electrons from reduced viologens in the inner layers the electron holes diffuse inward, oxidizing the active enzyme, which re-reduce the viologen groups via H2 oxidation (long-dashed gray line), In the outer layers the reduced viologen groups react with the incoming O2 and stop its penetration (Li et al. 2019). As soon as the O2 contamination is removed, the initial current is fully restored because, under anaerobic conditions, the enzyme that contributes to H2 oxidation is near the electrode surface (as seen in the left column of Fig 3), and has not been exposed to O2 (Li et al. 2019). The protection from O2 during catalysis is entirely dependent on the catalytic efficiency of H2-oxidation happening in the outer layer of the film, and the enzyme will inactivate entirely if the film is exposed to O2 in the absence of H2. However, owing to the intrinsic O2 protection mechanism of CbA5H, full protection of the hydrogenase is achieved not only during H2-oxidation catalysis (aerobic or anaerobic) but also during intermediate resting stages or storage.

[0360] DiscussionThe stability of the system understand-by conditions (open circuit potential, no hydrogen) in the presence of O2 is enabled by the unique intrinsic Ch-resistance of [FeFe]-hydrogenase CbA5H. Under oxidative conditions a strictly conserved cysteine which is an essential proton shuttle for the adt bridge of the 2Feu cluster is stabilized in a shifted position close enough to the substrate binding site to react with the Fea site. Here it transiently occupies the target site of O2 attack, which prevents an initiation of the H-cluster degradation process (Winkler et al.

[0361] 2021). This self-protection mechanism is maintained efficiently during long-term exposure to ambient air without the need to add external protectants like sulfide. This is confirmed by the maintained shape of the CVs before and after exposure to air. However, while being small, there is some decrease in catalytic current after each exposure (Fig. 2F and G). The native protection mechanism is not entirely tight: the fact that the enzyme responds with a slight delay to the oxidative conditions presumably resulting from O2 contact with the distal cluster of the [FeS]-cluster relay (Winkler et al. 2021) renders a certain fraction of the enzyme damaged after each O2 exposure (Fasano et al. 2024). Repeated exposure to air therefore leads to some irreversible enzyme inactivation leading to further decrease of the catalytic activity with each cycle as visible in Fig. 2H A similar loss of catalytic activity upon repeated air exposure was described for CbA5H by Rutz and coworkers following the impact repeated O2 exposure on residual activity by applying a methyl viologen dependent in vitro activity assay for H2-evolution after reductive reactivation. This suggests that the activity loss is primarily due to an inactivation of the enzyme itself presumably caused by the imperfect kinetics of the Ch-protection mechanism after O2 contact rather than by the entrapment of the enzyme in the hydrogel compromising its intrinsic stability. Furthermore, a substitution of M382 with Glu in CbA5H has been reported to affect the kinetics and flexibility of the protective loop, limiting the loss in residual activity during the course of repeated air exposures. However, these improvements were accompanied by a reduction in catalytic activity, highlighting the potential for further optimization (Rutz et al., 2022).

[0362] Rumbaugh et al. recently demonstrated the successful coupling of CbA5H with PsaE of PSI to generate a hybrid complex that can be used for H2-photoproduction (Rumbaugh et al., 2024) without the need to apply an external potential. The chimeric version of CbA5H also inactivates at oxidative potentials limiting the potential window for H2-oxidation, but this approach shows potential for future light driven H2 production applications if the kinetics of the O2 protection mechanism can be further optimized without largely decreasing the catalytic performance of CbA5H.

[0363] Oi-protection under aerobic / operative conditions was demonstrated for CbA5H embedded in a redox-polymer with a similar redox potential to DV2. The protection against O2 is enabled by the H2 oxidation activity of the enzyme yielding reduced-viologen groups / redox equivalents to reduce O2 in the outer layer before penetrating the redox-hydrogel. This sacrifice of a certainfraction of the catalytic current resulting from H2 oxidation catalysis is essential to get rid of the incoming O2 and is visible by a slight drop in catalytic current density (Fig. 2G) but when the exposure to O2 has been stopped / overcome, the catalytic current is retained (Plumere et al.

[0364] 2014, Li et al. 2019). In our earlier studies, we demonstrated how the thickness of the enzymatic film is decisive for the current response of the enzymatic film to the incoming O2 which can vary between minimally affected by O2 to a near complete collapse with a significant loss of catalytic activity. The latter is due to the deactivation of Hydrogenases by the incoming O2. This irreversible loss of catalyst can minimize the overall efficiency of the system. In the case of CbA5H, the O2 induced inactivation is reversible, meaning that in any case the fraction of the catalytic layer which is sacrificed during the stage of O2 protection is largely regained when the exposure to O2 is stopped.

[0365] The protection of CbA5H from high potential inactivation during operative conditions was afforded by using viologen-modified redox hydrogels as a potential buffer, as demonstrated before with both [NiFe]- (Plumeré et al. 2014) and other [FeFe]-hydrogenases (Oughli et al.

[0366] 2015). Here, the match between the sufficiently negative standard potential of dendrimer DV2 and the equilibrium potential Eeqof the 2H+ / H2 couple at neutral pH of 7.0 allows for a reversible H / H2 conversion (Fig. 2C and 2D) with the additional important feature of a pH-control-lable catalytic bias (Fig. 2E) as demonstrated by (Hardt et al. 2021). We evidenced the twodirectional protection of the current system in two different experiments. First, CV and CA experiments with CbA5H undergoing DET with the electrode showing full inactivation of the enzyme at potentials above ~ -100 mV, whereas under conditions of MET (DV2 / CbA5H) no inactivation occurs (Fig. 2, panels C and D). Second, MET control experiments with the enzyme embedded in a hydrogel with a redox potential higher than the inactivation potential of the enzyme (DV1 in Fig. 2) show inactivation at high potentials, confirming that the protection is indeed afforded on condition that the potential of the viologen is low enough. The detailed simulation data presented in Fig. 3 offer a comprehensive explanation. Only in a very narrow layer near the electrode surface is the effective Nernst potential of the viologen close to the electrode potential. Away from the electrode, this potential is close to the standard potential of either the viologen groups or of the H H2 couple with new concentration profiles explaining the protection mechanism against high potentials under anaerobic and aerobic conditions. At pH 7, the standard potential window for viologens is broad ranging between -350 and -200 mV, being oxidizing enough to efficiently catalyze H2 oxidation, but still remains below the threshold for oxidative inactivation. With the redox potential of the mediator in this range, the enzyme becomes an efficient H2 oxidation catalyst even though this is not the case under conditions of DET. The observation that the enzyme is a poor DET catalyst of H2 oxidation was previously described as the price the enzyme pays for being stable in air (Morra et al. 2016; Winkler et al.

[0367] 2021; Rutz et al. 2023; Corrigan, Tirsch, and Silakov 2020). Here the use of a carefully chosen mediator cancels out this downside and reveals that the enzyme's active site is perfectly capable of effective H2-oxidation catalysis.In conclusion, we have successfully combined three formally identified mechanisms of hydrogenase protection under anaerobic / operating, aerobic / operating and aerobic / non-operating conditions in one system. All three mechanisms have been demonstrated by wet lab experiments and were corroborated and further explained by detailed in silico simulation data. In addition, matching the redox hydrogel potential to the 2H+ / H2 couple at neutral pH allows for bidirectional H2 conversion with a controllable bias which can be either shifted in favor of H2 production or H2 oxidation (Hardt et al. 2021). All of these features will be useful tools for the construction of robust and efficient electrocatalytic films in future electrolyzer systems and biofuel cells.

[0368] Methods

[0369] The viologen-modified dendrimer / polymers (DV1, DV2 and PV2) were synthesized and fully characterized according to previously reported procedures (Hardt et al. 2021; Li et al. 2018). All electrochemical measurements were carried out in a three-electrode set-up consisting of a glassy-carbon rotating disc electrode (GC-RDEs, d = 3 mm, for MET) and a pyrolytic graphite edge working electrode (PGE, for DET). Pt-wire was used as a counter electrode and an Ag / AgCl 3M KC1 as a reference electrode. All experiments were conducted in Tris / citrate buffer (50 mM) with KC1 (100 mM) as the supporting electrolyte.

[0370] Enzyme films for DET experiments were prepared by drop-casting 3 pl of the [FeFe]-hydro-genase stock solution (10 pM) on a PGE rotating disc electrode. After drop-casting the electrode was incubated at RT for 3 min and rinsed with MQ water to remove any unbound enzyme molecules.

[0371] Hydrogel films for MET experiments were prepared by drop-casting a dendrimer-enzyme mixture of DVl / CbA5H or DV2 / CbA5H onto the working electrode’s GC-surface of the RDE. The films were allowed to dry over 2 hours at 37°C under aerobic atmosphere. All CVs were recorded with an electrode rotating in a buffer pre-degassed with N2 to create Ch-free conditions and then saturated with H2.

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[0407] Example 2:

[0408] Results

[0409] The replacement of the previously used enzyme DdHydAB with the recently discovered and characterized [FeFe]-hydrogenase CbA5H offers an effective approach to extend the O2 protection to operational pauses that were not previously addressed. CbA5H withstands oxygen exposure without damage through an intrinsic protection mechanism, wherein it, similar to the sulfide-protected DdHydAB enzyme, adopts the catalytically inactive Hinact state. However, in the case of CbA5H, the protective group is a thiol group from an enzyme's own cysteine, which, unlike the externally added sulfide ligand, is not lost after the enzyme's initial activation. As a result, CbA5H, unlike DdHydAB and other [FeFe]-hydrogenases, can switch completely and reversibly between the Ch-protected Hinact state and the catalytically active mode through air exposure or depending on the applied potential, without the need for external additives.

[0410] A more detailed examination of the protection mechanism initially showed that this protected but inactive state forms even at mildly oxidative potentials (> -0.3V), rendering the enzyme barely capable of hydrogen oxidation when being free in solution (Winkler et al. 2021). Since the protective effect of the redox-active matrix used for enzyme embedding relies on the reduction of oxygen, which again depends on the provision of electrons from enzymatically catalyzed hydrogen oxidation, these two protection mechanisms could not be combined until now.

[0411] To prevent the inactivation of the enzyme (CbA5H) at high potentials without losing its intrinsic oxygen protection, a new redox-active matrix (DV2) was developed, tailored to the redox potential of the enzyme's redox-active groups. The use of this redox-active matrix allows us to utilize the biocatalyst for efficient hydrogen production and electricity generation in the presence of oxygen, while also ensuring that the enzyme is protected from oxygen during storage or operational pauses. Due to the intrinsic properties of the selected biocatalyst, the cycle of activity and oxygen protection can be repeated multiple times without significant enzyme damage. This effectively resolves the two main conceptual problems associated with the use of [FeFe]-hydrogenases in hydrogen energy conversion systems.

[0412] Our invention thus provides a fully protected biocatalyst without relying on the addition of external substances. With the simple system presented here, comprising a suitable enzyme and a matching redox-active matrix, we were able to eliminate the key weaknesses of previous concepts (see Fig. 4):

[0413] 1. The appropriate redox-active matrix protects the enzyme from damage or efficiency loss in high voltage ranges (Fig.2a).2. The potential protection enables both hydrogen production and hydrogen electricity generation within the same cycle (Fig. 2a & b).

[0414] 3. Due to its intrinsic protection mechanism, the enzyme is protected from oxygen during storage (Fig. 2c).

[0415] 4. By embedding the enzyme in the chemically optimized redox-active matrix DV2, it is protected from oxygen during operation (Fig. 2g).

[0416] The system presented here allows, for the first time, the unification of all problem areas into a single system, thereby developing a future-proof prototype for regenerative enzyme-based hydrogen conversion (Fig. 5).

[0417] In our research, we also demonstrated that the enzyme can be effectively protected by a redoxactive matrix which has a reduction potential matched to the inactivation potential of the enzyme as explained above (Fig. 2 a & b). A chemically similar redox-active matrix, but with a different redox potential, described here as DV1, was unable to protect CbA5H from activity loss due to the high voltage required for hydrogen-dependent electricity generation (Fig. 2e).

[0418] However, DV1 is capable of protecting enzymes and CbA5H from oxygen and high potentials under more acidic conditions.

[0419] The chronoamperometric response of CbA5H during potential cycling between -0.6 V and 0.2 V. CbA5H maintains consistent catalytic activity at higher potentials when protected by DV2, whereas activity declines sharply without the redox-active matrix (Fig.2d). The comparison of residual activities of DdHydAB and CbA5H embedded in DV2 after O2 exposure during a transient operational pause of 3 hours shows that while DdHydAB loses nearly all its catalytic activity after oxygen exposure, CbA5H retains around 90% of its activity and maintains approximately 75% even after 18 hours of exposure, highlighting its superior resilience (Fig. 2f).

[0420] Discussion

[0421] The current state of the art provides oxygen protection for [FeFe]-hydrogenases under operational conditions and, with the addition of sulfide, offers a one-time protection during the limited period of enzyme production or application onto operational surfaces (see all references listed below). In contrast, by combining the intrinsically protected enzyme CbA5H with our newly developed redox-active matrix, we can link oxygen protection during storage conditions with oxygen protection during operational conditions.

[0422] The chemical fine-tuning of the redox group type in the redox-active matrix enables the prevention of efficiency losses under oxidative potentials, thereby allowing the effective use of the enzyme for both catalytic directions (hydrogen production and hydrogen oxidation).

[0423] Surprisingly, the precise adjustment of the redox-active matrix's redox potential allows the full activity of the enzyme to be utilized for hydrogen oxidation, while the conventional, previouslydescribed redox polymers lead to a rapid and complete deactivation of the enzyme for hydrogen oxidation. Our new concept of precisely matching the redox potential to oxygen-resistant hydrogenases like CbA5H enables, for the first time, the availability of biohybrid systems for any hydrogen oxidation-dependent applications, such as H2 / O2 fuel cells, hydrogen-driven biotechnology, or hydrogen biosensors.

[0424] References of Example 2:

[0425] Rodriguez-Macia P, Reijerse EJ, van Gastel M, DeBeer S, Lubitz W, Rudiger O, Birrell JA.

[0426] 2020. “Sulfide Protects [FeFe] Hydrogenases From 02.” J Am Chem Soc 140 (30): 9346- 9350.

[0427] Fourmond, Vincent, Stefanie Stapf, Huaiguang Li, Darren Buesen, James Birrell, Olaf Rudiger, Wolfgang Lubitz, Wolfgang Schuhmann, Nicolas Plumere, and Christophe Leger. 2015. “Mechanism of Protection of Catalysts Supported in Redox Hydrogel Films.” Journal of the American Chemical Society 137 (16): 5494-5505.

[0428] Hardt, Steffen, Stefanie Stapf, Dawit T. Filmon, James A. Birrell, Olaf Rudiger, Vincent Fourmond, Christophe Leger, and Nicolas Plumere. 2021. “Reversible H 2 Oxidation and Evolution by Hydrogenase Embedded in a Redox Polymer Film.” Nature Catalysis 4 (3): 251— 58.

[0429] Li, Huaiguang, Darren Buesen, Sebastien Dementin, Christophe Leger, Vincent Fourmond, and Nicolas Plumere. 2019. “Complete Protection of O2-Sensitive Catalysts in Thin Films.” Journal of the American Chemical Society 141 (42): 16734-42.

[0430] Oughli, Alaa A., Steffen Hardt, Olaf Rudiger, James A. Birrell, and Nicolas Plumere. 2020. “Reactivation of Sulfide-Protected [FeFe] Hydrogenase in a Redox-Active Hydrogel.” Chemical Communications 56 (69): 9958-61.

[0431] Oughli, Alaa Alsheikh, Felipe Conzuelo, Martin Winkler, Thomas Happe, Wolfgang Lubitz, Wolfgang Schuhmann, Olaf Rudiger, and Nicolas Plumere. 2015. “A Redox Hydrogel Protects the O2-Sensitive [FeFe]-Hydrogenase from Chlamydomonas Reinhardtii from Oxidative Damage.” Angewandte Chemie 54 (42): 12329-33.

[0432] Oughli, A. A., Velez, M., Birrell, J. A., Schuhmann, W., Lubitz, W., Plumere, N. & Rudiger, O. 2018. “Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.” Dalton Transactions 47: 10685-10691.

[0433] Plumere, Nicolas, Olaf Rudiger, Alaa Alsheikh Oughli, Rhodri Williams, Jeevanthi Vivekanan- than, Sascha Poller, Wolfgang Schuhmann, and Wolfgang Lubitz. 2014. “A Redox Hydrogel Protects Hydrogenase from High-Potential Deactivation and Oxygen Damage.” Nature Chemistry 6 (9): 822-27.

[0434] Ruff, A., Szczesny, J., Zacarias, S., Pereira, I. A. C., Plumere, N. & Schuhmann, W. 2017. “Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hil- denborough under Oxidative Conditions.” ACS Energy Letters 2: 964-968.

[0435] Winkler, Martin, JifuDuan, Andreas Rutz, Christina Felbek, Lisa Scholtysek, Oliver Lampret, Jan Jaenecke, et al. 2021. “A Safety Cap Protects Hydrogenase from Oxygen Attack.” Nature Communications 12 (1): 756.Example 3: Optimization of the CbA5H enzyme by mutagenesis of the enzyme CbA5H

[0436] As described in the invention disclosure, the enzyme (CbA5H) becomes deactivated at potentials higher than about 0.170 V vs. RHE, depending on the exact experimental conditions such as buffer or electrolyte used, limiting its use in ^-conversion (H2~ 2H++ 2e'). When embedded in a redox dendrimer with redox-active groups that have a relatively high redox potential of 0.194 V vs. RHE (DV1), the enzyme is not protected from this deactivation. However, it would may be possible to protect the hydrogenase by DV1 from oxygen and high potentials under more acidic conditions. DV1 would also successfully protect hydrogenases that have more positive inactivation potential than the redox-active matrix DV1 itself. When a redox dendrimer with redox-active groups possessing a lower redox potential of 0.014 V vs. RHE (DV2) is used, the enzyme is protected from deactivation and can be employed without limitation for hydrogen conversion.

[0437] Optimization of the CbA5H enzyme

[0438] In previous measurements CbA5H was used in its wild-type form, so no modifications were made directly to the enzyme. The present inventors and other groups made efforts to optimize the already effectively protected enzyme to enable an even more efficient protective mechanism via site-directed mutagenesis. As of yet, there is only one study that reports about a mutagenesis variant (M382E) that, exhibiting increased stability against O2 under repeated exposure and reactivation cycles compared to the wild type (Rutz et al., 2023). However, it exhibits a 33-50% reduction in catalytic performance due to its higher tendency to switch to the Ch-protected inactive Hinact state compared to the wild type as shown in Fig. 2 and Fig. 2S of Rutz et al., 2023. Figures from the article of Rutz et al., (2023) show the increased relative residual activity of variant M382E after repeated cycles of O2 exposure and reductive reactivation (see Fig. 2 of Rutz et al., 2023) and the decreased catalytic performance of M382E compared to the wild type of CbA5H (see Fig. S2 of Rutz et al., 2023).

[0439] These results show that variants of the CbA5H enzyme comprising one or more mutated amino acids compared to the WT enzyme may have equal or improved stability against O2 and / or catalytic performance. Accordingly, it is plausible that also slight variations of the hydrogenases provided herein, e.g. having a few amino acid changes, will work in the sense of the present invention, i.e. can be protected and catalyze the reduction of protons and oxidation of hydrogen as described herein.

[0440] References of Example 3:

[0441] Andreas Rutz, Chandan K. Das, Andrea Fasano, Jan Jaenecke, Shanika Yadav, Ulf-Peter Apfel, Vera Engelbrecht, Vincent Fourmond, Christophe Leger, Lars V. Schafer, and Thomas Happe.

[0442] 2023. “Increasing the 02 Resistance of the [FeFe] -Hydrogenase CbA5H through Enhanced Protein Flexibility”. ACS Catalysis 13(2): 856-865.Example 4: Three subtypes of O2 resistant [FeFeJ-Hydrogenases

[0443] 1. The three subtypes of O2 resistant [FeFel-Hydrogenases

[0444] The [FeFe] -Hydrogenase CbA5H of Clostridium beijerinckii is an exemplary hydrogenase that may be used in the composition of the present invention. The function of the enzyme can be inactivated under oxidative conditions, however, the enzymatic function of CbA5H of Clostridium beijerinckii can be protected from O2 and high potentials as described herein, see above and Examples 1 and 2. This inhibition of an enzyme under oxidative conditions and protection from O2 and high oxidative potentials is not solely limited to [FeFe]-Hydrogenase CbA5H of Clostridium beijerinckii.

[0445] The Ch-protection mechanism enfolds when an O2 resistant [FeFe]-hydrogenase is exposed to air or oxidative conditions and results from the binding of a nearby Cys residue to the [FeFe]- hydrogenase active site (open substrate binding site at Fea in the 2FeH subcluster of the H- cluster). The presence of this specific active site confirmation can be confirmed by an accumulation of a unique H-cluster state, either termed " Hinact" or “Hox+i”, exhibiting a characteristic Fourier Transform Infrared (FTIR) spectroscopy signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1. This spectroscopic feature has previously been described for representatives of three distinct subtypes of O2 resistant [FeFe]-Hydrogenases, including CbA5H of Clostridium beijerinckii (Morra et al., 2016, Winkler et al., 2021), Hnd of Desulfovibrio fructosovorans (Kpebe et al., 2018) and Cp III of Clostridium pasteurianum (Artz et al., 2020) (see Table 3). This allows to generalize the principle of the O2 protection to any hydrogenases showing this specific active site conformation when exposed to O2 or air.Table 3: [FeFeJ-hydrogenases experimentally verified to adopt the Hinact / Hox+1state under air / oxdiative conditions and the indicator bands of the corresponding state specific FTIR-absorbance spectrum. The signal ranges of the consensus spectrum serves as a reference to identify the O2 protected Hinact state in related O2 resistant [FeFe] -hydrogenases of the three subtypes (A-C).

[0446]

[0447] t-CO; t-CN: terminal ligands of the 2FeH cluster; g-CO: bridging CO ligand of the 2FeH cluster

[0448] 2, Further information on the representatives of the three O2 resistant FeFe-hydrogenases

[0449] Representative of subtype A:

[0450] The air-stable [FeFe] hydrogenase CbA5H from Clostridium beijerincki. (phylogenetic group A), where the binding of the cysteine upon exposure to air was previously demonstrated by the observation of theFTIR " Hinact" state, X ray crystallography, and electrochemical investigation of the kinetics of anaerobic inhibition. The relation between cysteine binding formation of the Hinact state and protection from O2 was established by investigating a site-directed mutant, where the cysteine is replaced with an aspartate, see Winkler et al. 2021.

[0451] Representative of subtype B:

[0452] The electron-bifurcating hydrogenase [FeFe]-hydrogenase Hnd of Solide sulfovibrio fruc-tosivorans (formerly Desulfovibrio fructosovorans) retains activity, when purified aerobically due to formation of the inactive state Hinact, protecting against O2 damage, see Kpebe et al. 2018.

[0453] Representative of subtype C:

[0454] The third [FeFe]-hydrogenase of Clostridium pasteurianum (CpIII) is oxidatively transformed into the inactive Hinact state (Hox+i in Fasano et al. 2024) and whose slow anaerobic oxidativeinactivation and moderate resistance to O2 both depend on the presence of a supernumerary cysteine (triple C motif) in the active site environment, see Fasano et al. 2024.

[0455] The above Example illustrates that the three subtypes (A-C) of O2 resistant [FeFe] -Hydrogenases all exhibit an O2 protection mechanism and are, thus, protected from O2 inactivation. This mechanism can be taken advantage of when using these hydrogenases in the composition of the present invention, which will allow the intrinsic protection mechanism to be maintained in operating and non-operating conditions of proton reduction, hydrogen oxidation or storage / resting phases. The above also demonstrates that the specific conformation of the active site is responsible throughout the different classes of hydrogenases. It is therefore plausible that all hydrogenases having such an active site conformation, e.g. confirmed through the specific FTIR spectroscopy signature would work in the sense of the present invention, i.e., can be protected entirely in the composition of the present invention from oxygen and oxidative potentials during catalysis of proton reduction and hydrogen oxidation as well as storage and resting phases. In particular, this has been demonstrated for hydrogenases belonging to the three different subtypes A, B and C above.

[0456] References of Example 4:

[0457] Artz J. H., Zadvornyy O. A., Mulder D. W. et al. 2020. “Tuning Catalytic Bias of Hydrogen Gas Producing Hydrogenases.” Journal of the American Chemical Society 142 (3): 1227-1235. Fasano, Andrea, et al. " Catalytic bias and redox-driven inactivation of the group B FeFe hydro¬ genase CpIII." ACS Catalysis 14.9 (2024): 7001-7010.

[0458] Morra, S., Arizzi, M. C., Valetti F. and Gilardi, G. 2016. “Oxygen Stability in the New [FeFe]- Hydrogenase from Clostridium beijerinckii SM10 (CbA5H).” Biochemistry 55 (42): 5897- 5900.

[0459] Kpebe A., Benvenuti M., Guendon C., Rebai A. et. al. 2018 “A new mechanistic model for an 02-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans." Biochimica et Biophysica Acta (BBA) - Bioenergetics 1859(12): 1302-1312.

[0460] Winkler, M., Duan, J., Rutz, A. et al. 2021. „A safety cap protects hydrogenase from oxygen attack.” Nat Commun 12, 756.

[0461] Example 5: pH-dependence of enzymatic inactivation and of redox-active matrices Fig. 6 is a comparison between the redox-potential of the redox-active matrices and the inactivation potential of CbA5H depending on the pH. Fig. 6a shows that if the potentials are plotted against RHE, the potential of the redox-active matrices changes with the pH, but the inactivation potential of the hydrogenase remains relatively constant. This plot shows best how any redox-active matrix can be used for the hydrogenase protection at a specific pH. For instance, DV1 would protect the hydrogenase at acidic conditions, while at basic pHs, the enzyme is not protected. Fig.6b shows that if the potentials are plotted against SHE, the potential of the redoxactive matrices remains constant and the inactivation potential of the enzyme changes with the pH.Example 6: Redox-active lipidic mesophases successfully enables hydrogen oxidation and evolution with the CbA5H hydrogenase

[0462] The results are shown in Figures 7 and 10. By using the viologens and phytantriol, stable films formed by the redox-active matrix RLM with the hydrogenase embedded can be prepared, which has never been achieved before. The CVs with apo-CbA5H at different pHs demonstrate that the redox-active matrix films comprising the RLM and the hydrogenase are stable at different pHs and therefore can be used for catalysis of hydrogen oxidation and hydrogen evolu-tion at different pHs. The films with CbA5H embedded into the RLM are protected from high potential inactivation in the presence of H2. Taken together, the data confirm that the RLM can successfully protect the hydrogenase, comparable for example with the redox-active matrices PV2 and DV2.

Claims

New PCT Application based on EP 25 305 533.9Technische Universität München, in Vertretung des Freistaates Bayern; Centre National de la Recherche Scientifique (CNRS); Aix-Marseille UniversitéVossius Ref.: AJ2983 PCT S3CLAIMS1. A composition for catalyzing reduction of protons and / or oxidation of hydrogen comprising a hydrogenase and a redox-active matrix having a reduction potential of about 0.13 V relative to a reversible hydrogen electrode (V vs. RHE) or less,wherein the hydrogenase is a hydrogenase having a cysteine side chain capable of reversibly coordinating its substrate binding di-iron active site,wherein the hydrogenase is a [FeFe] -hydrogenase, wherein the active site of the hydrogenase is a H-cluster comprising a [4Fe4S] -cluster (4FeH) coupled to a uniquely ligated dinuclear FeFe-subcluster (2FeH), andwherein the active site of the hydrogenase exhibits a Fourier Transform Infrared (FTIR) spectroscopy signature with bands at 2107±5, 2079±10, 2007±10, 1990±10 and 1830±20 cm’1in the presence of oxygen, oxidative stress and / or oxidative potentials.

2. The composition of claim 1, wherein the hydrogenase is selected from the group of CbA5H of Clostridium beijerinckii. Hnd of Desulfovibrio fructosovorans and CpIII of Clostridium pasteurianum or variants thereof having a sequence identity of at least about 90%, preferably wherein the hydrogenase is a hydrogenase having an amino acid sequence set forth in any one of SEQ ID NOs: 1-3, or variants thereof having a sequence identity of at least about 90%.

3. The composition of claim 1 or 2, wherein the redox-active matrix comprises a redox- polymer, a redox-dendrimer and / or a redox-active lipidic mesophase.

4. The composition of claim 3, wherein the redox-polymer comprises polyvinyl alcohol (PVA), polyvinyl imidazole (PVI), polyvinyl imine (PEI) and / or polyvinyl pyridine (PVP),wherein the redox-dendrimer comprises a symmetrical or asymmetrical dendrimer with 8, 16 or 32 linking positions, selected from a poly(amidoamine) (PAMAM) dendrimer, a polyester dendrimer, an ester functionalized dendrimer, a polypropylene imine (PPI) dendrimer, a poly(aryl ether) dendrimers, a poly(benzyl ether) dendrimer and / or a peptide dendrimer,wherein the redox-active lipidic mesophase comprises a carboxylic acid and a hydro- phobic hydrocarbon.

5. The composition of claim 3 or 4, wherein the redox-polymer, the redox-dendrimer and / or the redox-active lipidic mesophase further comprises a viologen moiety.

6. The composition of claim 5, wherein the viologen moiety is selected fromwherein R is selected from CH3, (CH2)nR2, or R2,wherein R2 is selected from H, OH, COOH, SOAc or CN, andwherein n can be any number, and / orwherein R is selected from H, CH3, (CH2)nR2, or R2,wherein R2 is selected from OH, COOH, SOAc or CN, andwherein n can be any number.

7. The composition of claim 5, wherein the viologen moiety is selected from Table 1, or wherein the redox-active matrix is selected from Table 2.

8. The composition of any one of claims 1 to 6, wherein the redox-active matrix comprises a 2,2’ -viologen moietylinked to a polyvinyl alcohol (PV2).

9. The composition of any one of claims 1 to 6, wherein the redox-active matrix comprises a poly(amidoamine) (PAMAM) dendrimer corewherein R is a 2,2’ -viologen moiety (DV2),10. The composition of any one of claims 1 to 6, wherein the redox-active matrix comprises a redox-active lipidic mesophase (RLM) with the formula11. A redox-active matrix comprising a redox-active lipidic mesophase (RLM) with the formula12. A method for protecting a hydrogenase from oxidative stress induced by oxygen and / or oxidative potentials, comprising formulating a hydrogenase with the redox-active matrix of claim 11, ora redox-active matrix comprising a 2,2’ -viologen moietylinked to a polyvinyl alcohol (PV2), ora redox-active matrix comprising a poly (amidoamine) (PAMAM) dendrimer corewherein R is a 2,2’ -viologen moiety (DV2),

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