RcoM protein-based carbon monoxide scavengers and preparations for the treatment of carbon monoxide poisoning

Abstract

A method is described for the rapid clearance of carbon monoxide (CO) from CO-bound hemoglobin, myoglobin, and cytochrome c oxidase in subjects with CO poisoning. The disclosed therapeutic method involves the use of rationally designed and engineered regulator of CO metabolism (RcoM) proteins and pharmaceutical compositions thereof, which scavenge CO from poisoned tissues. The recombinant RcoM composition is infused into the blood, where it rapidly sequesters CO and limits the toxic effects of CO on cellular respiration, oxygen transport, and oxygen utilization.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 022,821, filed May 11, 2020, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to recombinant regulator of carbon monoxide metabolism (RcoM) proteins and pharmaceutical compositions thereof. The present disclosure further relates to the use of recombinant RcoM proteins and compositions for the treatment of carbon monoxide (CO) poisoning, cyanide poisoning, and hydrogen sulfide poisoning, and as blood substitutes.

[0003] Government support approval This invention was made with government support under grant numbers HL098032, HL125886, HL136857, HL103455, HL110849 and HL007563 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] background Exposure to carbon monoxide via inhalation is a major cause of environmental poisoning. Individuals can be exposed to airborne carbon monoxide in a variety of different situations, including house fires, indoor generators or outdoor barbecue grills, or during suicide attempts in confined spaces. Carbon monoxide binds intracellularly to hemoglobin and heme proteins, particularly enzymes of the respiratory transport chain. Accumulation of carbon monoxide bound to hemoglobin and other heme proteins impairs oxygen delivery and oxygen utilization for oxidative phosphorylation. This ultimately leads to severe hypoxic and ischemic injury to vital organs such as the brain and heart. Individuals who accumulate more than 5–10% carboxyhemoglobin in their blood are at risk for brain injury and neurocognitive impairment. Patients with extremely high carboxyhemoglobin levels typically suffer from irreversible brain injury, respiratory failure, and / or cardiovascular collapse.

[0005] Despite the availability of methods for rapidly diagnosing carbon monoxide poisoning through standard arterial and venous blood gas analysis and CO-oximetry, and despite awareness of the risk factors for carbon monoxide poisoning, no antidote is available for this toxic exposure. Current treatment involves administering 100% oxygen via face mask and, when possible, exposing the patient to hyperbaric oxygen. Hyperbaric oxygen therapy increases the release rate of carbon monoxide from hemoglobin and accelerates its natural clearance. However, this treatment has little effect on the carbon monoxide clearance rate, and due to the complexity of hyperbaric oxygen equipment, this treatment is not available in the art. Furthermore, hyperbaric oxygen therapy often involves significant treatment delays and transportation costs. Thus, there is a need for an effective, rapid, and easily available treatment for treating carbon monoxide poisoning, also known as carboxyhemoglobinemia. Summary of the Invention [Means for solving the problem]

[0006] Abstract The present disclosure describes recombinant regulator of carbon monoxide metabolism (RcoM) proteins that have a high affinity for CO and their use as CO scavengers. The RcoM proteins of the present disclosure can remove CO from CO-bound hemoglobin, myoglobin, and cytochrome c oxidase (in mitochondria), and thus can be used in methods for treating carboxyhemoglobinemia and as blood substitutes.

[0007] Recombinant RcoM proteins are provided herein. In some embodiments, the recombinant RcoM protein comprises a heme-binding domain (HBD) having an amino acid sequence at least 90% identical to SEQ ID NO: 2. In some examples, the amino acid sequence of the HBD is at least 90% identical to SEQ ID NO: 2 and comprises amino acid substitutions at one or more of H74, C94, M104, M105, C127, and C130. In other examples, the HBD has a wild-type amino acid sequence. The recombinant RcoM protein may be a full-length RcoM (e.g., the RcoM of SEQ ID NO: 1) or a truncated RcoM, such as an RcoM consisting of or consisting essentially of the HBD. In certain examples, the recombinant RcoM protein comprises an affinity tag, e.g., a cleavable affinity tag, at the N- or C-terminus.

[0008] Further provided are pharmaceutical compositions comprising the recombinant RcoM proteins disclosed herein. In some embodiments, the pharmaceutical composition further comprises a reducing agent or an oxidizing agent.

[0009] Also provided herein are in vitro methods for removing carbon monoxide from hemoglobin, myoglobin, or mitochondria (cytochrome c oxidase) in blood or animal tissue. In some embodiments, the methods comprise contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein.

[0010] Further provided are methods for treating carboxyhemoglobinemia in a subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent.

[0011] Also provided are methods for treating cyanide poisoning in a subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the RcoM protein is present in its oxidized form.

[0012] Also provided are methods for treating hydrogen sulfide (HS) poisoning in a subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the RcoM protein is present in its reduced form.

[0013] Further provided are methods for replacing blood in a subject, hi some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein.

[0014] The foregoing and other objects and requirements of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1]The amino acid sequence of the RcoM-1 ortholog from P. xenovorans (SEQ ID NO: 1) is shown. RcoM-1 contains a PAS domain (residues 1-154 of SEQ ID NO: 1) and a LytTR domain (residues 155-267 of SEQ ID NO: 1). Crystal structures of homologous PAS and LytTR domains from other bacteria are also shown. The PAS domain structure is derived from the E. coli direct oxygen sensor (DOS) protein (Kurokawa et al., J Biol Chem 279(19): 20186-20193, 2004), and the LytTR domain structure is derived from the S. aureus transcription factor AgrA (Sidote et al., Structure 16(5):727-735, 2008).

[0016] [Figure 2] Amino acid sequence of RcoM-1 from P. xenovorans (residues 2-154 of SEQ ID NO: 2) truncated to include only the PAS CO-binding domain and lack the N-terminal methionine. Heme-binding residues are indicated in bold. A schematic diagram outlining the heme coordination environment in RcoM-1 is also shown.

[0017] [Figure 3] Amino acid sequence of RcoM-1 from P. xenovorans (SEQ ID NO: 3), further truncated to include only the critical region of the PAS CO-binding domain. Heme-binding residues are in bold (H74, C75, and M104, numbered with reference to SEQ ID NO: 1). A structural alignment of the PAS domain from the E. coli DOS protein with a homology model of the RcoM-1 PAS domain developed using the I-TASSER online modeling server is also shown. Dashed lines indicate the location of the proposed cleavage site. Heme-binding residues are shown as sticks.

[0018] [Figure 4]Hemoglobin-CO transfer kinetics in the presence of WT full-length RcoM-1 under aerobic conditions at 37°C, measured using stopped-flow UV-Vis spectroscopy. The concentrations of hemoglobin-CO (Hb-CO) and Fe(II)RcoM-1 were 20 μM, and experiments were performed in triplicate. Data for Hb-CO loss were fitted to a biexponential curve, yielding a slow-phase half-life (t) of 1.4 seconds. Data for Fe(II)-CO RcoM increase were fitted to a monoexponential curve, yielding a half-life of 0.93 seconds.

[0019] [Figure 5] Hemoglobin-CO transfer kinetics in the presence of WT full-length RcoM-1 under anaerobic conditions at 37°C was measured using UV-Vis spectroscopy. The concentrations of hemoglobin-CO and Fe(II)RcoM-1 were 15 μM and 15.8 μM, respectively. The absorbance changes at 530, 562, and 583 nm followed the transition from Fe(II) to Fe(II)-CO RcoM and were fitted to a single-exponential curve with a half-life of 50 seconds.

[0020] [Figure 6] Amino acid alignment of P. xenovorans RcoM-1 (SEQ ID NO: 1) and the H. crassostreae RcoM homolog (SEQ ID NO: 4). Residues H74, C94, and M104 of P. xenovorans RcoM-1 correspond to residues H57, C75, and M85 from the H. crassostreae RcoM homolog.

[0021] [Figure 7]Comparison of UV-Vis spectra of WT RcoM-1 and HBD16 RcoM-1 containing the C94S substitution. Visible spectrum of full-length wild-type RcoM-1 (left). Visible spectrum of the isolated heme-binding domain (HBD) of RcoM-1 with the C94S mutation (right). Spectra of ferric iron (Fe(III)), deoxyferrous iron (Fe(II)), and ferrous iron-CO species (Fe(II)-CO) are shown. The table indicates the wavelength of peak maximum (in nm) for each species, along with the estimated molar absorptivity (in mM cm) of each peak.

[0022] [Figure 8] Evidence for a stable O2 adduct in the HBD C94S. The isolated heme-binding domain (HBD) of RcoM1 with the C94S mutation is capable of binding oxygen. The visible spectrum of the ferrous (Fe(II)) species in the presence of the reducing agent sodium dithionite is indicated by an *. When the reducing agent is removed, the spectrum after desalting of Fe(II) is obtained. After air exposure, the formation of an oxyferrous spectrum is observed with maxima near 540 and 575 nm (Fe(II), air exposed). Reoxidation of the protein yields a ferric spectrum (Fe(III), re-ox) that matches the ferric spectrum shown in Figure 7.

[0023] [Figure 9] Truncated HBD16 RcoM with a C94S substitution has the same CO on rate as WT RcoM. The kinetics of the reaction of the ferrous heme-binding domain (HBD) of RcoM1 with carbon monoxide (CO) was determined by stopped-flow techniques. (Top left) Detail of the Soret band of the protein; the arrows indicate the direction of absorbance change. (Top right) Detail of the visible region of the spectrum. The arrows indicate the direction of absorbance change. (Bottom left) Absorbance change versus time at selected wavelengths. Calculation of the rates at different CO concentrations yields an association rate for the reaction of 1.2 × 105 M-1 sec-1. Similar values ​​are obtained for the wild-type full-length protein.

[0024] [Figure 10] Determination of the CO dissociation rate for the heme-binding domain (HBD) of RcoM1 with the C94S mutation. The ferrous iron-CO complex dissociates in the presence of nitric oxide (NO), and the reaction was monitored by absorbance change. Upon CO dissociation, NO binds to heme, causing a change in the absorbance spectrum. Excess NO prevents CO from rebinding to heme. (Top left) Detail of the visible region of the spectrum. The arrow indicates the direction of the absorbance change. (Top right) The time course of the absorbance change allows the determination of a dissociation rate of 4.9 × 10-2 s-1.

[0025] [Figure 11] Thermal unfolding of Fe(III) HBD RcoM-1 with a C94S mutation. Unfolding is monitored by the absorbance change at the Soret maximum of heme at 420 nm. Each UV-Vis spectrum was recorded after the sample was equilibrated at each temperature for 5 minutes. The small loss in Soret intensity observed between 20 and 75 °C is likely due to a change in the heme coordination number. The loss in Soret intensity between 75 and 98 °C was attributed to the loss of heme from the protein due to thermal unfolding. (Top left) UV-Vis spectra of Fe(III) HBD RcoM-1 with a C94S mutation recorded at temperatures between 20 and 98 °C. (Top right) Absorbance values ​​at the Soret maximum (420 nm) as a function of temperature. (Bottom left) UV-Vis spectra recorded during thermal unfolding between 75 and 98 °C. (Bottom right) Absorbance at the Soret maximum as a function of temperature recorded during thermal unfolding between 75° C. and 98° C. Using these data, the melting temperature, Tm, was determined to be 91° C.

[0026] [Figure 12]Comparison of electronic absorption (UV-Vis) spectra for RcoM heme-binding domain (HBD) truncation species in WT (Figure 12A) and Cys-replaced protein variants CC HBD (Figure 12B), C94S (Figure 12C), and CCC HBD (Figure 12D). Spectra for ferric iron (Fe(III)), deoxyferrous iron (Fe(II)), and ferrous-CO species (Fe(II)-CO), and oxyferrous iron (Fe(II)-O) are displayed.

[0027] [Figure 13] Comparison of electronic absorption (UV-Vis) spectra for RcoM HBD truncation species in the Met104 variants CC M104A (Figure 13A) and CC M104H (Figure 13B), each bearing Cys94. Spectra for ferric iron (Fe(III)), deoxyferrous iron (Fe(II)), and ferrous-CO species (Fe(II)-CO), and oxyferrous iron (Fe(II)-O) are displayed. (Figure 13C) Schematic of the protein-based ligand switching mechanism for RcoM highlighting the coordination sphere changes in these variants.

[0028] [Figure 14] Comparison of electronic absorption (UV-Vis) spectra for RcoM HBD truncation species in Met104 variants CCC M104A (Figure 14A), CCC M104L (Figure 14B), and CCC M104H (Figure 14C), each with a Cys94→Ser substitution. Spectra for ferric iron (Fe(III)), deoxyferrous iron (Fe(II)), and ferrous-CO species (Fe(II)-CO), and oxyferrous iron (Fe(II)-O) are displayed (Figure 14D). Schematic of the protein-derived ligand switching mechanism for RcoM highlighting the coordination sphere changes in these variants.

[0029] [Figure 15]Quantification of oxygen-binding affinity (P50) of RcoM HBD truncations. The fraction of oxygen-bound heme protein was measured as a function of oxygen partial pressure using UV-Vis spectroscopy using a tonometer apparatus equipped with an optical cuvette. (Figure 15A) Representative spectral changes of UV-Vis signatures for CC HBD RcoM variants as a function of oxygen partial pressure (P0). Oxygen-binding curves for CC HBD (Figure 15B), C94S HBD (Figure 15C), and CCC HBD (Figure 15D) plotted in terms of the fraction of oxygen-free (deoxy) and oxygen-bound (oxy+Fe(III)) heme protein. Autooxidation at low oxygen tension likely accounts for the formation of some ferric heme. The curves were fitted to a nonlinear, single-site binding model to quantify P50.

[0030] [Figure 16] Determination of the second-order rate constant (k,CO) for CO binding to RcoM WT HBD (Figure 16A) and the HBD truncations CC HBD (Figure 16B), C94S (Figure 16C), and CCC HBD (Figure 16D). The CO binding rate at each concentration of CO was measured using stopped-flow UV-Vis spectroscopy and fitted to a monoexponential equation. Each data point represents the average of two to three replicate measurements of these rates. Linear regression was applied to each curve, and the second-order rate constant was estimated as the slope.

[0031] [Figure 17] Representative determination of autooxidation rate (koxid) for WT HBD RcoM cleavage products. (Figure 17A) Reference spectra of Fe(III) and Fe(II)-O2 proteins. (Figure 17B) Spectral changes of UV-Vis signatures for Fe(II)-O2 WT HBD. (Figure 17C) Spectral changes at 542 nm and 573 nm were fitted with a single exponential to determine koxid.

[0032] [Figure 18]Summary of ligand binding parameters and heme stability properties for WT RcoM and the RcoM HBD variants C94S, CC HBD, and CCC HBD. [Figure 19] Representative unfolding of Fe(III) CCC HBD RcoM in the presence of urea at 37 °C. (Figure 19A) Unfolding was monitored by the absorbance change at the heme Soret maximum at 415 nm. Samples were allowed to equilibrate for 10 min before recording each UV-Vis spectrum. (Figure 19B) The unfolding data were fitted to a sigmoidal curve to determine the denaturant concentration at which half of the protein sample was unfolded ([D]).

[0033] [Figure 20] Lack of reactivity between RcoM HBD truncations and hydrogen peroxide. The Fe(III)WT HBD (Figure 20A) and variants CCC HBD (Figure 20B), CCC M104A HBD (Figure 20C), and CCC M104H HBD (Figure 20D) were incubated with 500 μM hydrogen peroxide at pH 7.4 and 25°C and monitored by UV-Vis spectroscopy every 2 minutes for 30 minutes. Minimal spectral changes were observed for each variant, suggesting that hydrogen peroxide does not react with the Fe(III) heme center of the RcoM HBD truncations to generate highly oxidized species.

[0034] [Figure 21] Summary of nitrate reduction data for full-length and HBD-truncated RcoM variants. Ferrous protein (10–15 μM) was incubated with 1–5 mM sodium nitrite at 37°C in the presence of 2.5 mM sodium dithionite. (Figure 21A) UV-Vis spectroscopy was used to monitor the conversion of Fe(II) heme to Fe(II)-NO. (Figures 21B–21C) The changes in spectral features at 562 nm and 578 nm were fitted to single-exponential curves to determine the observed rates of nitrite reduction. The observed rates were plotted as a function of nitrite concentration, and linear regression was applied to each plot to estimate the second-order rate constant as the slope.

[0035] [Figure 22] Representative kinetic traces for in vitro CO transfer from hemoglobin (Hb) to WT RcoM HBD (Figure 22A) and RcoM HBD variants CC HBD (Figure 22B), C94S HBD (Figure 22C), and CCC HBD (Figure 22D) under aerobic conditions at 37°C. CO2-bound Hb (20 μM) was incubated with equimolar ferrous oxyferrous RcoM, and CO transfer from Hb to RcoM was monitored using UV-Vis spectroscopy. The fraction of each CO2-bound heme protein was determined using spectral deconvolution, and the corresponding kinetic traces were fitted to single- or double-exponential equations. The half-life of each CO2-bound species is displayed, and the half-life and amplitude of the fast species are displayed for the double-exponentially fitted curves.

[0036] [Figure 23] Kinetic traces monitoring CO transfer from HbCO2 encapsulated in red blood cells (RBCs) to extracellular RcoM HBD cleavage products under aerobic conditions at 37 °C. Heme proteins were incubated at equimolar concentrations (50–100 μM), and RBCs were separated from extracellular RcoM by centrifugation at each time point. UV-Vis spectroscopy was used to monitor CO2 transfer from Hb to WT HBD RcoM (Figure 23A) and C94S HBD RcoM (Figure 23B). The fraction of each CO2-bound heme protein was determined using spectral deconvolution, and the corresponding kinetic traces were fitted to a monoexponential equation. Data points represent the mean ± SEM of triplicates, and the half-life of COHb in each experiment is shown.

[0037] [Figure 24]In an in vivo lethal CO poisoning model, the C94S and CCC HBD RcoM variants capture CO from HbCO. Schematic diagram of the in vivo model of severe CO poisoning in mice (top). Anesthetized, mechanically ventilated mice were exposed to 3,000 ppm CO in air for 4.5 minutes, after which Fe(II)-O2 CCC HBD RcoM was intravenously infused at an injection volume of 10 μL per gram of body weight (heme protein concentrations are listed in the table, bottom). Blood samples (15 μL) were collected immediately before and after the infusion and 25 minutes after CO exposure. At each time point, RBCs were separated from plasma by centrifugation, and the separated RBC pellets and plasma samples were immediately frozen at -80°C. The fraction of CO-bound hemoglobin (%HbCO) and the fraction of CO-bound RcoM (%RcoM-CO) from RBCs were then determined using spectral deconvolution. Infusion of RcoM resulted in a greater decrease in the fraction of Hb bound to CO (Δ%HbCO) compared with infusion with PBS. DETAILED DESCRIPTION OF THE INVENTION

[0038] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by reference to either of the displayed strands. The Sequence Listing was submitted as an 18.7 KB ASCII text file created on May 3, 2021, and is incorporated herein by reference. In the accompanying Sequence Listing: SEQ ID NO: 1 is the amino acid sequence of full-length WT RcoM-1 from P. xenovorans. SEQ ID NO: 2 is the amino acid sequence of truncated RcoM-1 lacking the LytTR domain (HBD16). SEQ ID NO: 3 is the amino acid sequence of a truncated RcoM-1 lacking the LytTR domain and part of the PAS domain (HBD12). SEQ ID NO: 4 is the amino acid sequence of WT RcoM from H. crassostreae. SEQ ID NO:5 is the amino acid sequence of the cleavage site from tobacco etch virus (TEV). SEQ ID NO: 6 is the amino acid sequence of the cleavage site from thrombin. SEQ ID NO: 7 is the amino acid sequence of the RcoM variant C94S HBD. SEQ ID NO: 8 is the amino acid sequence of the RcoM variant C127S / C130S HBD. SEQ ID NO: 9 is the amino acid sequence of the RcoM variant CCC HBD. SEQ ID NO: 10 is the amino acid sequence of the RcoM variant CC M104A HBD. SEQ ID NO: 11 is the amino acid sequence of the RcoM variant CC M104H HBD. SEQ ID NO: 12 is the amino acid sequence of the RcoM variant CCC M104A HBD. SEQ ID NO: 13 is the amino acid sequence of the RcoM variant CCC M104H HBD. SEQ ID NO: 14 is the amino acid sequence of the RcoM variant CCC M104L HBD.

[0039] Detailed Description I. Abbreviations CO Carbon monoxide H2S Hydrogen sulfide Hb hemoglobin Hb-CO Carboxyhemoglobin HBOC hemoglobin-based oxygen carrier HBD heme-binding domain NO Nitric oxide RcoM: Regulator of carbon monoxide metabolism TEV tobacco etch virus WT wild type II. Terminology and Methods

[0040] Unless otherwise stated, terminology is used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0041] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" can include single or multiple antigens and be considered equivalent to the expression "at least one antigen." As used herein, the term "comprise" means "include." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are given for convenience unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0042] In order to facilitate review of the various embodiments, the following explanations of terms are provided:

[0043] Administration: Providing or giving an agent, such as a therapeutic agent (e.g., a recombinant RcoM protein), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection or infusion (subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraventricular, intrastriatal, intracranial, intraspinal, etc.), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0044] Affinity tag: A peptide sequence added to a recombinant protein or polypeptide to aid in purification using affinity-based purification techniques such as affinity chromatography. Examples of affinity tags include, but are not limited to, albumin binding protein, alkaline phosphatase, AU1 epitope, AU5 epitope, bacteriophage T7 epitope, bacteriophage V5 epitope, biotin carboxy carrier protein, bluetongue virus tag, calmodulin binding peptide, chloramphenicol acetyltransferase, cellulose binding domain, chitin binding domain, choline binding domain, dihydrofolate reductase, E2 epitope, FLAG epitope, galactose binding protein, green fluorescent binding protein, Glu-Glu (EE tag), glutathione S-transferase, influenza hemagglutinin, HaloTag ( , histidine affinity tag, horseradish peroxidase, HSV epitope, ketosteroid isomerase, KT3 epitope, LacZ, luciferase, maltose binding protein, Myc epitope, NusA, PDZ domain, PDZ ligand, polyarginine, polyaspartate, polycysteine, polyhistidine, polyphenylalanine, Profinity eXact, protein C, S1-tag, S-tag, Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep-tag, streptavidin, small ubiquitin-like modifier (SUMO), thioredoxin, TrpE, ubiquitin, and VSV-G (e.g., Kimple). (See, e.g., et al., Curr Protoc Protein Sci 73: 9.9.1-.9.9.23, 2013, doi:10.1002 / 0471140864.ps0909s73).

[0045] Anemia: A deficiency of red blood cells and / or hemoglobin. Anemia is the most common disorder of the blood, resulting in a reduced ability of the blood to transfer oxygen to tissues. Because all human cells depend on oxygen for survival, varying degrees of anemia can have a wide range of clinical consequences. The three major classifications of anemia include excessive blood loss (acute, as in hemorrhage, or chronic, as in small blood loss), excessive destruction of blood cells (hemolysis), or insufficient red blood cell production (hematopoiesis).

[0046] The term "anemia" refers to all types of clinical anemia, including, but not limited to, microcytic anemia, iron deficiency anemia, hemoglobinopathies, heme synthesis disorders, globin synthesis disorders, sideroblastic disorders, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, and cold agglutinin hemolytic anemia.

[0047] In severe cases of anemia or during ongoing blood loss, a blood transfusion may be required. Physicians use one of several clinically accepted criteria to determine whether a blood transfusion is necessary to treat a subject with anemia. For example, the currently accepted Rivers protocol for early targeted treatment of sepsis requires a sustained hematocrit level above 30.

[0048] Anoxia: a pathological condition in which the body as a whole or an area of ​​the body is completely deprived of oxygen.

[0049] antidote: an agent that neutralizes or counteracts the effects of a poison such as carbon monoxide.

[0050] Bleeding disorder: A general term for a wide range of medical problems that lead to impaired blood clotting and continued bleeding. Physicians may refer to bleeding disorders by terms such as coagulation abnormalities, abnormal bleeding, and coagulation disorders. Bleeding disorders include any congenital, acquired, or inducible defect that results in abnormal (or pathological) bleeding. Examples include, but are not limited to, disorders of inadequate clotting or hemostasis, such as hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency), hemophilia C (factor XI deficiency), other clotting factor deficiencies (such as factor VII or XIII), abnormal levels of clotting factor inhibitors, platelet disorders, thrombocytopenia, vitamin K deficiency, and von Willebrand's disease.

[0051] Bleeding episode: Refers to an occurrence of uncontrolled, excessive, and / or pathological bleeding. Bleeding episodes may result from, for example, drug-induced bleeding (such as bleeding induced by nonsteroidal anti-inflammatory drugs or warfarin), anticoagulant overdose or poisoning, aneurysm, vascular rupture, surgery, and trauma (including, for example, abrasions, contusions, lacerations, incisions, or gunshot wounds). Bleeding episodes may also result from diseases such as cancer, gastrointestinal ulcers, or infections.

[0052] Blood replacement products or blood substitutes: Compositions used to replenish fluid volume and / or carry oxygen and other blood gases in the cardiovascular system. Blood substitutes include, for example, volume expanders (to increase blood volume) and oxygen therapeutics (to transport oxygen in the blood). Oxygen therapeutics include, for example, hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbons (PFCs). Preferred blood substitutes mimic the oxygen-carrying capacity of hemoglobin, do not require cross-matching or compatibility testing, have a long shelf life, exhibit a long intravascular half-life (greater than days to weeks), and are free of side effects and pathogens.

[0053] Carbon monoxide (CO): A colorless, odorless, and tasteless gas that is toxic to humans and animals when encountered in sufficiently high concentrations. CO also occurs at low levels during normal animal metabolism.

[0054] Carboxyhemoglobin (HbCO): A stable complex of carbon monoxide (CO) and hemoglobin (Hb) formed in red blood cells when CO is inhaled or produced during normal metabolism.

[0055] Carboxyhemoglobinemia or carbon monoxide poisoning: A condition caused by excessive amounts of carbon monoxide in the blood. Typically, exposure to 100 parts per million (ppm) or more of CO is sufficient to cause carboxyhemoglobinemia. Symptoms of mild acute CO poisoning include dizziness, confusion, headache, dizziness, and flu-like effects, while higher exposures can lead to significant central nervous system and cardiac toxicity and even death. Long-term sequelae often occur after acute poisoning. Carbon monoxide can also have serious effects on the fetus in pregnant women. Chronic exposure to low levels of carbon monoxide can lead to depression, confusion, and memory loss. Carbon monoxide causes its harmful effects in humans primarily by combining with hemoglobin in the blood to form carboxyhemoglobin (HbCO). This prevents oxygen from binding to hemoglobin, reducing the blood's oxygen-carrying capacity and leading to hypoxia. Additionally, myoglobin and mitochondrial cytochrome oxidase are thought to be adversely affected. Carbonmonoxyhemoglobin can revert back to hemoglobin, but recovery takes time because the HbCO complex is fairly stable. Current treatment methods for CO poisoning include administering 100% oxygen or providing hyperbaric oxygen therapy.

[0056] Cerebral ischemia or ischemic stroke: A condition that occurs when the tissues' oxygen demand exceeds their oxygen supply as a result of partial or complete blockage of an artery to or within the brain. After an ischemic stroke, the brain suffers damage due to a lack of oxygen and other nutrients.

[0057] Coagulation disorder: A medical term for a defect in the body's mechanism for blood clotting.

[0058] Contacting: To bring into direct physical association; includes both solid and liquid forms. When used in connection with in vivo methods, "contacting" also includes administering.

[0059] Cyanide poisoning: A type of poisoning resulting from exposure to some forms of cyanide, such as hydrogen cyanide gas and cyanide salts. Cyanide poisoning can occur through smoke inhalation from house fires, exposure to metal abrasives, certain pesticides, and certain seeds (such as apple seeds). Early symptoms of cyanide poisoning include headache, dizziness, rapid heart rate, shortness of breath, and vomiting. Later symptoms include convulsions, slowed heart rate, low blood pressure, loss of consciousness, and cardiac arrest.

[0060] Cytochrome c oxidase: enzyme that is part of the respiratory electron transport chain. This enzyme is found in mitochondria.

[0061] Vicia faba: common name for glucose-6-phosphate dehydrogenase (G6PD) deficiency; an X-linked recessive disorder characterized by nonimmune hemolytic anemia responsive to several causes.

[0062] Fusion protein: A protein that contains at least part of two different (heterologous) proteins.

[0063] Gastrointestinal bleeding: Refers to any form of bleeding (blood loss) in the gastrointestinal tract from the pharynx to the rectum.

[0064] Hemoglobin (Hb): An iron-containing, oxygen-transporting metalloprotein in the red blood cells of vertebrates and other animals. In humans, the hemoglobin molecule is an assembly of four globular protein subunits. Each subunit consists of a protein chain tightly associated with a nonprotein heme group. Each protein chain is arranged into a set of alpha-helical structural segments connected together in a globin-fold configuration, so named because this is the same folding motif used in other heme / globin proteins. This folding pattern contains a pocket that tightly binds the heme group.

[0065] Hemoglobin-based oxygen carrier (HBOC): A transfusable liquid of purified, recombinant, and / or modified hemoglobin that functions as an oxygen carrier and can be used as a blood substitute. Several HBOCs are known and / or in clinical development. Examples of HBOCs include, but are not limited to, DCLHb (HEMASSIST™; Baxter), MP4 (HEMOSPAN™; Sangart), pyridoxylated Hb POE-conjugate (PHP) + catalase & SOD (Apex Biosciences), OR-PolyHbA0 (HEMOLINK™; Hemosol), PolyBvHb (HEMOPURE™; Biopure), PolyHb (POLYHEME™; Northfield), rHb1.1 (OPTRO™; Somatogen), PEG-Hemoglobin (Enzon), OXYVITA™, and HBOC-201 (Greenburg and Kim, Crit Care 8(Suppl 2):S61-S64, 2004; te Lintel Hekkert et al., Am J Physiol Heart Circ Physiol 298:H1103-H1113, 2010; Eisenach, Anesthesiology 111:946-963, 2009).

[0066] Hemophilia: the name for several inherited genetic diseases that impair the body's ability to control clotting.

[0067] Hemorrhage: Loss of blood from the circulatory system. Bleeding can occur internally, when blood leaks from blood vessels inside the body, or externally, either from a natural opening such as the vagina, mouth, or rectum, or from a break in the skin.

[0068] Heterologous: A heterologous protein or polypeptide refers to a protein or polypeptide that is derived from a different source or species.

[0069] Hydrogen sulfide poisoning: A type of poisoning resulting from excessive exposure to hydrogen sulfide (H2S). H2S binds to iron in mitochondrial cytochrome enzymes, preventing cellular respiration. Exposure to low levels of H2S can cause eye irritation, sore throat, coughing, nausea, shortness of breath, pulmonary edema, fatigue, loss of appetite, headache, irritability, memory loss, and dizziness. Higher levels of exposure can cause immediate collapse, inability to breathe, and death.

[0070] Hemorrhagic shock: A state of reduced tissue perfusion resulting in inadequate delivery of oxygen and nutrients necessary for cellular function. Hypovolemic shock, the most common type, results from loss of circulatory blood volume due to clinical etiologies such as penetrating and blunt trauma, gastrointestinal bleeding, and obstetric hemorrhage.

[0071] Hypoxemia: An abnormal deficiency in oxygen concentration in arterial blood.

[0072] Hypoxia: a condition in which the body as a whole (systemic hypoxia) or an area of ​​the body (tissue hypoxia) is deprived of an adequate supply of oxygen.

[0073] Ischemia: A vascular phenomenon in which a reduction in blood supply to an organ, tissue, or part of the body is caused, for example, by constriction or occlusion of one or more blood vessels. Ischemia can also result from vasoconstriction or thrombosis or embolism. Ischemia can lead to direct ischemic injury, tissue damage due to cell death caused by reduced oxygen supply.

[0074] Ischemia / reperfusion injury: Ischemia / reperfusion injury involves the immediate injury that occurs during the lack of blood flow, as well as tissue injury that occurs after blood flow is restored. Much of this injury is now understood to be caused by chemicals and free radicals released in the ischemic tissue.

[0075] When tissue is exposed to ischemia, a series of chemical events can be initiated, ultimately leading to cellular dysfunction and necrosis. If ischemia terminates with the restoration of blood flow, a second series of adverse events ensues, resulting in further injury. Thus, whenever blood flow is transiently reduced or blocked in a subject, the resulting injury involves two components: direct injury occurring during the ischemic period and indirect or reperfusion injury occurring afterward. During prolonged ischemic periods, direct ischemic injury caused by hypoxia dominates. During relatively brief periods of ischemia, indirect or reperfusion-mediated injury becomes increasingly important. In some cases, reperfusion-induced injury can be more severe than injury induced by ischemia itself. This pattern of relative contributions of direct and indirect mechanisms of injury has been shown to occur in all organs.

[0076] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified from other biological components, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells, in the cells, blood, or tissues of the organism in which it naturally occurs, or in the organism itself. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0077] Methemoglobin: An oxidized form of hemoglobin in which the iron in the heme component has been oxidized from the ferrous (+2) to the ferric (+3) state. This renders the hemoglobin molecule unable to effectively transport and release oxygen to tissues. Normally, about 1% of total hemoglobin is present in the methemoglobin form.

[0078] Microcytosis: a blood disorder characterized by the presence of microcytocytes (abnormally small red blood cells) in the blood.

[0079] Myoglobin: a heme-containing globin protein found in the muscle tissue of vertebrates and most mammals. Myoglobin transports and stores oxygen in muscle cells.

[0080] Oxidizing agent: A substance capable of accepting electrons from another substance (also referred to as "oxidizing" a substance). An oxidizing agent gains electrons in a chemical reaction and is reduced. An oxidizing agent is also known as an "electron acceptor." In some embodiments herein, the oxidizing agent is a quinone, such as benzoquinone or naphthaquinone. In other embodiments, the oxidizing agent is an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof. In some examples, an electron carrier (e.g., TMPD or crystal violet) is used in combination with the oxidizing agent to facilitate electron transfer. In some embodiments herein, oxidation of RcoM is achieved by exposure to visible light.

[0081] Paraburkholderia xenovorans: A species of purple bacteria found in soil. P. xenovorans is a gram-negative aerobic bacterium. At 9.7 Mb, P. xenovorans has one of the largest known prokaryotic genomes. This bacterium is capable of efficiently degrading polychlorinated biphenyls (PCBs). P. xenovorans is also known as Burkholderia xenovorans.

[0082] Peptide or polypeptide: A polymer whose monomers are amino acid residues joined together by amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, with the L-isomer being preferred. The terms "peptide," "polypeptide," or "protein," as used herein, encompass any amino acid sequence and are intended to include modified sequences, including modified RcoM proteins. The terms "peptide" and "polypeptide" are specifically intended to encompass naturally occurring proteins as well as those produced recombinantly or synthetically.

[0083] Conservative amino acid substitutions are those substitutions that, when made, minimally interfere with the properties of the original protein, i.e., such substitutions preserve and do not significantly alter the structure, and particularly the function, of the protein. Examples of conservative substitutions are shown in the table below. [Table 6]

[0084] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains.

[0085] Substitutions generally expected to produce the greatest changes in protein properties will be non-conservative, for example, (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) a cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue with an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamine or aspartic acid; or (d) a residue with a bulky side chain, e.g., phenylalanine, is substituted for (or by) a residue without a side chain, e.g., glycine.

[0086] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of the proteins and other compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually contain an injectable fluid containing a pharmaceutically and physiologically acceptable fluid, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0087] Preventing, treating, or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of the disease. "Treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or condition after they have begun to develop, for example, reducing HbCO in the blood of a subject with CO poisoning. "Amelioration" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0088] Purified: The term purified does not require absolute purification, but rather is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more abundant than the peptide or protein in its natural environment within a cell. In one embodiment, the preparation is purified such that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Thus, in a specific, non-limiting example, a substantially purified protein is 90% free from other proteins or cellular components.

[0089] Recombinant: A recombinant nucleic acid or protein is a nucleic acid or protein having a sequence that is not found in nature or that is created by the artificial combination of two naturally separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid, e.g., genetic engineering techniques. The term recombinant includes nucleic acids and proteins that have been altered by the addition, substitution, or deletion of portions of naturally occurring nucleic acid molecules or proteins.

[0090] Reducing Agent: An element or compound that loses (or "donates") electrons to another chemical species in a chemical redox reaction. A reducing agent is typically in one of its lower possible oxidation states and is known as an electron donor. Because it loses electrons in a redox reaction, it becomes oxidized. Exemplary reducing agents include, but are not limited to, sodium dithionite, ascorbic acid, N-acetylcysteine, methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, earth metals, formic acid, and sulfite compounds.

[0091] Regulator of carbon monoxide metabolism (RcoM): a protein found in some prokaryotes involved in CO sensing and transcriptional regulation. RcoM proteins contain an N-terminal PAS domain and a DNA-binding LytTR domain. The PAS domain contains a hexacoordinate b-type heme moiety and binds strongly to CO and nitric oxide (NO). Residues His74 and Met104 in the PAS domain serve as axial ligands for the heme Fe(II), with Met104 displacing upon CO or NO binding. The aerobic Gram-negative bacterium Paraburkholderia xenovorans (also known as Burkholderia xenovorans) expresses two homologous proteins, RcoM-1 and RcoM-2, which share approximately 93% sequence identity and have a very high affinity for CO. RcoM-1 and RcoM-2 act as CO sensors capable of regulating aerobic and anaerobic CO oxidation. The wild-type amino acid sequence of RcoM-1 from P. xenovorans is set forth herein as SEQ ID NO: 1. RcoM homologues (and UniProt IDs) from various bacterial species are listed in Table 3.

[0092] Rhabdomyolysis: Rapid destruction of skeletal muscle tissue due to mechanical, physical, or chemical trauma. The primary consequence is acute renal failure due to the massive release of creatine phosphokinase enzyme and other cellular by-products into the blood system and the accumulation of muscle breakdown products, some of which are toxic to the kidneys.

[0093] Sequence identity / similarity: Identity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more significant when orthologous proteins or cDNAs are derived from more closely related species (such as human and mouse sequences) compared to more distantly related species (such as human and C. elegans sequences).

[0094] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, which presents a detailed discussion of sequence alignment methods and homology calculations.

[0095] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0096] Spherocytosis: an autolytic hemolytic anemia characterized by the production of red blood cells (or erythrocytes) that are spherical rather than doughnut-shaped.

[0097] Subject: Living multicellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

[0098] Thalassemia: an inherited, autosomal recessive blood disorder. In thalassemia, a genetic defect results in a reduced rate of synthesis of one of the globin chains that make up hemoglobin. The reduced synthesis of one of the globin chains leads to the formation of abnormal hemoglobin molecules, resulting in anemia, a symptom characteristic of thalassemia.

[0099] Therapeutically effective amount: An amount of a compound or composition, e.g., isolated or recombinant RcoM protein, sufficient to achieve a desired effect in a treated subject. For example, this may be the amount necessary to scavenge carbon monoxide in blood or tissue, reduce HbCO levels in the blood, and / or reduce one or more signs or symptoms associated with carbon monoxide poisoning.

[0100] Ulcer: An open wound on the skin, eye, or mucous membrane, often, but not exclusively, caused by an initial abrasion and generally maintained by inflammation, infection, and / or a medical condition that prevents healing.

[0101] Vasospasm: A cause of stroke secondary to spasm of the blood vessels supplying the brain. This type of stroke typically follows a subarachnoid aneurysm, with delayed onset of vasospasm occurring within 2 to 3 weeks of the hemorrhagic event. This type of stroke may be associated with sickle cell disease. IV. Recombinant RcoM Protein

[0102] There is a need for an effective, rapid, and easily available therapy for treating carboxyhemoglobinemia. The present disclosure provides a recombinant regulator of carbon monoxide metabolism (RcoM) protein that exhibits very high affinity for carbon monoxide and can therefore be used as a CO scavenger. The RcoM protein of the present disclosure may be used to treat hydrogen sulfide or cyanide poisoning, or may be used as a blood substitute.

[0103] The RcoM protein was first identified as a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS-fold domain to a C-terminal DNA-binding LytTR domain (see Figure 1). RcoM proteins contain a hexacoordinate b-type heme moiety that tightly binds CO and nitric oxide (NO). PAS domain residues His74 and Met104 (relative to SEQ ID NO: 1) function as heme Fe(II) axial ligands, with Met104 displacing upon CO or NO binding. Two RcoM homologs from P. xenovorans (RcoM-1 and RcoM-2) are functional in vivo and act as CO sensors capable of regulating aerobic and anaerobic CO oxidation.

[0104] RcoM exhibits a very high affinity for CO and is selective for CO over oxygen. Given these properties, the RcoM proteins of the present disclosure are ideal for treating carbon monoxide poisoning by directly capturing CO from CO-bound hemoglobin, myoglobin, and cytochrome c oxidase. The RcoM proteins of the present disclosure can also be used to treat cyanide or H2S poisoning or as a blood substitute. Directed mutations that enhance stability, increase CO affinity, and / or decrease the oxygen affinity of the RcoM proteins are also described herein.

[0105] The wild-type (WT) and modified RcoM proteins are described below. In the WT amino acid sequence (SEQ ID NO: 1), the LytTR domain (DNA binding) is underlined, and the remainder of the sequence is the PAS domain (see Figure 1). The truncated RcoM proteins disclosed herein (SEQ ID NOs: 2, 3, and 7-14) do not contain the LytTR domain (see Figures 2 and 3). In all RcoM sequences (SEQ ID NOs: 1-3, and 7-14), the bolded residues correspond to H74, C94, M104, C127, C130, and M105, as numbered relative to SEQ ID NO: 1. WT RcoM-1 (29 kDa) from P. xenovorans: [ka] HBD16 RcoM (16kDa) cleavage product: [ka] HBD12 RcoM (12kDa) cleavage product: [ka]

[0106] Throughout this disclosure, unless otherwise indicated, specific amino acid residues are numbered with reference to full-length WT RcoM-1 in SEQ ID NO: 1. Table 1 lists the corresponding residue positions in SEQ ID NOs: 1-3, respectively. Table 1. Important residues in the WT and truncated RcoM sequences [Table 1]

[0107] Eight RcoM HBD variants were generated based on HBD16 of SEQ ID NO: 2. Table 2 lists each variant along with their respective amino acid substitutions and the complete amino acid sequence (bold residues indicate substitutions). Table 2. RcoM HBD16 variants [Table 2-1] [Table 2-2] [Table 2-3]

[0108] Provided herein is a recombinant regulator of carbon monoxide metabolism (RcoM) protein that exhibits a very high affinity for CO. In some embodiments, the recombinant RcoM protein comprises a heme-binding domain (HBD), and the amino acid sequence of the HBD is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2. In some embodiments, the amino acid sequence of the HBD is a wild-type sequence (e.g., SEQ ID NO:2). In other embodiments, the amino acid sequence of the HBD comprises an amino acid substitution at one or more of H74, C94, M104, M105, C127, and C130. In some examples, the amino acid sequence of the HBD is at least 90% or at least 95% identical to SEQ ID NO:2 and comprises an amino acid substitution at one or more of C94, M104, C127, and C130.

[0109] The RcoM proteins of the present disclosure may be modified, such as by amino acid substitutions at various residues, to alter heme ligand affinity and / or specificity and / or enhance protein stability. In some embodiments, the RcoM protein contains a single amino acid substitution. In other embodiments, the RcoM protein contains at least two, at least three, at least four, at least five, or at least six amino acid substitutions. In some examples, the amino acid substitutions are conservative substitutions.

[0110] In some examples, the recombinant RcoM protein includes a substitution at H74, which is a heme-coordinating histidine. In specific, non-limiting examples, the substitution is selected from H74S, H74T, H74M, H74W, H74A, H74L, H74I, H74V, and H74G.

[0111] In some examples, the recombinant RcoM protein includes a substitution at C94, which is the Fe(II) heme-coordinating cysteine. In specific, non-limiting examples, the substitution is selected from C94S, C94T, C94H, C94W, C94M, C94A, C94L, C94I, C94V, and C94G.

[0112] In some examples, the recombinant RcoM protein includes a substitution at M104, which is an Fe(II) heme-coordinating methionine. In specific, non-limiting examples, the substitution is selected from M104S, M104T, M104H, M104W, M104A, M104L, M104I, M104V, and M104G.

[0113] In some examples, the recombinant RcoM protein comprises a substitution at M105, which is a non-heme-coordinating methionine. In specific, non-limiting examples, the substitution is selected from M105S, M105T, M105H, M105W, M105A, M105L, M105I, M105V, and M105G.

[0114] In some examples, the recombinant RcoM protein comprises a substitution at C127, which is a non-heme-coordinating cysteine. In specific, non-limiting examples, the substitution is selected from C127S, C127T, C127M, C127A, C127L, C127I, C127V, and C127G.

[0115] In some examples, the recombinant RcoM protein comprises a substitution at C130, which is a non-heme-coordinating cysteine. In specific, non-limiting examples, the substitution is selected from C130S, C130T, C130M, C130A, C130L, C130I, C130V, and C130G.

[0116] In some examples, the recombinant RcoM protein comprises a single amino acid substitution in C94, a single amino acid substitution in M104, two amino acid substitutions in C94 and M104, two amino acid substitutions in C127 and C130, three amino acid substitutions in C94, C127 and C130, three amino acid substitutions in M104, C127 and C130, three amino acid substitutions in H74, C94 and M104, four amino acid substitutions in C94, M104, C127 and C130, five amino acid substitutions in C94, M104, M105, C127 and C130, five amino acid substitutions in H74, C94, M104, C127 and C130, or six amino acid substitutions in H74, C94, M104, M105, C127 and C130. In specific, non-limiting examples, the recombinant RcoM protein has a C94S substitution; a C127S substitution and a C130S substitution; a C94S substitution, a C127S substitution and a C130S substitution; a C94S substitution and a M104L substitution; a M104A substitution, a C127S substitution and a C130S substitution; a M104H substitution, a C127S substitution and a C130S substitution; a M104L substitution, a C127S substitution and a C130S substitution; a C94S substitution, a M104A substitution, a C127S substitution and a C130S substitution; and a C130S substitution; a C94S substitution, an M104H substitution, a C127S substitution and a C130S substitution; a C94S substitution, an M104L substitution, a C127S substitution and a C130S substitution; a H74S substitution, a C94S substitution and an M104L substitution; a C94S substitution, an M104L substitution, an M105L substitution, a C127S substitution and a C130S substitution; or a H74S substitution, a C94S substitution, an M104L substitution, an M105L substitution, a C127S substitution and a C130S substitution.

[0117] In particular examples, the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

[0118] In some embodiments, the RcoM protein has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-3. In some examples, the RcoM protein comprises or consists of any one of SEQ ID NOs: 1-3.

[0119] In some examples, the amino acid sequence of the RcoM protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, except for amino acid substitutions at one or more of H74, C94, M104, C127, C130 and M105.

[0120] In a specific example, the amino acid sequence of the RcoM protein consists of SEQ ID NO: 1, except for the H74S substitution, the C94S substitution, the M104 substitution selected from M104A, M104H, and M104L, the M105L substitution, the C127S substitution, the C130S substitution, or any combination thereof. In another example, the amino acid sequence of the protein consists of SEQ ID NO: 2, except for the H74S substitution, the C94S substitution, the M104 substitution selected from M104A, M104H, and M104L, the M105L substitution, the C127S substitution, the C130S substitution, or any combination thereof. In yet another specific example, the amino acid sequence of the protein consists of SEQ ID NO: 3, except for the H74S substitution, the C94S substitution, the M104 substitution selected from M104A, M104H, and M104L, the M105L substitution, the C127S substitution, the C130S substitution, or any combination thereof.

[0121] Using bioinformatics analysis, 112 rcoM genes from various microorganisms were identified, 44 of which are associated with aerobic CO metabolism. One of the identified rcoM genes is from a mesophilic microorganism (Hydrogenophaga crassostreae), which is believed to express an RcoM protein with enhanced thermostability. Thus, in some embodiments, the recombinant RcoM protein is from one of the species listed in Table 3 and has the listed UniProt ID. Table 3. Microorganisms with RcoM gene homologs [Table 3-1] [Table 3-2] [Table 3-3]

[0122] The amino acid sequences of the RcoM homologs listed above are incorporated herein by reference as they appeared in the UniProt database on May 11, 2020.

[0123] In some embodiments, the RcoM protein is derived from Hydrogenophaga crassostreae. In some examples, the RcoM protein has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some examples, the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO: 4. Full-length RcoM sequence from H. crassostreae

[0124] [ka]

[0125] In a specific, non-limiting example, the RcoM protein is at least 90% identical to SEQ ID NO: 4 and contains one or more of the amino acid substitutions described above for the RcoM-1 homologue from P. xenovorans (see Figure 6 for alignment).

[0126] In some embodiments, the recombinant RcoM protein comprises a tag at the N-terminus, C-terminus, or both. In some examples, the tag is an affinity tag, such as an affinity tag to aid in protein purification. Any suitable affinity tag can be used, such as one or more of His6, FLAG, glutathione S-transferase (GST), influenza virus hemagglutinin (HA), c-Myc, maltose-binding protein (MBP), protein A, or protein G. In a specific example, the affinity tag is a His6 tag. In some examples, the affinity tag is cleavable. In a specific example, the cleavage tag comprises a TEV-derived cleavage site having the amino acid sequence ENLYFQ[G / S] (SEQ ID NO: 5). In another specific example, the cleavage tag comprises a thrombin-derived cleavage site having the amino acid sequence LVPRGS (SEQ ID NO: 6).

[0127] In some embodiments, the recombinant RcoM protein does not contain a tag.

[0128] In some embodiments, the recombinant RcoM protein is present in an oxidized form (the heme bound to the CO of RcoM, which is Fe(II), is oxidized to Fe(III)). Oxidation of RcoM can be achieved, for example, by exposure to an oxidizing agent. In some embodiments, the oxidizing agent is an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof. In other embodiments, the oxidizing agent is a quinone, such as benzoquinone or naphthaquinone. In some instances, an electron carrier (e.g., TMPD or crystal violet) is used in combination with the oxidizing agent to facilitate electron transfer. In other embodiments, oxidation of RcoM is accomplished by exposure to visible light. For example, RcoM with a heme bound to CO that is Fe(II) is oxidized in the presence of air at 0.15 W / cm for a period of about 1-12 hours. 2 ~140W / cm 2 The cells can be exposed to white light (e.g., by exposure to an incandescent bulb such as a halogen lamp) using either fiber optics or a heat-dissipating screen, with intensities ranging from 100 to 1500 nm. Similar methods are described by Kerby et al. (J. Bacteriol 190:3336-3343, 2008), Bouzhir-Sima et al. (J Phys Chem B 120:10686-10694, 2016), and Salman et al. (Biochem 58:4028-4034, 2019). V. Pharmaceutical Compositions

[0129] The recombinant RcoM protein described herein can be administered as an isolated protein or as part of a pharmaceutical composition. Accordingly, provided herein are pharmaceutical compositions comprising the recombinant RcoM disclosed herein, or a derivative thereof, and one or more pharmaceutically acceptable excipients, and optionally one or more other active (therapeutic) ingredients. An excipient is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation of a pharmaceutical composition depends on several factors, such as the chosen route of administration. Any of the well-known techniques and excipients may be used as suitable and as understood in the art. The pharmaceutical compositions disclosed herein may be manufactured by any method known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, collecting, or compressing processes.

[0130] In some embodiments, pharmaceutical compositions are disclosed that comprise one or more recombinant RcoM proteins disclosed herein, together with one or more pharmaceutically acceptable carriers thereof and, optionally, one or more other therapeutic ingredients. An excipient / carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Appropriate formulation of a pharmaceutical composition will depend on the route of administration chosen. Any of the well-known techniques and excipients may be used as suitable and as understood in the art. In some embodiments, the composition comprises one or more of the following excipients: N-acetylcysteine, sodium citrate, glycine, histidine, glutamic acid, sorbitol, maltose, mannitol, trehalose, lactose, glucose, raffinose, dextrose, dextran, ficoll, gelatin, hydroxyethyl starch, benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, m-cresol, myristyl gamma-picolinium chloride, methylparaben, propylparaben, 2-penoxythanol, phenylmercuric nitrate, thimerosal, acetone sodium bisulfite, argon, palmitate Ascorbyl phosphate, ascorbic acid (sodium / acid), sodium bisulfite, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), cysteine / cysteic acid HCl, sodium dithionite (sodium hydrosulfite, sodium sulfoxylate), gentisic acid, gentisic acid ethanolamine, monosodium glutamate, glutathione, sodium formaldehyde sulfoxylate, potassium metabisulfite, sodium metabisulfite, methionine, monothioglycerol (thioglycerol), nitrogen, propyl gallate, sodium sulfite, alpha tocopherol, alpha tocopherol hydrogen succinate, and sodium thioglycolate. The present disclosure also contemplates other excipients, including any disclosed in Pramanick et al., Pharma Times 45(3): 65-77, 2013, which is incorporated herein by reference.

[0131] In some embodiments, the RcoM protein of the pharmaceutical composition is pegylated, polymerized, or crosslinked.

[0132] In some embodiments, the pharmaceutical composition comprises a native or recombinant globin molecule, such as native or recombinant hemoglobin or neuroglobin, or further comprises a hemoglobin-based oxygen carrier (HBOC). In some examples, the HBOC comprises DCLHb (HEMASSIST™; Baxter), MP4 (HEMOSPAN™; Sangart), pyridoxylated Hb POE-conjugate (PHP) + catalase & SOD (Apex Biosciences), OR-PolyHbA0 (HEMOLINK™; Hemosol), PolyBvHb (HEMOPURE™; Biopure), PolyHb (POLYHEME™; Northfield), rHb1.1 (OPTRO™; Somatogen), PEG-Hemoglobin (Enzon), OXYVITA™, or HBOC-201, or any combination thereof.

[0133] The pharmaceutical compositions disclosed herein may be administered by a variety of routes depending on whether local or systemic treatment is desired and on the area to be treated.

[0134] Pharmaceutical compositions include those suitable for parenteral (including subcutaneous, transdermal, intramuscular, intravenous, intraarterial, and intramedullary) or intraperitoneal administration, although the most suitable route may depend, for example, on the condition and disorder of the recipient. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or by injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration may be in the form of a single bolus dose or may be, for example, via a continuous perfusion pump. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like, may be necessary or desirable. In some embodiments, the compound may be contained in such pharmaceutical compositions together with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water-soluble vehicles, emulsifiers, buffers, humectants, humectants, solubilizers, preservatives, and the like. Those skilled in the art can refer to various pharmacological references for guidance. For example, see Modern Pharmaceutics, 5th Edition, Banker & Rhodes, CRC Press (2009); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 13th Edition, McGraw Hill, New York (2018). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Typically, these methods include the step of bringing into association the isolated recombinant RcoM molecule or derivative thereof disclosed herein (the "active ingredient") with the carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately associating the active ingredient with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired composition.

[0135] Recombinant RcoM protein may be formulated for parenteral administration by injection. Injectable compositions may be presented in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. Pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, or may be stored in powder or freeze-dried (lyophilized) form requiring only the addition of a sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind described above.

[0136] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of active compounds, which may contain antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions may also contain suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. If necessary, the suspension may contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions.

[0137] It should be understood that in addition to the ingredients particularly mentioned above, the pharmaceutical compositions may contain other agents conventional in the art having regard to the type of pharmaceutical composition in question (e.g., flavoring agents may be included as being suitable for oral administration).

[0138] Unit-dose pharmaceutical compositions contain an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0139] RcoM proteins may be effective over a wide dosage range and may generally be administered in a therapeutically effective amount, although it will be understood that the amount of compound actually administered will usually be determined by a physician depending on the relevant circumstances, including the condition being treated, the route of administration chosen, the actual compound being administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, etc.

[0140] In some embodiments, the recombinant RcoM protein of the present disclosure can be administered in a therapeutically effective amount of about 0.01 g to about 1000 g per day. In some examples, the dose of recombinant RcoM protein is about 0.1 g to about 900 g, about 0.1 g to about 800 g, about 0.1 g to about 700 g, about 0.1 g to about 600 g, about 0.1 g to about 500 g, about 0.1 g to about 400 g, about 0.1 g to about 300 g, about 0.1 g to about 200 g, about 0.1 g to about 100 g, about 1 g to about 900 g, about 1 g to about 800 g, about 1 g to about 700 g, about 1 g to about 600 g, about 1 g to about 1000 g, about 1 g to about 1000 g, about 1 g to about 1500 g, about 1 g to about 200 g, about 1 g to about 250 g, about 1 g to about 300 g, about 1 g to about 400 g, about 1 g to about 500 g, about 1 g to about 6 ... about 500, about 1 g to about 400, about 1 g to about 300 g, about 1 g to about 200 g, about 1 g to about 100 g, about 10 g to about 900, about 10 g to about 800 g, about 10 g to about 700 g, about 10 g to about 600 g, about 10 g to about 500 g, about 10 g to about 400 g, about 10 g to about 300 g, about 10 g to about 200 g, or about 10 g to about 100 g, or a range between any two of these values.

[0141] The amount of active ingredient that is combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. In some embodiments disclosed herein, pharmaceutical compositions comprise one or more of the RcoM proteins of the present disclosure (as an active ingredient) in combination with one or more pharmaceutically acceptable carriers (excipients).

[0142] In some embodiments, the one or more recombinant RcoM proteins comprise about 0.01% to about 50% of the pharmaceutical composition. In some embodiments, the one or more recombinant RcoM proteins comprise about 0.01% to about 50%, about 0.01% to about 45%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.05% to about 50%, about 0.05% to about 45%, about 0.05% to about 40%, or Approximately 0.05% to approximately 30%, approximately 0.05% to approximately 20%, approximately 0.05% to approximately 10%, approximately 0.1% to approximately 50%, approximately 0.1% to approximately 45%, approximately 0.1% to approximately 40%, approximately 0.1% to approximately 30%, approximately 0.1% to approximately 20%, approximately 0.1% to approximately 10%, approximately 0.1% to approximately 5%, approximately 0.5% to approximately 50%, approximately 0.5% to approximately 45%, approximately 0.5% to approximately 40%, approximately 0. 5% to about 30%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% to about 5%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 45%, about 5% to about 40%, about It comprises 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, or a value within one of these ranges. Specific non-limiting examples include about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or a range between any two of these values, all of the foregoing representing a weight percentage of the pharmaceutical composition.

[0143] The amount of recombinant RcoM protein administered to a patient will vary depending on what is being administered, the purpose of the administration, e.g., prophylaxis or therapy, the condition of the patient, the method of administration, etc. In therapeutic applications, compositions can be administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.

[0144] In some embodiments, pharmaceutical compositions can be sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is or lyophilized, with lyophilized preparations being combined with a sterile aqueous carrier prior to administration. In some embodiments, the pH of the RcoM protein preparation is about 3 to about 11, about 5 to about 9, about 5.5 to about 6.5, or about 5.5 to about 7.5. The use of certain of the above excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

[0145] In certain embodiments, the pharmaceutical composition includes a reducing agent. In some examples, the reducing agent is selected from ascorbic acid, N-acetylcysteine, sodium dithionite, methylene blue, glutathione, B5 / B5-reductase / NADH, tris(2-carboxyethyl)phosphine, dithiothreitol, or a combination thereof. Other agents that have the property of reducing iron containing heme molecules can also be used.

[0146] In other specific embodiments, the pharmaceutical composition comprises an oxidizing agent, hi some examples, the oxidizing agent is selected from an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

[0147] In certain embodiments, a pharmaceutical composition may be deoxygenated by producing and maintaining the RcoM protein or pharmaceutical composition in an oxygen-free environment. VI. Methods for Treating CO, H2S, and Cyanide Poisoning

[0148] The recombinant RcoM protein disclosed herein (see Section IV) exhibits extremely high affinity for carbon monoxide. Based on this property, the RcoM protein of the present disclosure can be used in a variety of in vivo and in vitro methods, such as as an antidote for carbon monoxide poisoning. The use of the RcoM protein of the present disclosure to treat cyanide and hydrogen sulfide (HS) poisoning is also described.

[0149] Also provided herein are methods for treating carboxyhemoglobinemia (carbon monoxide poisoning) in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant RcoM protein or a pharmaceutical composition containing the recombinant RcoM protein disclosed herein. In some embodiments, the method comprises selecting a subject with carboxyhemoglobinemia (carbon monoxide poisoning) prior to administration of the RcoM protein or pharmaceutical composition thereof. In some examples, the subject has at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% carboxyhemoglobin in their blood. In some embodiments, the RcoM protein is present in its reduced form. In some examples, the reducing agent includes sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), trehalose, a reducing carbohydrate (such as sorbitol or mannitol), or any combination thereof.

[0150] Further provided herein are methods for removing carbon monoxide from native hemoglobin, myoglobin, or mitochondria (i.e., derived from mitochondrial cytochrome c oxidase) in a subject's blood or tissue by contacting the subject's blood or tissue with a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some embodiments, the method includes selecting a subject with carboxyhemoglobinemia (carbon monoxide poisoning) before contacting the subject's blood or tissue with the disclosed RcoM protein or pharmaceutical composition thereof. In some examples, the subject has at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% carboxyhemoglobin in their blood. In some embodiments, the RcoM protein is present in its reduced form. In some examples, the reducing agent includes sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), trehalose, a reducing carbohydrate (such as sorbitol or mannitol), or any combination thereof.

[0151] Also provided herein are methods for removing hydrogen sulfide from native hemoglobin, myoglobin, or mitochondria (such as intramitochondrial cytochrome c oxidase) in a subject's blood or tissue by contacting the subject's blood or tissue with a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the methods further comprise selecting a subject with hydrogen sulfide poisoning before contacting the subject's blood or tissue with the RcoM protein or pharmaceutical composition. Further provided are methods for treating hydrogen sulfide poisoning in a subject by administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the methods further comprise selecting a subject with hydrogen sulfide poisoning before administering the RcoM protein or pharmaceutical composition. In some embodiments of these methods, the RcoM protein is present in its reduced form. Examples of reducing agents for inclusion in the pharmaceutical composition include, but are not limited to, sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), trehalose, a reducing carbohydrate (such as sorbitol or mannitol), or any combination thereof.

[0152] Further provided herein are methods for removing cyanide derived from native hemoglobin, myoglobin, or mitochondria (e.g., derived from mitochondrial cytochrome c oxidase) in a subject's blood or tissue by contacting the subject's blood or tissue with a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the method further comprises selecting a subject with cyanide poisoning before contacting the subject's blood or tissue with the RcoM protein or pharmaceutical composition. Also provided are methods for treating cyanide poisoning in a subject by administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein. In some examples, the method further comprises selecting a subject with cyanide poisoning before administering the RcoM protein or pharmaceutical composition. In some embodiments of these methods, the RcoM protein is present in its oxidized form. In some examples, the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

[0153] In some embodiments of the in vivo methods disclosed herein, the RcoM protein or pharmaceutical composition is administered intravenously or intramuscularly, hi some examples, the RcoM protein or pharmaceutical composition is administered by intravenous infusion, intraperitoneal injection, or intramuscular injection.

[0154] In some embodiments, RcoM protein is administered at a dose of about 0.1 to about 300 g per day, either alone or as part of a pharmaceutical composition. Additional dose ranges are described above in Section V.

[0155] Also provided herein is an in vitro method for removing carbon monoxide from hemoglobin, myoglobin, or mitochondria (e.g., from cytochrome c oxidase in mitochondria) in blood or animal tissue, the method comprising contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein. In some embodiments, the RcoM protein is present in its reduced form.

[0156] Further provided herein is an in vitro method for removing hydrogen sulfide derived from hemoglobin, myoglobin, or mitochondria (e.g., derived from cytochrome c oxidase in mitochondria) in blood or animal tissue, the method comprising contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein. In some embodiments, the RcoM protein is present in its reduced form.

[0157] Also provided herein is an in vitro method for removing cyanide derived from hemoglobin, myoglobin, or mitochondria (e.g., derived from cytochrome c oxidase within mitochondria) in blood or animal tissue, the method comprising contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein. In some embodiments, the RcoM protein is present in its oxidized form.

[0158] In some embodiments of the disclosed methods, the recombinant RcoM protein is pegylated, polymerized, or cross-linked. VII. Recombinant RcoM as a blood substitute

[0159] The recombinant RcoM protein disclosed herein is capable of binding and transporting oxygen (see Figures 8 and 15A-15D; Examples 3 and 4). Thus, it is contemplated that the RcoM protein of the present disclosure can be used as a blood substitute.

[0160] Provided herein are methods for replacing blood and / or increasing oxygen delivery to tissues in a subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein, thereby replacing blood and / or increasing oxygen delivery in the subject.

[0161] The subject to be treated may be, for example, any subject that needs to increase blood volume or increase oxygen delivery to tissue.In some embodiments, the subject has or is at risk of developing the disease, disorder or injury that is related to the deficiency of red blood cells and / or hemoglobin or that is related to the reduction of oxygen delivery to tissue.In some examples, the disease, disorder or injury includes bleeding disorder, bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, burns, ulcer, ectopic pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke or yellow fever.

[0162] In some embodiments, the bleeding episode in the subject treated with the recombinant RcoM protein results from anticoagulant overdose, aneurysm, blood vessel rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or infection.

[0163] In some embodiments, the bleeding disorder in the subject treated with the recombinant RcoM protein comprises hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, platelet disorders, coagulation disorders, faba bean poisoning, thrombocytopenia, vitamin K deficiency, or von Willebrand's disease.

[0164] In some embodiments, the anemia in the subject being treated comprises microcytic anemia, iron deficiency anemia, heme synthesis disorders, globin synthesis disorders, sideroblastic disorders, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia.

[0165] In some embodiments, the shock in the subject being treated comprises septic shock, hemorrhagic shock, or hypovolemic shock.

[0166] In some embodiments, the subject to be treated suffers from or is at risk of suffering from a disease or condition associated with reduced blood flow, and thus, increasing oxygen delivery is beneficial for the treatment of the subject.Examples of diseases or conditions that can be treated using the methods of the present disclosure include, but are not limited to, ischemia, myocardial infarction, stroke, ischemia-reperfusion injury, elevated blood pressure, pulmonary hypertension (including neonatal pulmonary hypertension, primary pulmonary hypertension, and secondary pulmonary hypertension), systemic hypertension, skin ulcer, acute renal failure, chronic renal failure, intravascular thrombosis, ischemic central nervous system events, vasospasm (such as cerebral artery vasospasm), hemolytic conditions, peripheral vascular disease, trauma, cardiac arrest, systemic surgery, or organ transplantation.

[0167] In some embodiments, the recombinant RcoM protein is administered to the subject intravenously.

[0168] In some embodiments, the method further comprises administering to the subject a second blood replacement product, a blood product, or whole blood. In some examples, the second blood replacement product comprises a hemoglobin-based oxygen carrier, an artificial red blood cell, or an oxygen-releasing compound. In some examples, the blood product comprises packed red blood cells, plasma, or serum.

[0169] In some examples, the subject is a human. In other examples, the subject is a non-human animal.

[0170] Also provided are compositions comprising the RcoM protein of the present disclosure and a native or recombinant globin molecule (native or recombinant hemoglobin or neuroglobin), or an oxygen carrier such as a hemoglobin-based oxygen carrier (HBOC). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient, or both. In some examples, the RcoM protein in the composition is pegylated, polymerized, or crosslinked. VIII. Embodiments

[0171] Embodiment 1. A recombinant regulator of carbon monoxide metabolism (RcoM) protein, comprising a heme-binding domain (HBD), wherein the amino acid sequence of the HBD is at least 90% identical to SEQ ID NO:2 and comprises an amino acid substitution at one or more of H74, C94, M104, M105, C127 and C130.

[0172] Embodiment 2. the substitution at H74 is selected from H74S, H74T, H74M, H74W, H74A, H74L, H74I, H74V and H74G; the substitution at C94 is selected from C94S, C94T, C94H, C94W, C94M, C94A, C94L, C94I, C94V and C94G; the substitution at M104 is selected from M104S, M104T, M104H, M104W, M104A, M104L, M104I, M104V and M104G; the substitution at M105 is selected from M105S, M105T, M105H, M105W, M105A, M105L, M105I, M105V and M105G; the substitution at C127 is selected from C127S, C127T, C127M, C127A, C127L, C127I, C127V and C127G; and / or 2. The recombinant RcoM protein of embodiment 1, wherein the substitution at C130 is selected from C130S, C130T, C130M, C130A, C130L, C130I, C130V and C130G.

[0173] Embodiment 3. The recombinant RcoM protein of embodiment 1 or embodiment 2, wherein the amino acid sequence of the HBD is at least 95% identical to SEQ ID NO:2 and comprises an amino acid substitution at one or more of C94, M104, C127 and C130.

[0174] Embodiment 4. The HBD comprises: C94S replacement, C127S and C130S substitutions, C94S substitution, C127S substitution and C130S substitution, M104A substitution, C127S substitution and C130S substitution, M104H substitution, C127S substitution and C130S substitution, M104L substitution, C127S substitution and C130S substitution, C94S substitution, M104A substitution, C127S substitution and C130S substitution, C94S substitution, M104H substitution, C127S substitution, and C130S substitution, or C94S substitution, M104L substitution, C127S substitution, and C130S substitution 5. The recombinant RcoM protein according to any one of embodiments 1 to 4, comprising:

[0175] Embodiment 5. the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, or the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 2, except for amino acid substitutions at one or more of H74, C94, M104, C127, C130 and M105; A recombinant RcoM protein according to any one of embodiments 1 to 4.

[0176] Embodiment 6. The recombinant RcoM protein of any one of embodiments 1 to 5, wherein the RcoM protein comprises an N-terminal tag or a C-terminal tag.

[0177] Embodiment 7. The recombinant RcoM protein of embodiment 6, wherein the tag is an affinity tag.

[0178] Embodiment 8. The recombinant RcoM protein of embodiment 7, wherein the affinity tag is His6, FLAG, glutathione S-transferase (GST), influenza virus hemagglutinin (HA), c-Myc, maltose binding protein (MBP), protein A, or protein G.

[0179] Embodiment 9. The recombinant RcoM protein according to any one of embodiments 6 to 8, wherein the tag is cleavable.

[0180] Embodiment 10. An in vitro method for removing carbon monoxide from hemoglobin, myoglobin, or mitochondria in blood or animal tissue, comprising contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein of any one of embodiments 1 to 9, thereby removing carbon monoxide from hemoglobin in the blood or animal tissue.

[0181] Embodiment 11. A method of treating carboxyhemoglobinemia in a subject, comprising administering to the subject a therapeutically effective amount of an RcoM protein of any one of embodiments 1 to 9.

[0182] Embodiment 12 The method of embodiment 11, further comprising selecting the subject with carboxyhemoglobinemia prior to administering the recombinant RcoM protein.

[0183] Embodiment 13 The method of embodiment 11 or embodiment 12, wherein the subject has at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% carboxyhemoglobin in their blood.

[0184] Embodiment 14 The method of any one of embodiments 11 to 13, wherein the recombinant RcoM protein is administered by intravenous infusion, intraperitoneal injection, or intramuscular injection.

[0185] Embodiment 15. The method of any one of embodiments 11 to 14, wherein the recombinant RcoM protein is administered at a dose of about 0.1 g to about 300 g per day.

[0186] Embodiment 16 The method of any one of embodiments 11 to 15, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent.

[0187] Embodiment 17. The method of embodiment 16, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

[0188] Embodiment 18. A method of treating cyanide poisoning in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant RcoM protein of any one of embodiments 1 to 9, wherein the RcoM protein is present in its oxidized form, thereby treating cyanide poisoning in the subject.

[0189] Embodiment 19 The method of embodiment 18, further comprising selecting a subject with cyanide poisoning before administering the recombinant RcoM protein.

[0190] Embodiment 20 The method of embodiment 18 or embodiment 19, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising an oxidizing agent.

[0191] Embodiment 21. The method of embodiment 20, wherein the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

[0192] Embodiment 22. A method of treating hydrogen sulfide (H2S) poisoning in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant RcoM protein of any one of embodiments 1 to 9, wherein the RcoM protein is present in its reduced form, thereby treating H2S poisoning in the subject.

[0193] Embodiment 23 The method of embodiment 22, further comprising selecting a subject with H2S poisoning before administering the recombinant RcoM protein.

[0194] Embodiment 24 The method of embodiment 22 or embodiment 23, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent.

[0195] Embodiment 25. The method of embodiment 24, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), trehalose, dithiothreitol (DTT), or any combination thereof.

[0196] Embodiment 26. A method for replacing blood in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant RcoM protein described in any one of embodiments 1 to 9, thereby replacing blood in the subject.

[0197] Embodiment 27. The method of embodiment 26, wherein the subject has or is at risk of developing a disease, disorder, or injury associated with a deficiency of red blood cells and / or hemoglobin, or associated with reduced oxygen delivery to tissues.

[0198] Embodiment 28. The method of embodiment 27, wherein the disease, disorder, or injury comprises a bleeding disorder, bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, burns, an ulcer, ectopic pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever.

[0199] Embodiment 29. Whether the bleeding episode results from anticoagulant overdose, aneurysm, vascular rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or infection; bleeding disorders include hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, platelet disorders, coagulation disorders, faba bean poisoning, thrombocytopenia, vitamin K deficiency, or von Willebrand disease; anemia includes microcytic anemia, iron deficiency anemia, heme synthesis disorders, globin synthesis disorders, sideroblastic disorders, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia; or 29. The method of embodiment 28, wherein the shock comprises septic shock, hemorrhagic shock, or hypovolemic shock.

[0200] Embodiment 30. The method of embodiment 26, wherein the subject is suffering from or at risk of suffering from myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary hypertension, or vasospasm.

[0201] Embodiment 31 The method of any one of embodiments 26 to 30, wherein the recombinant RcoM protein is administered intravenously to the subject.

[0202] Embodiment 32. The embodiment of any one of claims 26 to 31, wherein the recombinant RcoM protein is pegylated, polymerized, or cross-linked.

[0203] Embodiment 33. The method of any one of embodiments 26-32, further comprising administering to the subject a second blood replacement product, blood product, or whole blood.

[0204] Embodiment 34 The method of embodiment 33, wherein the second blood replacement product comprises a hemoglobin-based oxygen carrier, artificial red blood cells, or an oxygen-releasing compound.

[0205] Embodiment 35. The method of embodiment 33, wherein the blood product comprises packed red blood cells, plasma, or serum.

[0206] Embodiment 36. The method of any one of embodiments 11 to 35, wherein the subject is a human.

[0207] Embodiment 37. The method of any one of embodiments 11 to 35, wherein the subject is a non-human animal.

[0208] Embodiment 38. A pharmaceutical composition comprising a recombinant RcoM protein according to any one of embodiments 1 to 9 and a pharmaceutically acceptable carrier.

[0209] Embodiment 39. The pharmaceutical composition of embodiment 38, further comprising a reducing or oxidizing agent.

[0210] Embodiment 40. The pharmaceutical composition of embodiment 39, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

[0211] Embodiment 41. The pharmaceutical composition of embodiment 39, wherein the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, a quinone, or any combination thereof.

[0212] Embodiment 42 The pharmaceutical composition of any one of embodiments 38 to 41, wherein the recombinant RcoM protein is pegylated, polymerized, or crosslinked. [Example]

[0213] Example 1 Transfer of CO from HbCO to RcoM-1 in an aerobic environment Hemoglobin-CO (Hb-CO) transfer rates were assessed in the presence of WT full-length RcoM-1 (SEQ ID NO: 1) under aerobic conditions at 37°C and measured using stopped-flow UV-Vis spectroscopy and standard deconvolution methods based on the extinction coefficients for different ligand-bound species of RcoM-1 and hemoglobin. The concentrations of Hb-CO and Fe(II)RcoM-1 were 20 μM, and experiments were performed in triplicate. Data for hemoglobin-CO loss were fitted with a biexponential curve, revealing a slow-phase half-life (t ) of 1.4 seconds. 1 / 2 ) The data for the increase in Fe(II)-CO RcoM were fitted to a single exponential curve, yielding a half-life of 0.93 seconds. The results are shown in Figure 4. The data demonstrate that RcoM has a high affinity for CO, allowing for the rapid and efficient transfer of CO from Hb to RcoM. Example 2 Transfer of CO from Hb-CO to RcoM-1 in an anaerobic environment

[0214] The hemoglobin-CO transfer rate was measured using UV-Vis spectroscopy in the presence of wild-type full-length RcoM-1 (SEQ ID NO: 1) under anaerobic conditions at 37°C. The concentrations of Hb-CO and Fe(II) RcoM-1 were 15 μM and 15.8 μM, respectively. The absorbance changes at 530, 562, and 583 nm tracking the THE transition from Fe(II) to Fe(II)-CO RcoM were fitted to a single exponential curve, which revealed a half-life of 50 seconds. The results are shown in Figure 5. These results demonstrate that RcoM-1 can capture CO from Hb-CO species and therefore can be used as a CO scavenger in vivo. Example 3 Characterization of truncated RcoM (HBD C94S) with a C94C substitution

[0215] This example describes studies to characterize a modified RcoM protein (SEQ ID NO: 7) lacking the PAS domain and containing a C94S substitution. The results of these studies demonstrate that the gas-binding properties of RcoM can be altered by modifying the heme-binding residues. stability

[0216] The HBD C94S mutant is much more stable than WT RcoM-1. This mutant form of RcoM can be stored at higher concentrations than WT RcoM (approximately 480 mM heme compared to approximately 130 mM for WT RcoM). HBD C94S can also be reduced using dithionite (e.g., DTT, TCEP) in the absence of a stabilizing reducing agent. Furthermore, both the oxidized and reduced forms of RcoM are stable to aggregation when stored at 4°C for more than a week. Studies have shown that the T for Fe(III)RcoM-1 HBD C94S m demonstrated that the RcoM-1 temperature was 90°C (recorded at a 7 μM RcoM concentration under anaerobic conditions in a septum-sealed cuvette). Under the same conditions, WT RcoM-1 irreversibly unfolded at approximately 40°C. UV-Vis spectrum comparison

[0217] Spectra for full-length wild-type RcoM-1 and HBD C94S RcoM were evaluated. Spectra for ferric iron (Fe(III)), deoxyferrous iron (Fe(II)), and ferrous iron-CO species (Fe(II)-CO) were determined. The wavelengths (in nm) for the various peak maxima are reported along with the estimated molar absorptivity (mM) for each peak. -1 cm -1) were calculated. The results are shown in Figure 7. As expected, the Fe(II) and Fe(II)-CO spectra were very similar between the WT and HBD C94S RcoM proteins. However, the Fe(III) spectrum appeared very different, demonstrating that the Fe(III) heme coordination environment was altered as a result of the C94S substitution.

[0218] Further studies provided evidence for stable O2 adducts in HBD C94S. HBD C94S was reduced with excess dithionite to generate ferrous Fe(II) species. The reduced HBD C94S was then desalted, and UV-Vis samples were prepared under microaerobic conditions. After recording the UV-Vis spectrum under microaerobic conditions, the cuvette was uncapped to allow air to enter, and the spectrum was re-recorded to reveal oxygen-bound species (RcoM concentration = 8 μM). Figure 8 shows the visible spectra for the ferrous (Fe(II)) species in the presence of a reducing agent, the Fe(II) species after desalting, the Fe(II) species after air exposure, and the reoxidized Fe(III) species. Association and dissociation rates of HBD C94S RcoM-CO

[0219] The kinetics of the reaction of the ferrous heme-binding domain (HBD) of C94S RcoM with carbon monoxide (CO) was determined by stopped-flow techniques (Figure 9). The study was performed at an RcoM concentration of 10 μM, CO concentrations ranging from 55 to 287 μM, and a temperature of 37 °C. Calculation of the rates at different CO concentrations yielded a 1.2 × 10 5 M -1 seconds -1 The association rate (k on ) was obtained. Similar values ​​were obtained for the wild-type full-length protein. Thus, the CO on-rate was not affected by the C94S substitution.

[0220] The CO dissociation rate for HBD C94S was determined using a 10 μM RcoM concentration, a 2 mM nitric oxide (NO) concentration (generated using 1 mM ProliNONOate), and a temperature of 37°C. The reaction was monitored by the absorbance change as the ferrous-CO complex dissociated in the presence of NO. As CO dissociates, NO binds to heme, causing a change in the absorbance spectrum. Excess NO prevents CO from rebinding to heme. The time course of the absorbance change indicated a 4.9 x 10 -2 seconds -1 This enabled the determination of the dissociation rate of the protein (Figure 10). Thermal unfolding of HBD C94S

[0221] Unfolding was monitored by the change in absorbance at the heme Soret maximum at 420 nm (Figure 11). The sample was allowed to equilibrate at each temperature for 5 minutes before recording each UV-Vis spectrum. The small loss in Soret intensity observed between 20°C and 75°C is likely due to a change in the heme coordination number. The loss in Soret intensity between 75°C and 98°C was attributed to the loss of heme from the protein due to thermal unfolding. UV-Vis spectra for the Fe(III) HBD RcoM-1 with the C94S mutation were recorded at each temperature between 20°C and 98°C. The T of the HBD C94S m was determined to be 91°C. Example 4 RcoM heme-binding domain (HBD) variants

[0222] This example describes the generation and characterization of several truncated RcoM HBD variants.

[0223] Eight RcoM variants were generated. The variants listed in Table 4 were successfully cloned, expressed in E. coli, and purified to homogeneity. The variants encompass the heme-binding domain (HBD) of RcoM-1 from Paraburkholderia xenovorans and carry various mutations (at one or more of residues C94, M104, C127, and C130) to enhance solubility, stability, and CO2 capture properties. The expressed variants also contained a C-terminal 6-His tag. The variant containing the 6-His tag was 17 kDa. Table 4. RcoM HDB16 variants [Table 4-1] [Table 4-2]

[0224] Electronic absorption (UV-Vis) spectra for RcoM HBD WT and variants are shown in Figures 12A-12D, 13A-13B, and 14A-14C. A schematic diagram of the protein-driven ligand switching mechanism for RcoM highlighting the coordination sphere changes in the M104 variant is shown in Figure 13C. A schematic diagram of the protein-driven ligand switching mechanism for RcoM highlighting the coordination sphere changes in the CCC M104 variant is shown in Figure 14D.

[0225] Quantification of oxygen binding affinity (P) of RcoM HBD truncations 50 ) are shown in Figures 15A-15D. The fraction of heme protein bound to oxygen was measured as a function of oxygen partial pressure using UV-Vis spectroscopy using a tonometer apparatus equipped with an optical cuvette. Oxygen partial pressure (P O2Representative spectral changes in UV-Vis signatures for CC HBD RcoM variants as a function of k are shown in Figure 15A. Oxygen binding curves for CC HBD, C94S HBD, and CCC HBD are shown in Figures 15B-15D. The second-order rate constants (k) for CO binding to RcoM WT HBD and HBD variants CC HBD, C94S HBD, and CCC HBD were on,CO ) was determined (Figures 16A-16D). The CO binding rate at each concentration of CO was measured using stopped-flow UV-Vis spectroscopy and fitted with a monoexponential fit. Linear regression was applied to each curve, and the second-order rate constant was estimated as the slope. The results were as follows: [Table 7]

[0226] Autoxidation rate (k oxid ) is 0.87 hours -1 Figure 17A shows the reference spectra for the Fe(III) and Fe(II)-O2 proteins. The spectral changes of the UV-Vis signature for the Fe(II)-O2 WT HBD are shown in Figure 17B. The spectral changes at 542 nm and 573 nm were fitted with a single exponential, k oxid was determined (FIG. 17C). FIG. 18 shows a table providing a summary of the ligand binding parameters and heme stability properties for WT RcoM and the RcoM HBD variants C94S, CC HBD, and CCC HBD.

[0227] The unfolding of the Fe(III) CCC HBD RcoM was assessed in the presence of urea (0 M, 4 M, and 8 M urea). Unfolding was monitored by the absorbance change at the heme Soret maximum at 415 nm. After allowing the samples to equilibrate for 10 min, each UV-Vis spectrum was recorded (Figure 19A). The unfolding data were fitted with a sigmoidal curve to determine the concentration of denaturant at which half of the protein sample was unfolded ([D]). 50) was determined (Figure 19B). 50 was 4.6M.

[0228] The reactivity of RcoM HBD variants with hydrogen peroxide was also assessed. Fe(III) WT HBD and variants CCC HBD, CCC M104A HBD, and CCC M104H HBD were incubated with 500 μM hydrogen peroxide at pH 7.4 and 25°C and monitored by UV-Vis spectroscopy every 2 minutes over the course of 30 minutes (Figures 20A-20D). Minimal spectral changes were observed for each variant, suggesting that hydrogen peroxide does not react with the Fe(III) heme center of the truncated RcoM HBD to generate highly oxidized species.

[0229] Nitrite reduction was assessed for full-length and HBD-truncated RcoM variants. Ferrous protein (10–15 μM) was incubated with 1–5 mM sodium nitrite at 37°C in the presence of 2.5 mM sodium dithionite. UV-Vis spectroscopy was used to monitor the conversion of Fe(II) heme to Fe(II)-NO (Figure 21A). The changes in spectral features at 562 nm and 578 nm were fitted to single-exponential curves to determine the observed rates of nitrite reduction. The observed rates were plotted as a function of nitrite concentration, and linear regression was applied to each plot to estimate the second-order rate constant as the slope (Figures 21B–21C).

[0230] Additional studies were conducted to assess the CO2 capture capacity of RcoM. Kinetic traces were developed for in vitro CO2 transfer from hemoglobin (Hb) to wild-type RcoM HBD and the RcoM HBD variants CC HBD, C94S HBD, and CCC HBD under aerobic conditions at 37°C. CO2-bound Hb (20 μM) was incubated with equimolar ferrous oxyferrous RcoM, and CO2 transfer from Hb to RcoM was monitored using UV-Vis spectroscopy. The fraction of each CO2-bound heme protein was determined using spectral deconvolution, and the corresponding kinetic traces were fitted to single- or double-exponential equations. The half-lives of each CO2-bound species are displayed in Figures 22A–22D, and the half-lives and amplitudes of the fast species are displayed for the double-exponential fitted curves. Figures 23A-23B show kinetic traces monitoring CO transfer from HbCO encapsulated in red blood cells (RBCs) to extracellular RcoM HBD cleavage products under aerobic conditions at 37°C. Heme proteins were incubated at equimolar concentrations (50-100 μM), and RBCs were separated from extracellular RcoM by centrifugation at each time point. UV-Vis spectroscopy was used to monitor CO transfer from Hb to WT HBD RcoM (Figure 23A) and C94S HBD RcoM (Figure 23B). The fraction of each CO bound heme protein was determined using spectral deconvolution, and the corresponding kinetic traces were fitted to a monoexponential equation. The half-lives of COHb in the presence of WT HBD and C94S HBD were 24 ± 6 s and 23 ± 5 s, respectively.

[0231] These results demonstrate that RcoM HBD variants rapidly capture CO from RBC-encapsulated Hb under aerobic conditions similar to those likely occurring in vivo during acute CO poisoning. The RcoM HBD variants are selective for CO over oxygen, such that CO transfer from HbCO proceeds under aerobic conditions. Example 5 Virulence screening of RcoM HBD variants in mice

[0232] Recombinantly expressed RcoM truncations were introduced into healthy mice via tail vein catheters at concentrations of 1 mM or 10 mM and an injection volume of 10 μL per gram of body weight. Behavior (including nesting) was monitored over a 48-hour period, after which mice were sacrificed and blood collected for toxicity assessment. The results are shown in Table 5. Blood chemistry results, indicating liver function (AST and ALT) and kidney function (BUN and creatinine), for all mice treated with RcoM truncations were comparable to those of control mice given phosphate-buffered saline (PBS). These results indicate that intravenous injection of RcoM truncations did not induce organ-specific toxicity in mice. Table 5. Toxicity screening results [Table 5]

[0233] Example 6 RcoM CO scavenging in vivo The ability of the C94S and CCC HBD RcoM variants to capture CO from HbCO was evaluated in a mouse model of lethal CO poisoning. Anesthetized, mechanically ventilated mice were exposed to 3,000 ppm CO in air for 4.5 minutes, followed by intravenous infusion of Fe(II)-O2CCC HBD RcoM at an injection volume of 10 μL per gram of body weight (heme protein concentrations shown in Figure 24). Blood samples (15 μL) were collected immediately before and after infusion and 25 minutes after CO exposure. At each time point, RBCs were separated from plasma by centrifugation, and the separated RBC pellets and plasma samples were immediately frozen at -80°C. The fraction of CO-bound hemoglobin (%HbCO) and the fraction of CO-bound RcoM (%RcoM-CO) from RBCs were then determined using spectral deconvolution. Infusion of RcoM resulted in a greater decrease in the fraction of Hb bound to CO (Δ%HbCO) compared with infusion with PBS (FIG. 24), indicating that RcoM is capable of scavenging CO in vivo.

[0234] In view of the many possible embodiments to which the principles of the subject matter of this disclosure may be applied, it should be recognized that the illustrated embodiments are merely examples of the disclosure and should not be construed as limitations on the scope of the disclosure. Rather, the scope of the disclosure is defined by the claims that follow. Accordingly, the inventors claim all that comes within the scope and spirit of those claims. The present invention provides, for example, the following items. (Item 1) A recombinant regulator of carbon monoxide metabolism (RcoM) protein comprising a heme-binding domain (HBD), wherein the amino acid sequence of the HBD is at least 90% identical to SEQ ID NO: 2 and comprises amino acid substitutions at one or more of H74, C94, M104, M105, C127 and C130. (Item 2) the substitution at H74 is selected from H74S, H74T, H74M, H74W, H74A, H74L, H74I, H74V and H74G; the substitution at C94 is selected from C94S, C94T, C94H, C94W, C94M, C94A, C94L, C94I, C94V and C94G; the substitution in M104 is selected from M104S, M104T, M104H, M104W, M104A, M104L, M104I, M104V and M104G; the substitution at M105 is selected from M105S, M105T, M105H, M105W, M105A, M105L, M105I, M105V and M105G; the substitution at C127 is selected from C127S, C127T, C127M, C127A, C127L, C127I, C127V and C127G; and / or 2. The recombinant RcoM protein according to item 1, wherein the substitution in C130 is selected from C130S, C130T, C130M, C130A, C130L, C130I, C130V and C130G. (Item 3) 2. The recombinant RcoM protein of item 1, wherein the amino acid sequence of the HBD is at least 95% identical to SEQ ID NO: 2 and contains amino acid substitutions at one or more of C94, M104, C127 and C130. (Item 4) The HBD C94S replacement, C127S and C130S substitutions, C94S substitution, C127S substitution and C130S substitution, M104A substitution, C127S substitution and C130S substitution, M104H substitution, C127S substitution and C130S substitution, M104L substitution, C127S substitution and C130S substitution, C94S substitution, M104A substitution, C127S substitution and C130S substitution, C94S substitution, M104H substitution, C127S substitution, and C130S substitution, or 2. The recombinant RcoM protein according to item 1, comprising a C94S substitution, a M104L substitution, a C127S substitution, and a C130S substitution. (Item 5) the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, or 2. The recombinant RcoM protein of item 1, wherein the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 2, except for amino acid substitutions at one or more of H74, C94, M104, C127, C130, and M105. (Item 6) 2. The recombinant RcoM protein according to item 1, wherein the RcoM protein comprises an N-terminal tag or a C-terminal tag. (Item 7) 7. The recombinant RcoM protein according to item 6, wherein the tag is an affinity tag. (Item 8) 8. The recombinant RcoM protein according to item 7, wherein the affinity tag is His6, FLAG, glutathione S-transferase (GST), influenza virus hemagglutinin (HA), c-Myc, maltose-binding protein (MBP), protein A, or protein G. (Item 9) 7. The recombinant RcoM protein according to item 6, wherein the tag is cleavable. (Item 10) 1. An in vitro method for removing carbon monoxide from hemoglobin, myoglobin, or mitochondria in blood or animal tissue, comprising contacting the blood or animal tissue with an effective amount of the recombinant RcoM protein described in item 1, thereby removing carbon monoxide from hemoglobin in the blood or animal tissue. (Item 11) A method for treating carboxyhemoglobinemia in a subject, comprising administering to the subject a therapeutically effective amount of the RcoM protein described in item 1. (Item 12) 12. The method of claim 11, further comprising selecting a subject with carboxyhemoglobinemia before administering the recombinant RcoM protein. (Item 13) 12. The method of item 11, wherein the subject has at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% carboxyhemoglobin in their blood. (Item 14) 12. The method of claim 11, wherein the recombinant RcoM protein is administered by intravenous infusion, intraperitoneal injection, or intramuscular injection. (Item 15) Item 12. The method according to Item 11, wherein the recombinant RcoM protein is administered at a dose of about 0.1 g to about 300 g per day. (Item 16) 12. The method of claim 11, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent. (Item 17) Item 17. The method of item 16, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof. (Item 18) 1. A method for treating cyanide poisoning in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant RcoM protein according to item 1, wherein the RcoM protein is present in its oxidized form, thereby treating cyanide poisoning in the subject. (Item 19) 20. The method of claim 18, further comprising selecting a subject with cyanide poisoning before administering the recombinant RcoM protein. (Item 20) 19. The method of claim 18, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising an oxidizing agent. (Item 21) 21. The method of claim 20, wherein the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof. (Item 22) Hydrogen sulfide (H 2 S) A method for treating poisoning, comprising administering to the subject a therapeutically effective amount of the recombinant RcoM protein of item 1, wherein the RcoM protein is present in its reduced form, thereby reducing H in the subject. 2 Methods for treating S poisoning. (Item 23) Before administering the recombinant RcoM protein, 2 23. The method of claim 22, further comprising selecting a subject with S addiction. (Item 24) 23. The method of claim 22, wherein the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent. (Item 25) 25. The method of claim 24, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), trehalose, dithiothreitol (DTT), or any combination thereof. (Item 26) A method for replacing blood in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant RcoM protein described in item 1, thereby replacing blood in the subject. (Item 27) 27. The method of claim 26, wherein the subject has or is at risk of developing a disease, disorder, or injury associated with a deficiency of red blood cells and / or hemoglobin, or associated with reduced oxygen delivery to tissues. (Item 28) 28. The method of item 27, wherein the disease, disorder, or injury comprises a bleeding disorder, bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, burns, ulcers, ectopic pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever. (Item 29) the bleeding episode results from anticoagulant overdose, aneurysm, vascular rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or infection; the bleeding disorder comprises hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, a platelet disorder, a coagulation disorder, faba bean poisoning, thrombocytopenia, vitamin K deficiency, or von Willebrand's disease; the anemia comprises microcytic anemia, iron deficiency anemia, heme synthesis disorders, globin synthesis disorders, sideroblastic disorders, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia; or 29. The method of item 28, wherein the shock comprises septic shock, hemorrhagic shock, or hypovolemic shock. (Item 30) 27. The method of item 26, wherein the subject is suffering from or at risk of suffering from myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary hypertension or vasospasm. (Item 31) 27. The method of claim 26, wherein the recombinant RcoM protein is administered to the subject intravenously. (Item 32) 27. The method of claim 26, wherein the recombinant RcoM protein is pegylated, polymerized, or cross-linked. (Item 33) 27. The method of claim 26, further comprising administering to the subject a second blood replacement product, blood product, or whole blood. (Item 34) 34. The method of claim 33, wherein the second blood replacement product comprises a hemoglobin-based oxygen carrier, artificial red blood cells, or an oxygen-releasing compound. (Item 35) 34. The method of claim 33, wherein the blood product comprises packed red blood cells, plasma, or serum. (Item 36) Item 12. The method of item 11, wherein the subject is a human. (Item 37) 12. The method of claim 11, wherein the subject is a non-human animal. (Item 38) A pharmaceutical composition comprising the recombinant RcoM protein according to item 1 and a pharmaceutically acceptable carrier. (Item 39) 38. The pharmaceutical composition according to item 37, further comprising a reducing or oxidizing agent. (Item 40) 40. The pharmaceutical composition of item 39, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof. (Item 41) 40. The pharmaceutical composition of claim 39, wherein the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, a quinone, or any combination thereof. (Item 42) 39. The pharmaceutical composition of claim 38, wherein the recombinant RcoM protein is pegylated, polymerized, or crosslinked.

Claims

1. A recombinant regulator of carbon monoxide metabolism (RcoM) protein, wherein the recombinant RcoM protein comprises a heme-binding domain (HBD), and the amino acid sequence of the HBD is at least 90% identical to SEQ ID NO:2; (i) C94S substitution; (ii) a C127S substitution and a C130S substitution; or (iii) C94S substitution, C127S substitution, and C130S substitution A recombinant RcoM protein comprising: a) a recombinant RcoM protein having enhanced thermal stability compared to a wild-type RcoM protein comprising an HBD consisting of the amino acid sequence of SEQ ID NO: 2; and b) a recombinant RcoM protein capable of binding to heme and carbon monoxide.

2. A recombinant regulator of carbon monoxide metabolism (RcoM) protein, wherein the recombinant RcoM protein comprises a heme-binding domain (HBD), and the amino acid sequence of the HBD is at least 90% identical to SEQ ID NO:2; (i) C94S, M104A, C127S, and C130S substitutions; (ii) a C94S substitution, a M104H substitution, a C127S substitution, and a C130S substitution; or (iii) C94S substitution, M104L substitution, C127S substitution, and C130S substitution A recombinant RcoM protein comprising: a) a recombinant RcoM protein having enhanced nitrite reduction compared to a wild-type RcoM protein comprising an HBD consisting of the amino acid sequence of SEQ ID NO: 2; and b) a recombinant RcoM protein that binds to heme and carbon monoxide.

3. 3. The recombinant RcoM protein of claim 1, wherein the amino acid sequence of the HBD is at least 95% identical to SEQ ID NO:

2.

4. 2. The recombinant RcoM protein of claim 1, wherein the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

5. 3. The recombinant RcoM protein of claim 2, wherein the amino acid sequence of the RcoM protein comprises or consists of SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

6. The recombinant RcoM protein of claim 1 or 2, wherein the RcoM protein comprises an N-terminal tag or a C-terminal tag.

7. The recombinant RcoM protein of claim 6, wherein the tag is an affinity tag.

8. The recombinant RcoM protein of claim 7, wherein the affinity tag is His6, FLAG, glutathione S-transferase (GST), influenza virus hemagglutinin (HA), c-Myc, maltose-binding protein (MBP), protein A, or protein G.

9. The recombinant RcoM protein of claim 6 , wherein the tag is cleavable.

10. An in vitro method for removing carbon monoxide from hemoglobin, myoglobin or mitochondria in blood or animal tissue, comprising contacting the blood or animal tissue with an effective amount of the recombinant RcoM protein described in claim 1 or 2, thereby removing carbon monoxide from hemoglobin in the blood or animal tissue.

11. A composition comprising the RcoM protein of claim 1 or 2 for use in a method for treating carboxyhemoglobinemia in a subject.

12. 12. The composition of claim 11, wherein the method further comprises selecting a subject with carboxyhemoglobinemia prior to administering the composition.

13. 12. The composition of claim 11, wherein the subject has at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% carboxyhemoglobin in their blood.

14. The composition of claim 11 , wherein the composition is administered by intravenous infusion, intraperitoneal injection, or intramuscular injection.

15. The composition of claim 11, wherein the recombinant RcoM protein is administered at a dose of 0.1 g to 300 g per day.

16. The composition of claim 11 , wherein the composition is administered as a pharmaceutical composition comprising a reducing agent.

17. 17. The composition of claim 16, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

18. 10. A composition comprising the recombinant RcoM protein of claim 1 or 2 for use in a method for treating cyanide poisoning in a subject, wherein the RcoM protein is present in its oxidized form.

19. 20. The composition of claim 18, wherein the method further comprises selecting a subject with cyanide poisoning prior to administering the composition.

20. 20. The composition of claim 18, wherein the composition is administered as a pharmaceutical composition comprising an oxidizing agent.

21. 21. The composition of claim 20, wherein the oxidizer comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

22. Hydrogen sulfide (H ２ S) A composition comprising the recombinant RcoM protein of claim 1 or 2 for use in a method for treating poisoning, wherein the RcoM protein is present in its reduced form.

23. The method further comprises administering, prior to administering the composition, ２ 23. The composition of claim 22, further comprising selecting a subject with S poisoning.

24. 23. The composition of claim 22, wherein the composition is administered as a pharmaceutical composition comprising a reducing agent.

25. 25. The composition of claim 24, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), trehalose, dithiothreitol (DTT), or any combination thereof.

26. A composition comprising the recombinant RcoM protein of claim 1 or 2 for replacing blood in a subject, wherein the blood is replaced with the composition.

27. 27. The composition of claim 26, wherein the subject has or is at risk of developing a disease, disorder, or injury associated with a deficiency of red blood cells and / or hemoglobin, or associated with reduced oxygen delivery to tissues.

28. 28. The composition of claim 27, wherein the disease, disorder, or injury comprises a bleeding disorder, bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, burns, ulcers, ectopic pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever.

29. the bleeding episode results from anticoagulant overdose, aneurysm, vascular rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or infection; the bleeding disorder comprises hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, a platelet disorder, a coagulation disorder, faba bean poisoning, thrombocytopenia, vitamin K deficiency, or von Willebrand's disease; the anemia comprises microcytic anemia, iron deficiency anemia, heme synthesis disorders, globin synthesis disorders, sideroblastic disorders, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia; or 29. The composition of claim 28, wherein the shock comprises septic shock, hemorrhagic shock, or hypovolemic shock.

30. 27. The composition of claim 26, wherein the subject is suffering from or at risk of suffering from myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary hypertension, or vasospasm.

31. 27. The composition of claim 26, wherein the composition is administered to the subject intravenously.

32. 27. The composition of claim 26, wherein the recombinant RcoM protein is pegylated, polymerized, or cross-linked.

33. 27. The composition of claim 26, wherein the composition is administered to the subject in combination with a second blood replacement product, a blood product, or whole blood.

34. 34. The composition of claim 33, wherein the second blood replacement product comprises a hemoglobin-based oxygen carrier, artificial red blood cells, or an oxygen-releasing compound.

35. 34. The composition of claim 33, wherein the blood product comprises packed red blood cells, plasma, or serum.

36. The composition of claim 11 , wherein the subject is a human.

37. The composition of claim 11 , wherein the subject is a non-human animal.

38. A pharmaceutical composition comprising the recombinant RcoM protein of claim 1 or 2 and a pharmaceutically acceptable carrier.

39. 39. The pharmaceutical composition of claim 38, further comprising a reducing or oxidizing agent.

40. 40. The pharmaceutical composition of claim 39, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetylcysteine ​​(NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

41. 40. The pharmaceutical composition of claim 39, wherein the oxidizing agent comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, a quinone, or any combination thereof.

42. 39. The pharmaceutical composition of claim 38, wherein the recombinant RcoM protein is pegylated, polymerized, or crosslinked.

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