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

Abstract

Methods for rapid removal of carbon monoxide (CO) from CO-bound hemoglobin, myoglobin, and cytochrome c oxidase in a subject poisoned with CO are described. The disclosed therapy involves the use of a rationally designed, modified CO metabolic regulator (RcoM) protein and pharmaceutical compositions thereof that clear carbon monoxide from the poisoned tissue. Recombinant RcoM compositions are infused into the blood, where they rapidly sequester carbon monoxide and limit the toxic effects of carbon monoxide on cellular respiration, oxygen delivery, and oxygen utilization.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 022,821, filed May 11, 2020, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to recombinant carbon monoxide metabolism regulator (RcoM) proteins and pharmaceutical compositions thereof. The present disclosure further relates to the use of recombinant RcoM proteins and compositions for treating carbon monoxide (CO) poisoning, cyanide poisoning, and hydrogen sulfide poisoning, and as a blood substitute.

[0004] Statement Regarding Government-Sponsored Research

[0005] 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 the invention. BACKGROUND

[0006] Inhalational exposure to carbon monoxide is a leading cause of environmental poisoning. Individuals can be exposed to carbon monoxide in the air in a variety of different situations, such as a house fire, a generator or outdoor barbecue used indoors, or an attempt at suicide in an enclosed space. Carbon monoxide binds to hemoglobin and heme proteins in cells, 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 results in severe hypoxia and ischemic injury to vital organs such as the brain and heart. Individuals with more than 5-10% carboxyhemoglobin in their blood are at risk for brain injury and neurocognitive dysfunction. Patients with very high levels of carboxyhemoglobin often suffer irreversible brain injury, respiratory failure, and / or cardiovascular collapse.

[0007] Despite the availability of methods to rapidly diagnose carbon monoxide poisoning using standard arterial and venous blood gas analysis and oximetry, and despite the understanding of risk factors for carbon monoxide poisoning, there is no available antidote for such toxic exposures. Current treatment methods are to administer 100% oxygen via a facemask and, where possible, to expose the patient to hyperbaric oxygen. Hyperbaric oxygen therapy increases the rate of carbon monoxide release from hemoglobin and accelerates the natural clearance of carbon monoxide. However, this therapy has only a small effect on the rate of carbon monoxide clearance, and the therapy is not available in the field based on the complexity of hyperbaric oxygen facilities. In addition, hyperbaric oxygen therapy is often associated with significant treatment delays and transportation costs. Thus, there is a need for an effective, rapid, and readily available therapy to treat carbon monoxide poisoning, also known as carboxyhemoglobinemia. SUMMARY

[0008] The present disclosure describes recombinant carbon monoxide metabolism regulator (RcoM) proteins with high affinity for CO and their use as CO scavengers. The disclosed RcoM proteins are capable of removing CO from CO-bound hemoglobin, myoglobin, and cytochrome c oxidase (in mitochondria), and thus are useful in methods of treating carboxyhemoglobinemia and as blood substitutes.

[0009] Provided herein are recombinant RcoM proteins. In some embodiments, the recombinant RcoM protein comprises a heme-binding domain (HBD) having an amino acid sequence that is 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 an amino acid substitution at one or more of H74, C94, M104, M105, C127, and C130. In other examples, the HBD has the wild-type amino acid sequence. The recombinant RcoM protein can be a full-length RcoM (such as the RcoM of SEQ ID NO: 1), or can be a truncated RcoM, such as an RcoM consisting of or consisting essentially of an HBD. In some particular examples, the recombinant RcoM protein comprises an affinity tag, such as a cleavable affinity tag, at the N-terminus or C-terminus.

[0010] 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.

[0011] Also provided herein is an in vitro method of removing carbon monoxide from hemoglobin, myoglobin, or mitochondria (cytochrome c oxidase) in blood or animal tissue. In some embodiments, the method comprises contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein.

[0012] Further provided is a method of treating carboxyhemoglobinemia 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 disclosed herein. In some examples, the recombinant RcoM protein is administered as a pharmaceutical composition comprising a reducing agent.

[0013] Also provided is a method of treating cyanide 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 disclosed herein. In some examples, the RcoM protein is in its oxidized form.

[0014] Further provided are methods of treating hydrogen sulfide (H2S) poisoning in a subject. In some embodiments, the method comprises 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 in its reduced form.

[0015] Further provided are methods of replacing blood in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant RcoM protein or pharmaceutical composition disclosed herein.

[0016] The foregoing and other objects and features of the present disclosure will become more fully apparent from the following detailed description, taken in conjunction with the accompanying figures. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : Amino acid sequence of 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 from the direct oxygen sensor (DOS) protein in E. coli (Kurokawa et al., J Biol Chem 279(19): 20186-20193, 2004), and the LytTR domain structure is from the transcription factor AgrA in S. aureus (Sidote et al., Structure 16(5): 727-735, 2008).

[0018] Figure 2 : Amino acid sequence of RcoM-1 from P. xenovorans is truncated to include only the PAS CO-binding domain and without the N-terminal methionine (residues 2-154 of SEQ ID NO: 2). The heme-binding residues are shown in bold. A schematic of the heme coordination environment in RcoM-1 is also shown.

[0019] Figure 3The amino acid sequence of RcoM-1 from P. xenovorans was further truncated to include only the key region of the PAS CO-binding domain (SEQ ID NO: 3). The heme-binding residues are shown in bold (H74, C75, and M104, numbered with reference to SEQ ID NO: 1). Also shown is a structural alignment of the PAS domain from the E. coli DOS protein with a homology model of the RcoM-1 PAS domain obtained using the I-TASSER online modeling server. Dashes indicate the location of the proposed truncation site. The heme-binding residues are shown as dashed lines.

[0020] Figure 4 : Hemoglobin-CO transfer kinetics measured using stopped-flow UV-Vis spectroscopy in the presence of WT full-length RcoM-1 under aerobic conditions at 37°C. Concentrations of hemoglobin-CO (Hb-CO) and Fe(II) RcoM-1 were 20 μΜ. Experiments were performed in triplicate. Data for Hb-CO loss were fit to a bi-exponential curve, which showed a slow phase half-life (t 1 / 2 ) of 1.4 seconds. Data for Fe(II)-CO RcoM increase were fit to a single-exponential curve, which showed a half-life of 0.93 seconds.

[0021] Figure 5 : Hemoglobin-CO transfer kinetics measured using UV-Vis spectroscopy in the presence of WT full-length RcoM-1 under anaerobic conditions at 37°C. Concentrations of hemoglobin-CO and Fe(II) RcoM-1 were 15 μΜ and 15.8 μΜ, respectively. Changes in absorbance at 530, 562, and 583 nm were tracked for the transition from Fe(II) to Fe(II)-CO RcoM and fit to a single-exponential curve, which showed a half-life of 50 seconds.

[0022] Figure 6 : Amino acid alignment of P. xenovorans RcoM-1 (SEQ ID NO: 1) and 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.

[0023] 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 carrying the C94S mutation (right). Spectra of trivalent iron (Fe(III), deoxyferrous iron (Fe(II)) and ferrous-CO species (Fe(II)-CO) are shown. Table shows the maximum peak wavelength (nm) for each species and the estimated molar absorbance (mM -1 cm -1 ) for each peak.

[0024] Figure 8 : Evidence for the stabilization of the O2 adduct in the HBD C94S. The isolated heme-binding domain (HBD) of RcoM1 carrying the C94S mutation can bind oxygen. The visible spectrum of the ferrous (Fe(II) species in the presence of the reducing agent hydrosulfite is indicated by *. Upon removal of the reducing agent, the desalted Fe(II) spectrum is obtained. Upon exposure to air, the formation of the oxidized ferrous spectrum is observed, with maxima at about 540 nm and 575 nm (Fe(II), air exposure). Re-oxidation of the protein yields the trivalent iron spectrum (Fe(III), re-ox), which is in agreement with the trivalent iron spectrum shown in Figure 7 .

[0025] Figure 9 : Truncated HBD16 RcoM with C94S substitution has the same CO onrate as WT RcoM. The reaction kinetics of the ferrous heme-binding domain (HBD) of RcoM1 with carbon monoxide (CO) was determined using the stopped-flow technique. (Top left) Detail of the protein Soret band; arrow indicates direction of absorbance change. (Top right) Detail of the visible range of the spectrum. Arrow indicates direction of absorbance change. (Bottom left) Absorbance at selected wavelengths as a function of time. The rate at different CO concentrations was calculated to yield a reaction on-rate of 1.2 x 10 5 M -1 s -1 . A similar value was obtained for the wild-type full-length protein.

[0026] Figure 10 : Determination of the CO off-rate of the heme-binding domain (HBD) of RcoM1 carrying the C94S mutation. The reaction was monitored by absorbance changes as the ferrous-CO complex dissociated in the presence of nitric oxide (NO). As CO dissociates, NO binds to the heme, causing a change in the absorption spectrum. Excess NO prevents CO from rebinding to the heme. (Top left) Detail of the visible range of the spectrum. Arrow indicates direction of absorbance change. (Top right) Time course of the absorbance change allows determination of the 4.9 x 10-2 s -1 dissociation rate.

[0027] Figure 11 Thermal unfolding of Fe(III) HBD RcoM-1 carrying the C94S mutation. Unfolding was monitored by the absorbance change at the heme Soret maximum at 420 nm. The samples were equilibrated at each temperature for 5 min before each UV-Vis spectrum was recorded. The small loss in Soret intensity observed between 20 °C and 75 °C can be due to changes in the heme coordination number. The loss in Soret intensity between 75 °C and 98 °C is attributed to thermal unfolding leading to loss of heme from the protein. (Top left) UV-Vis spectra of Fe(III) HBD RcoM-1 carrying the C94S mutation recorded at each temperature between 20 °C and 98 °C. (Top right) Absorbance values at the 420 nm Soret maximum as a function of temperature. (Bottom left) UV-Vis spectra recorded during thermal unfolding between 75 °C and 98 °C. (Bottom right) Absorbance values at the Soret maximum as a function of temperature recorded during thermal unfolding between 75 °C and 98 °C. These data were used to determine the melting stability, T m 91 °C.

[0028] Figures 12A-12D Comparison of the electronic absorption (UV-Vis) spectra of the RcoM heme binding domain (HBD) truncates in WT Figure 12A ) and Cys-substituted protein variant CC HBD Figure 12B ), C94S Figure 12C ) and CCCHBD Figure 12D . Spectra of the trivalent iron (Fe(III)), deoxy ferrous (Fe(II)), and ferrous-CO species (Fe(II)-CO) and oxidized ferrous (Fe(II)-O2) are shown.

[0029] Figures 13A-13C Comparison of the electronic absorption (UV-Vis) spectra of the RcoM HBD truncates in Met104 variant CC M104A Figure 13A ) and CC M104H Figure 13B , both carrying Cys94. Spectra of the trivalent iron (Fe(III)), deoxy ferrous (Fe(II)), and ferrous-CO species (Fe(II)-CO) and oxidized ferrous (Fe(II)-O2) are shown. Figure 13C Schematic of the protein-derived ligand switching mechanism of RcoM highlighting the changes in the coordination sphere in these variants.

[0030] Figures 14A-14D Comparison of the electronic absorption (UV-Vis) spectra of the RcoM HBD truncates in Met104 variant CCC M104A Figure 14A), CCC M104L( Figure 14B ), and CCC M104H( Figure 14C ) RcoM HBD truncates. Electronic absorption (UV-Vis) spectra comparison of RcoM HBD truncates, all with Cys94→Ser substitution. Spectra of ferric (Fe(III), deoxy ferrous (Fe(II)), and ferrous-CO species (Fe(II)-CO) and ferrous-02 (Fe(II)-02) are shown. Figure 14D ) Schematic of the protein-derived ligand switching mechanism of RcoM highlighting the coordination sphere changes in these variants.

[0031] Figures 15A-15D : Quantification of oxygen binding affinity (P 50 ) in RcoM HBD truncates. The fraction of hemoglobin bound to oxygen as a function of oxygen partial pressure was measured using UV-Vis spectroscopy with a tonometer device equipped with an optical cuvette. Figure 15A ) Representative spectral changes in UV-Vis characteristics of CC HBD RcoM variants as a function of oxygen partial pressure (P O2 ). Oxygen binding curves of CC HBD( Figure 15B ), C94S HBD( Figure 15C ), and CCC HBD( Figure 15D ) plotted as fraction of deoxy (depleted of oxygen) and oxygen bound (oxygen + Fe(III)) hemoglobin. Autoxidation under low oxygen tension can be the cause of some ferryl hemoglobin formation. Curves were fit to a non-linear one-site binding model to quantify P 50 .

[0032] Figures 16A-16D : CO binding (k on , CO) of WT HBD RcoM( Figure 16A ) and HBD truncates CC HBD( Figure 16B ), C94S( Figure 16C ), and CCC HBD( Figure 16D ). CO binding rates at each CO concentration were measured using stopped-flow UV-Vis spectroscopy and fit to a single exponential. Each data point represents the average of 2 to 3 replicate measurements of these rates. Linear regression was applied to each curve and the second-order rate constant was estimated as the slope.

[0033] Figures 17A-17C : Representative determination of autoxidation rate (k oxid ) of WT HBD RcoM truncates. Figure 17A ) Reference spectra of Fe(III) and Fe(II)-02 proteins. Figure 17BSpectral changes in the UV-Vis features of the Fe(II)-02WT HBD. Figure 17C )Fitting of spectral changes at 542 nm and 573 nm to a single exponential to determine k oxid .

[0034] Figure 18 Summary of ligand binding parameters and heme stability properties of WT RcoM and RcoM HBD variants C94S, CC HBD, and CCC HBD.

[0035] Figures 19A-19B Representative unfolding of Fe(III) CCC HBD RcoM in the presence of urea at 37 °C. Figure 19A Unfolding was monitored by changes in absorbance at the heme Soret maximum at 415 nm. The samples were allowed to equilibrate for 10 minutes prior to recording each UV-Vis spectrum. Figure 19B Unfolding data were fitted to a sigmoidal curve to determine the denaturant concentration at which half of the protein sample was unfolded ([D 50 ).

[0036] Figures 20A-20D Lack of reactivity between RcoM HBD truncates and hydrogen peroxide. Fe(III) WT HBD Figure 20A ) and variant CCC HBD Figure 20B ), CCC M104A HBD Figure 20C ), and CCC M104H HBD Figure 20D ) were incubated with 500 μΜ hydrogen peroxide at pH 7.4, 25 °C and monitored by UV-Vis spectroscopy every 2 minutes for 30 minutes. Minimal spectral changes were observed for each variant, indicating that hydrogen peroxide does not react with the Fe(III) heme center of RcoM HBD truncates to produce highly oxidized species.

[0037] Figures 21A-21C Summary of nitrite reduction data for full-length and HBD-truncated RcoM variants. Diron proteins (10-15 μΜ) were incubated with 1-5 mM sodium nitrite in the presence of 2.5 mM sodium hydrosulfite at 37 °C. Figure 21A Conversion of Fe(II) heme to Fe(II)-NO was monitored by UV-Vis spectroscopy. Figures 21B-21C Changes in spectral features at 562 nm and 578 nm were fitted to a single exponential curve to determine the observed rate of nitrite reduction. Observed rates were plotted as a function of nitrite concentration, and a linear regression was applied to each plot, estimating the second-order rate constant as the slope.

[0038] Figures 22A-22DRepresentative kinetic traces of CO transfer from hemoglobin (Hb) to WT RcoM HBDs Figure 22A ) and RcoM HBD variants CCHBD Figure 22B ), C94SHBD Figure 22C ), and CCC HBD Figure 22D ) under aerobic conditions at 37°C. CO-bound Hb (20 μΜ) was incubated with equimolar oxygenated iron RcoM and CO transfer from Hb to RcoM was monitored using UV-Vis spectroscopy. The fraction of each CO-bound hemoglobin was determined using spectral deconvolution and the corresponding kinetic traces were fit to a single exponential or double exponential equation. The half-life of each CO-bound species is shown, with the fast species half-life and amplitude shown for curves fit to a double exponential.

[0039] Figures 23A-23B Representative kinetic traces of CO transfer from HbCO encapsulated in red blood cells (RBCs) to extracellular RcoM HBD truncates were monitored under aerobic conditions at 37°C. Hemoglobin was incubated at equimolar concentrations (50-100 μΜ) and RBCs were isolated from extracellular RcoM at each time point by centrifugation. CO transfer from Hb to WT HBD RcoM Figure 23A ) and C94S HBD RcoM Figure 23B ) was monitored using UV-Vis spectroscopy. The fraction of each CO-bound hemoglobin was determined using spectral deconvolution and the corresponding kinetic traces were fit to a single exponential equation. Data points represent the mean ± SEM of 3 experiments and the half-life of COHb is shown for each experiment.

[0040] Figure 24 C94S and CCC HBD RcoM variants clear CO from HbCO in an in vivo lethal CO poisoning model. Schematic of a mouse severe CO poisoning in vivo model (top panel). Anesthetized, mechanically ventilated mice were exposed to 3,000 ppm CO in air for 4.5 minutes and then infused intravenously with Fe(II)-02 CCC HBD RcoM (hemoglobin concentrations listed in table, bottom panel) at an injection volume of 10 μΐ^ / g body weight. Blood samples (15 μΐ^) were drawn immediately before and after infusion, and at 25 minutes after CO exposure. At each time point, RBCs were isolated from plasma by centrifugation and the isolated RBC pellet and plasma samples were immediately frozen at -80°C. Subsequently, the fraction of CO-bound hemoglobin from RBCs (% HbCO) and the fraction of CO-bound RcoM (% RcoM-CO) were determined using spectral deconvolution. Infusion of RcoM resulted in a greater decrease in the fraction of CO-bound Hb (Δ % HbCO) compared to infusion of PBS.

[0041] SEQUENCE LISTING

[0042] Nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The sequence listing was created on May 3, 2021, and submitted in ASCII text file, size 18.7 KB, which is incorporated by reference herein. In the accompanying sequence listing:

[0043] SEQ ID NO: 1 is the amino acid sequence of full-length WT RcoM-1 from P. xenovorans.

[0044] SEQ ID NO: 2 is the amino acid sequence of truncated RcoM-1 (HBD16) without the LytTR domain.

[0045] SEQ ID NO: 3 is the amino acid sequence of truncated RcoM-1 (HBD12) without the LytTR domain and part of the PAS domain.

[0046] SEQ ID NO: 4 is the amino acid sequence of WT RcoM from H. crassostreae.

[0047] SEQ ID NO: 5 is the amino acid sequence of a cleavage site from Tobacco Etch Virus (TEV).

[0048] SEQ ID NO: 6 is the amino acid sequence of a cleavage site from Thrombin.

[0049] SEQ ID NO: 7 is the amino acid sequence of RcoM variant C94S HBD.

[0050] SEQ ID NO: 8 is the amino acid sequence of RcoM variant C127S / C130S HBD.

[0051] SEQ ID NO: 9 is the amino acid sequence of RcoM variant CCC HBD.

[0052] SEQ ID NO: 10 is the amino acid sequence of RcoM variant CC M104A HBD.

[0053] SEQ ID NO: 11 is the amino acid sequence of RcoM variant CC M104H HBD.

[0054] SEQ ID NO: 12 is the amino acid sequence of RcoM variant CCC M104A HBD

[0055] SEQ ID NO: 13 is the amino acid sequence of RcoM variant CCC M104H HBD.

[0056] SEQ ID NO: 14 is the amino acid sequence of RcoM variant CCC M104L HBD. DETAILED DESCRIPTION

[0058] I. Abbreviations

[0059] CO carbon monoxide

[0060] H2S hydrogen sulfide

[0061] Hb hemoglobin

[0062] Hb-CO carboxyhemoglobin

[0063] HBOC hemoglobin-based oxygen carrier

[0064] HBD heme-binding domain

[0065] NO nitric oxide

[0066] RcoM carbon monoxide metabolic regulator

[0067] TEV tobacco etch virus

[0068] WT wild type

[0069] II. Terms and Methods

[0070] Technical terms are used according to their customary usage unless otherwise indicated. Definitions for commonly used terms in molecular biology can be found in Benjamin Lewin, Genes X, 2009, published by Jones & Bartlett Publishers; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Vol. 16, 2008, published by Wiley-VCH; and other similar references.

[0071] As used herein, the singular forms "a," "an," and "the" refer to both the singular as well as plural, unless the context clearly dictates otherwise. By way of example, the term "an antigen" includes a single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for a nucleic acid or polypeptide are approximate, and are provided for description. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the patent specification, including the

[0072] To facilitate review of the various embodiments, the following explanations of terms are provided:

[0073] Administration: The provision or giving of a pharmaceutical 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 (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, intrastriatal, intracranial, and into the spinal cord), oral, intra-cordal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0074] 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-carrier protein, blue tongue 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 (E-E tag), glutathione S-transferase, influenza hemagglutinin, Halo 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, poly cysteine, 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 (see, e.g., Kimple et al., Curr Protoc Protein Sci 73:9.9.1-9.9.23, 2013, doi:10.1002 / 0471140864.ps0909s73).

[0075] Anemia: A deficiency in red blood cells and / or hemoglobin. Anemia is the most common blood disorder, which results in a decreased ability of blood to transport oxygen to tissues. Because the survival of all human cells depends on oxygen, anemia of varying degrees produces a wide range of clinical consequences. The three main types of anemia include excessive blood loss (acute blood loss such as hemorrhage or chronic blood loss from low volume loss), excessive destruction of blood cells (hemolysis), or insufficient production of red blood cells (ineffective hematopoiesis).

[0076] The term "anemia" refers to all types of clinical anemia, including but not limited to: microcytic anemia, iron deficiency anemia, hemoglobinopathy, heme synthesis defect, globin synthesis defect, sideroblastic defect, 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, cold agglutinin hemolytic anemia.

[0077] In the case of severe anemia or persistent blood loss, blood transfusion can be required. A physician can use any of a number of clinically accepted criteria to determine that blood transfusion is necessary for the treatment of a subject with anemia. For example, the Rivers protocol, which is currently accepted for early goal-directed therapy in sepsis, requires that the hematocrit be maintained above 30.

[0078] Hypoxia: A pathological state in which the body as a whole or a region of the body is completely deprived of oxygen supply.

[0079] Detoxifying agent: An agent that neutralizes or counteracts the effects of a toxic substance, such as carbon monoxide.

[0080] Bleeding disorder: A general term for various medical problems that result in poor blood clotting and persistent bleeding. Physicians also use terms such as coagulopathy, abnormal bleeding, and coagulation disorder to indicate bleeding disorders. Bleeding disorders include any congenital, acquired, or induced defect that results in abnormal (or pathological) bleeding. Examples include, but are not limited to, disorders of coagulation or hemostasis, such as hemophilia A (deficiency in factor VIII), hemophilia B (deficiency in factor IX), hemophilia C (deficiency in factor XI), other coagulation factor deficiencies (such as factor VII or factor XIII), abnormal levels of coagulation factor inhibitors, platelet disorders, thrombocytopenia, vitamin K deficiency, and von Willebrand’s disease.

[0081] Bleeding episode: Refers to the occurrence of uncontrolled, excessive, and / or pathological bleeding. Bleeding episodes can result from, for example, drug-induced bleeding (such as bleeding induced by non-steroidal anti-inflammatory drugs or warfarin), overdose or poisoning of anticoagulants, aneurysm, blood vessel rupture, surgery, and trauma (including, for example, abrasions, contusions, lacerations, incisions, or gunshot wounds). Bleeding episodes can also result from diseases such as cancer, gastrointestinal ulcers, and the like, or from infection.

[0082] Blood replacement or blood substitute: A composition used to fill fluid volume and / or carry oxygen and other blood gases in the cardiovascular system. Blood substitutes include, for example, volume expanders (used to increase blood volume) and oxygen therapeutics (used to deliver oxygen in 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 (over days and weeks), and are free of side effects and pathogens.

[0083] Carbon monoxide (CO): A colorless, odorless, tasteless gas that is toxic to humans and animals at sufficiently high concentrations. Low levels of CO are also produced during normal animal metabolism.

[0084] Carboxyhemoglobin (HbCO): A stable complex of carbon monoxide (CO) and hemoglobin (Hb) that forms in red blood cells when CO is inhaled or produced during normal metabolism.

[0085] Carboxyhemoglobinemia or carbon monoxide poisoning: A disorder caused by the presence of excess carbon monoxide in the blood. Typically, exposure to 100 parts per million (ppm) or greater of CO is sufficient to cause carboxyhemoglobinemia. Symptoms of mild acute CO poisoning include dizziness, confusion, headache, dizziness, and flu-like reactions; larger amounts of exposure can cause significant toxicity to the central nervous system and heart, and even death. Long-term sequelae often occur after acute poisoning. Carbon monoxide also has serious effects on the fetus of a pregnant woman. Long-term exposure to low levels of carbon monoxide can cause depression, confusion, and memory loss. Carbon monoxide primarily causes adverse effects in humans by binding to hemoglobin in the blood to form carboxyhemoglobin (HbCO). This prevents oxygen from binding to hemoglobin, reducing the blood's oxygen-carrying capacity, resulting in hypoxia. In addition, myoglobin and mitochondrial cytochrome oxidase are thought to be adversely affected. Carboxyhemoglobin can be reduced to hemoglobin, but this reduction takes time because the HbCO complex is quite stable. Current methods of treating CO poisoning include administration of 100% oxygen or providing hyperbaric oxygen therapy.

[0086] Cerebral ischemia or ischemic stroke: A disorder that occurs when an artery to or in the brain is partially or completely blocked, causing the oxygen demand of the tissue to exceed the oxygen supply. The brain is damaged by the stroke due to the lack of oxygen and other nutrients after an ischemic stroke.

[0087] Coagulopathy: A medical term for a defect in the body's clotting mechanism.

[0088] Contact: To be placed in direct physical association; both in solid and liquid form. When used in the context of in vivo methods, "contacting" also includes administration.

[0089] Cyanide poisoning: A form of poisoning caused by exposure to some forms of cyanide, such as hydrogen cyanide gas and cyanide salts. Cyanide poisoning can occur from inhaling smoke produced by a house fire, exposure to metal polishes, certain pesticides, and certain seeds, such as apple seeds. Early symptoms of cyanide poisoning include headache, dizziness, rapid heart rate, rapid breathing, and vomiting. Later symptoms include seizures, slow heart rate, low blood pressure, loss of consciousness, and cardiac arrest.

[0090] Cytochrome c oxidase: An enzyme that is part of the respiratory electron transport chain. The enzyme is found in mitochondria.

[0091] Favism: A colloquial term for glucose-6-phosphate dehydrogenase (G6PD) deficiency; an X-linked recessive disorder characterized by non-immune hemolytic anemia in response to multiple etiologies.

[0092] Fusion protein: A protein that comprises at least a portion of two different (heterologous) proteins.

[0093] Gastrointestinal bleeding: refers to any form of bleeding (hemorrhage) in the gastrointestinal tract from the pharynx to the rectum.

[0094] Hemoglobin (Hb): an iron-containing oxygen transport metalloprotein in the red blood cells of vertebrate and other animals. In humans, the hemoglobin molecule is a collection of four globular protein subunits. Each subunit is composed of a protein chain tightly bound to a non-protein heme group. Each protein chain is arranged in a set of alpha-helical structural segments connected together in a globin fold arrangement, so named because of the fold motif used in other heme / globin proteins. This fold pattern contains a pocket that tightly binds the heme group.

[0095] Hemoglobin-based oxygen carrier (HBOC): an infusible, purified, recombinant and / or modified hemoglobin fluid that functions as an oxygen carrier and can be used as a blood substitute. Numerous HBOCs are known and / or are in clinical development. Examples of HBOCs include, but are not limited to, DCLHb (HemAssist TM ; Baxter), MP4 (Hemospas TM ; Sangart), pyoxylated Hb POE-conjugate (PHP) + catalase & SOD (Apex Biosciences), O-R-PolyHbA0 (Hemolink TM ; Hemosol), PolyBvHb (Hemopure TM ; Biopure), PolyHb (Polyheme TM ; Northfield), rHbl.l (Optro™; Somatogen), PEG-hemoglobin (Enzon), Oxyvita TM , 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).

[0096] Hemophilia: the name for several inherited genetic diseases that impair the body's ability to control blood clotting.

[0097] Hemorrhage: Loss of blood from the circulatory system. Bleeding can occur internally, with blood leaking from vessels in the body, or externally, through natural openings such as the vagina, mouth, or rectum, or through breaks in the skin.

[0098] Heterologous: Heterologous proteins or polypeptides refer to proteins or polypeptides derived from different sources or species.

[0099] Hydrogen sulfide poisoning: A poisoning caused by excessive exposure to hydrogen sulfide (H2S). H2S binds to iron in mitochondrial cytochrome enzymes and prevents cellular respiration. Exposure to lower concentrations of H2S can result in eye irritation, sore throat, cough, nausea, shortness of breath, pulmonary edema, fatigue, loss of appetite, headache, irritability, poor memory, and dizziness. Higher levels of exposure can result in immediate collapse, inability to breathe, and death.

[0100] Hemorrhagic shock: A condition in which reduced tissue perfusion results in inadequate delivery of oxygen and nutrients essential for cellular function. Hypovolemic shock is the most common type, resulting from reduced circulating blood volume caused by clinical causes such as penetrating and blunt trauma, gastrointestinal bleeding, and obstetric hemorrhage.

[0101] Hypoxemia: An abnormally insufficient arterial blood oxygen concentration.

[0102] Hypoxia: A pathological condition in which the body as a whole (systemic hypoxia) or a body part (tissue hypoxia) lacks an adequate supply of oxygen.

[0103] Ischemia: A vascular phenomenon in which the blood supply to an organ, tissue, or part of the body is reduced, caused by, for example, constriction or obstruction of one or more blood vessels. Ischemia is sometimes caused by vasoconstriction or thrombosis or embolism. Ischemia can lead to direct ischemic injury, tissue damage due to cell death caused by reduced oxygen supply.

[0104] Ischemia / reperfusion injury: In addition to the direct injury that occurs during blood flow deprivation, ischemia / reperfusion injury also involves tissue damage that occurs after blood flow is restored. Current understanding is that this injury is largely caused by chemical products and free radicals released into the ischemic tissue.

[0105] When tissue is subjected to ischemia, a series of chemical events is triggered that can ultimately result in cell dysfunction and necrosis. If the ischemia is ended by restoration of blood flow, a second series of damaging events then occurs, resulting in additional injury. Thus, whenever blood flow to a subject is reduced or interrupted for a brief period, the resulting injury involves two components - direct injury that occurs during the ischemic interval and subsequent indirect or reperfusion injury. When the duration of ischemia is long, direct ischemic injury caused by hypoxia predominates. For shorter durations of ischemia, indirect or reperfusion-mediated injury becomes increasingly important. In some cases, the injury produced by reperfusion can be more severe than that caused by ischemia itself. The relative contribution of injury by these direct and indirect mechanisms has been shown to occur in all organs.

[0106] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components of the cells, blood, or tissue of an organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells. Nucleic acid molecules and proteins that have been "isolated" include those that have been purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

[0107] Met-Hb: Methemoglobin: An oxidized form of hemoglobin in which the iron in the heme component has been oxidized from the ferrous (+2) state to the ferric (+3) state. This renders the hemoglobin molecule incapable of efficiently transporting and releasing oxygen to tissues. Normally, the methemoglobin form accounts for about 1% of total hemoglobin.

[0108] Microcytosis: A blood disorder characterized by the presence of microcytes (abnormally small red blood cells) in the blood.

[0109] Myoglobin: A heme-containing globin found in muscle tissue of vertebrates and most mammals. Myoglobin carries and stores oxygen in muscle cells.

[0110] Oxidant: A substance that is able to accept an electron from another substance (also known as an "oxidizing" substance). The oxidant gains an electron and is reduced in the chemical reaction. Oxidants are also known as "electron acceptors". In some embodiments herein, the oxidant is a quinone, such as benzoquinone or naphthoquinone. In other embodiments, the oxidant is an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof. In some examples, an electron mediator (e.g., TMPD or crystal violet) is used in combination with the oxidant to facilitate electron transfer. In some embodiments herein, oxidation of RcoM is carried out by exposure to visible light.

[0111] Paraburkholderia xenovorans: a proteobacterium found in soil. P. xenovorans is a gram-negative, aerobic bacterium. P. xenovorans has one of the largest known prokaryotic genomes at 9.7 Mb. The bacterium is capable of efficiently degrading polychlorinated biphenyls (PCBs). P. xenovorans is also known as Burkholderia xenovorans.

[0112] Peptide or polypeptide: a polymer in which the monomers are amino acid residues joined together by amide bonds. When the amino acids are a-amino acids, either the L-optical isomer or the D-optical isomer can be used, with the L-isomer being preferred. As used herein, the terms "peptide," "polypeptide," or "protein" are intended to encompass any sequence of amino acids and include modified sequences, including modified RcoM proteins. The terms "peptide" and "polypeptide" are specifically intended to encompass naturally occurring proteins, as well as proteins produced recombinantly or synthetically.

[0113] Conservative amino acid substitutions are those that have the least disturbance to the properties of the original protein when made, i.e., the structure and, in particular, the function of the protein is conserved and does not change significantly as a result of such substitutions. Examples of conservative substitutions are shown in the table below.

[0114]

[0115] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, e.g., as a turn or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0116] Substitutions that are generally expected to produce the greatest changes in protein properties are non-conservative, e.g., changes in which (a) a hydrophilic residue, e.g., serine or threonine, is substituted (or replaced with) for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) a cysteine or proline is substituted (or replaced with) for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted (or replaced with) for (or by) an electronegative residue, e.g., glutamine or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted (or replaced with) for (or by) one not having a side chain, e.g., glycine.

[0117] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers used are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of the proteins and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional nontoxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0118] Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to therapeutic intervention after a disease or pathological condition has begun to develop, such as reducing HbCO in the blood of a subject with CO poisoning. "Ameliorating" refers to reducing the number or severity of signs or symptoms of a disease.

[0119] Purified: The term purified does not require absolute purity; rather it is a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is enriched relative to the peptide or protein in its natural environment within a cell. In one embodiment, a purified preparation is such that the protein or peptide comprises at least 50% of the total peptide or protein content of the preparation. Substantially purified 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 one particular non-limiting example, 90% of a substantially purified protein is free of other proteins or cellular components.

[0120] Recombinant: A recombinant nucleic acid or protein is one having a sequence not found in nature or having a sequence created by artificially combining two otherwise separate segments of sequence. Such artificial combination is typically accomplished by chemical synthesis or by the artificial manipulation of isolated nucleic acid segments, for example by genetic engineering techniques. The term recombinant includes nucleic acids and proteins that have been altered by the addition, substitution, or deletion of a portion of a naturally occurring nucleic acid molecule or protein.

[0121] Reducing agent: An element or compound that loses (or "donates") electrons to another chemical substance in a redox chemical reaction. Reducing agents are usually in one of their possible lower oxidation states and are called electron donors. A reducing agent is oxidized because it loses electrons in a redox reaction. Exemplary reducing agents include, but are not limited to, sodium hydrosulfite, ascorbic acid, N-acetyl cysteine, methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, earth metals, formic acid, and sulfite compounds.

[0122] Carbon monoxide metabolism regulator (RcoM): A protein found in some prokaryotes that is involved in CO sensing and transcriptional regulation. The RcoM protein contains an N-terminal PAS domain and a LytTR domain that binds DNA. The PAS domain contains a six-coordinate heme moiety of type b that binds tightly to CO and nitric oxide (NO). Residues His74 and Met104 of the PAS domain serve as axial ligands to the heme Fe(II), with Met104 being displaced upon binding of CO or NO. The aerobic gram-negative bacterium Paraburkholderia xenovorans (also known as Burkholderia xenovorans) expresses two homologous proteins, RcoM-1 and RcoM-2, that have about 93% sequence identity and have very high affinity for CO. RcoM-1 and RcoM-2 serve as CO sensors that are able to regulate aerobic CO oxidation and anaerobic CO oxidation. The wild-type amino acid sequence of P. xenovorans RcoM-1 is set forth herein as SEQ ID NO: 1. RcoM homologs from multiple bacterial species (and UniProt IDs) are listed in Table 3.

[0123] Rhabdomyolysis: Rapid breakdown of skeletal muscle tissue due to traumatic injury, including mechanical, physical, or chemical injury. The main result is the release of large amounts of creatine phosphokinase and other cellular byproducts into the blood system, and acute kidney failure due to accumulation of muscle breakdown products, some of which are harmful to the kidneys.

[0124] Sequence identity / similarity: Identity between two or more nucleic acid sequences or between 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 sequence identity. Sequence similarity can be measured in terms of percentage similarity (allowing for conservative amino acid substitutions); the higher the percentage, the more sequence similarity. Homologues or orthologues of a nucleic acid or amino acid sequence, when aligned for maximum correspondence using standard methods, have a relatively high degree of sequence identity / similarity. This homology is more pronounced when the orthologous proteins or cDNAs are derived from closely related species (such as human and mouse sequences) than when the relatedness is more remote (such as human sequences and C. elegans sequences).

[0125] Sequence alignment methods 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 shows detailed consideration of sequence alignment methods and homology calculations.

[0126] 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 Biotechnology Information (NCBI) and on the internet, in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. More information can be found at the NCBI website.

[0127] Spherocytosis: An autohemolytic anemia characterized by the production of spheroidal rather than donut-shaped red blood cells (or erythrocytes).

[0128] Subject: A living multicellular organism, including a vertebrate organism, which category includes humans and non-human mammals.

[0129] Thalassemia: A genetic, autosomal recessive blood disorder. In thalassemia, a genetic defect results in a decreased rate of synthesis of one of the globin chains that make up hemoglobin. The decreased synthesis of one of the globin chains results in the formation of abnormal hemoglobin molecules, which in turn results in anemia, which is the characteristic symptom of thalassemia.

[0130] Therapeutically effective amount: An amount of a compound or composition, such as an isolated or recombinant RcoM protein, sufficient to achieve a desired effect in a subject receiving treatment. For example, it can be the amount required to scavenge carbon monoxide in blood or tissue, reduce HbCO levels in blood, and / or reduce one or more signs or symptoms associated with carbon monoxide poisoning.

[0131] Ulcer: An open sore of the skin, eye, or mucous membrane, usually but not exclusively caused by an initial abrasion, often maintained by inflammation, infection, and / or medical conditions that impede healing.

[0132] Vasospasm: One of the causes of stroke; secondary to vasospasm of blood vessels supplying the brain. This type of stroke often occurs after subarachnoid aneurysm hemorrhage and develops as vasospasm delayed for 2 to 3 weeks after the hemorrhagic event. A similar type of stroke can complicate sickle cell disease.

[0133] IV. Recombinant RcoM Proteins

[0134] There is a need for an effective, fast, and easily accessible therapy to treat carboxyhemoglobinemia. The present disclosure provides recombinant carbon monoxide metabolism regulator (RcoM) proteins, which exhibit very high affinity for carbon monoxide and thus can be used as CO scavengers. The disclosed RcoM proteins can also be used to treat hydrogen sulfide or cyanide poisoning, or can be used as blood substitutes.

[0135] RcoM proteins were first identified as CO-sensing bacterial transcriptional regulators that couple an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain (see Figure 1). The RcoM protein contains a hexa-coordinated b-type heme moiety that binds tightly to CO and nitric oxide (NO). Residues His74 and Met104 (relative to SEQ ID NO: 1) of the PAS domain serve as axial ligands to the heme Fe(II), which is displaced by Met104 upon binding of CO or NO. Two RcoM homologs from P. xenovorans (RcoM-1 and RcoM-2) are functional in vivo and serve as CO sensors that can regulate aerobic CO oxidation and anaerobic CO oxidation.

[0136] RcoM exhibits very high affinity for CO and a selectivity for CO over oxygen. Given these properties, the disclosed RcoM proteins are ideal for scavenging CO from CO-bound hemoglobins, myoglobins, and cytochrome c oxidases to treat carbon monoxide poisoning. The disclosed RcoM proteins can also be used to treat cyanide or H2S poisoning, or as a blood substitute. Directed mutations to enhance the stability of the RcoM protein, increase CO affinity, and / or decrease oxygen affinity are further described herein.

[0137] Wild-type (WT) and modified RcoM proteins are described below. In the WT amino acid sequence (SEQ ID NO: 1), the LytTR domain (binds DNA) is underlined; 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 include the LytTR domain (see Figure 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, numbered relative to SEQ ID NO: 1.

[0138] WT RcoM-1 from P. xenovorans (29 kDa):

[0139]

[0140] HBD16 RcoM (16 kDa) truncated sequence:

[0141]

[0142] HBD12 RcoM (12 kDa) truncated sequence:

[0143]

[0144] Throughout the disclosure, unless otherwise specified, a particular amino acid residue is numbered with reference to the full-length WT RcoM-1 of SEQ ID NO: 1. Table 1 lists the position of each corresponding residue in SEQ ID NOs: 1-3.

[0145] Table 1. Key residues in WT and truncated RcoM sequences

[0146]

[0147]

[0148] Eight RcoM HBD variants were generated based on HBD16 of SEQ ID NO: 2. Table 2 lists each variant, as well as their respective amino acid substitutions and full amino acid sequence (bolded residues indicate substitutions).

[0149] Table 2. RcoM HBD16 variants

[0150]

[0151]

[0152] Provided herein are recombinant carbon monoxide metabolic regulator (RcoM) proteins that exhibit very high affinity for CO. In some embodiments, the recombinant RcoM protein includes a heme-binding domain (HBD), and the amino acid sequence of the HBD has 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% identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the HBD is a wild-type sequence, such as SEQ ID NO: 2. In other embodiments, the amino acid sequence of the HBD includes 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 has at least 90% or at least 95% identity to SEQ ID NO: 2 and includes an amino acid substitution at one or more of C94, M104, C127, and C130.

[0153] The disclosed RcoM proteins can be modified, such as by making amino acid substitutions at multiple residues, to alter heme ligand affinity and / or specificity, and / or to enhance protein stability. In some embodiments, the RcoM protein includes a single amino acid substitution. In other embodiments, the RcoM protein includes at least two, at least three, at least four, at least 5, or at least 6 amino acid substitutions. In some examples, the amino acid substitutions are conservative substitutions.

[0154] In some examples, the recombinant RcoM protein includes a substitution at H74, which is a heme-coordinating histidine. In some particular non-limiting examples, the substitution is selected from the group consisting of H74S, H74T, H74M, H74W, H74A, H74L, H74I, H74V, and H74G.

[0155] In some examples, the recombinant RcoM protein includes a substitution at C94, which is a Fe(II) heme-coordinating cysteine. In some particular non-limiting examples, the substitution is selected from the group consisting of C94S, C94T, C94H, C94W, C94M, C94A, C94L, C94I, C94V, and C94G.

[0156] In some examples, the recombinant RcoM protein includes a substitution at M104, which is a Fe(II) heme-coordinating methionine. In some particular non-limiting examples, the substitution is selected from the group consisting of M104S, M104T, M104H, M104W, M104A, M104L, M104I, M104V, and M104G.

[0157] In some examples, the recombinant RcoM protein includes a substitution at M105, which is a non-heme-coordinating methionine. In some particular non-limiting examples, the substitution is selected from the group consisting of M105S, M105T, M105H, M105W, M105A, M105L, M105I, M105V, and M105G.

[0158] In some examples, the recombinant RcoM protein includes a substitution at C127, which is a non-heme-coordinating cysteine. In some particular non-limiting examples, the substitution is selected from the group consisting of C127S, C127T, C127M, C127A, C127L, C127I, C127V, and C127G.

[0159] In some examples, the recombinant RcoM protein includes a substitution at C130, which is a non-heme-coordinating cysteine. In some particular non-limiting examples, the substitution is selected from the group consisting of C130S, C130T, C130M, C130A, C130L, C130I, C130V, and C130G.

[0160] In some examples, the recombinant RcoM protein comprises: a single amino acid substitution at C94; a single amino acid substitution at M104; two amino acid substitutions at C94 and M104; two amino acid substitutions at C127 and C130; three amino acid substitutions at C94, C127, and C130; three amino acid substitutions at M104, C127, and C130; three amino acid substitutions at H74, C94, and M104; four amino acid substitutions at C94, M104, C127, and C130; five amino acid substitutions at C94, M104, M105, C127, and C130; five amino acid substitutions at H74, C94, M104, C127, and C130; or six amino acid substitutions at H74, C94, M104, M105, C127, and C130. In some particular non-limiting examples, the recombinant RcoM protein comprises 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; a C94S substitution, a M104H substitution, a C127S substitution, and a C130S substitution; a C94S substitution, a M104L substitution, a C127S substitution, and a C130S substitution; a H74S substitution, a C94S substitution, and a M104L substitution; a C94S substitution, a M104L substitution, a M105L substitution, a C127S substitution, and a C130S substitution; or a H74S substitution, a C94S substitution, a M104L substitution, a M105L substitution, a C127S substitution, and a C130S substitution.

[0161] In some 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.

[0162] In some embodiments, the amino acid sequence of the RcoM protein has 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% identity 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.

[0163] 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 an amino acid substitution at one or more of H74, C94, M104, C127, C130, and M105.

[0164] In particular examples, the amino acid sequence of the RcoM protein consists of SEQ ID NO: 1, except for an H74S substitution, a C94S substitution, an M104 substitution selected from M104A, M104H, and M104L, an M105L substitution, a C127S substitution, a C130S substitution, or any combination thereof. In other examples, the amino acid sequence of the protein consists of SEQ ID NO: 2, except for an H74S substitution, a C94S substitution, an M104 substitution selected from M104A, M104H, and M104L, an M105L substitution, a C127S substitution, a C130S substitution, or any combination thereof. In other particular examples, the amino acid sequence of the protein consists of SEQ ID NO: 3, except for an H74S substitution, a C94S substitution, an M104 substitution selected from M104A, M104H, and M104L, an M105L substitution, a C127S substitution, a C130S substitution, or any combination thereof.

[0165] Using bioinformatic analysis, 112 rcoM genes were identified in a variety of microorganisms, 44 of which are associated with aerobic CO metabolism. One of the identified rcoM genes is from a thermophilic microorganism (Hydrogenophaga crassostreae), which is believed to express a RcoM protein with enhanced thermal stability. Accordingly, in some embodiments, the recombinant RcoM protein is a protein from one of the species listed in Table 3 and having the listed UniProt ID.

[0166] Table 3. Microorganisms with homologous sequences to rcoM genes

[0167]

[0168]

[0169] The amino acid sequences of the RcoM homologs listed above are incorporated by reference as they appeared in the UniProt database on May 11, 2020.

[0170] In some embodiments, the RcoM protein is from Hydrogenophaga crassostreae. In some examples, the amino acid sequence of the RcoM protein 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.

[0171] Full-length Rcom sequence from H. crassostreae

[0172] MEAEVANKSPLYLLEKFEVGVIHLDAKRTVLAMNDFARKVLPVGEKQPFDKLVSSFHPARSKPKVDFLLDQASSCPMVSAVPMTMIINIPEQVLLIKVTRLADHMGKTTGFVLVFYDVTQVVSQEVAASEPPSTSVRLTRIPMVANHKVAFVDTQDVLCLESQAHSTRILTRDGFHFCNLSIGDLESRLDPEQFMRIHRCFIVNLQGVAELGREGSKTHVVLKGKNKEPVPVARGDVLRLRKALGLLSRH (SEQ ID NO: 4).

[0173] In particular non-limiting examples, the RcoM protein is at least 90% identical to SEQ ID NO: 4 and contains one or more amino acid substitutions as described above for the RcoM-1 homolog from P. xenovorans (see Figure 6 ) for alignment).

[0174] In some embodiments, the recombinant RcoM protein includes a tag at the N-terminus, the C-terminus, or both. In some examples, the tag is an affinity tag, such as an affinity tag that facilitates purification of the protein. Any suitable affinity tag can be used, such as one or more of His6, FLAG, glutathione S-transferase (GST), influenza hemagglutinin (HA), c-Myc, maltose binding protein (MBP), protein A, or protein G. In particular examples, the affinity tag is a His6 tag. In some examples, the affinity tag is cleavable. In particular examples, the cleavage tag includes a cleavage site from TEV having the amino acid sequence ENLYFQ[G / S] (SEQ ID NO: 5). In other particular examples, the cleavage tag includes a cleavage site from thrombin having the amino acid sequence LVPRGS (SEQ ID NO: 6).

[0175] In some embodiments, the recombinant RcoM protein does not include a tag.

[0176] In some embodiments, the recombinant RcoM protein is in an oxidized form (heme with Fe(ll) bound to CO is oxidized to Fe(lll) in RcoM). 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 naphthoquinone. In some examples, an electron mediator (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 Fe(ll) bound to CO can be exposed to white light (e.g., by exposure to an incandescent bulb such as a halogen lamp) using a fiber optic or heat sink screen with an intensity of 0.15 W / cm2to 140 W / cm2for about 1 to 12 hours. Similar methods are described in 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). 2 2 RcoM with Fe(ll) bound to CO can be exposed to white light (e.g., by exposure to an incandescent bulb such as a halogen lamp) using a fiber optic or heat sink screen with an intensity of 0.15 W / cm2to 140 W / cm2for about 1 to 12 hours. Similar methods are described in 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).

[0177] V. Pharmaceutical Compositions

[0178] The recombinant RcoM proteins described herein can be administered as isolated proteins or as part of a pharmaceutical composition. Accordingly, provided herein are pharmaceutical compositions comprising a recombinant RcoM or a derivative thereof disclosed herein, and one or more pharmaceutically acceptable excipients, and optionally comprising one or more additional 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 can be used as appropriate and as understood in the art. The pharmaceutical compositions disclosed herein can be prepared in any of the ways known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0179] ​In some embodiments, pharmaceutical compositions are disclosed that include one or more recombinant RcoM proteins disclosed herein, along with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. The excipient(s) / carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient thereof. Proper formulation of the pharmaceutical composition depends on the selected route of administration. Any of the known techniques and excipients can be used as appropriate and as understood in the art. In some embodiments, the composition includes one or more of the following excipients: N-acetyl cysteine, sodium citrate, glycine, histidine, glutamic acid, sorbitol, maltose, mannitol, trehalose, lactose, glucose, raffinose, dextrose, dextran, sucrose, gelatin, hydroxyethyl starch, benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, m-cresol, myristyl gamma-picolinium chloride, methyl paraben, propyl paraben, 2-pentyl alcohol, phenylmercuric nitrate, thiomersal, sodium acetone bisulfite, argon, ascorbic acid palmitate, ascorbate (sodium / acid), sodium bisulfite, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), cysteine / cysteine hydrochloride, sodium dithionite (sodium bisulfite, sodium hyposulfite), gentisic acid, gentisic acid ethanolamine, monosodium glutamate, glutathione, formaldehyde sodium sulfite, potassium metabisulfite, sodium metabisulfite, methionine, monothioglycerol (thioglycerol), nitrogen, propyl gallate, sodium sulfite, tocopherol alpha, alpha tocopherol succinate hydrogen salt, and sodium mercaptoacetate. Other excipients are encompassed by the present disclosure, including any of the excipients disclosed in Pramanick et al., Pharma Times 45(3):65-77, 2013, which is incorporated by reference herein.

[0180] In some embodiments, the RcoM protein of the pharmaceutical composition is pegylated, polymerized, or cross-linked.

[0181] In some embodiments, the pharmaceutical composition further comprises a native or recombinant globin molecule, such as native or recombinant hemoglobin or neuroglobin, or comprises a hemoglobin-based oxygen carrier (HBOC). In some examples, the HBOC includes DCLHb (HEMASST TM ; Baxter), MP4 (HEMOSPAN TM ; Sangart), pyridoxalated Hb POE-conjugate (PHP) + catalase & SOD (Apex Biosciences), O-R-PolyHbA0 (HEMOLINK TM ; Hemosol), PolyBvHb (HEMOPURE TMBiopure), PolyHb (POLYHEME TM rHb1.1 (OPTRO TM Somatogen), PEG-hemoglobin (Enzon), OXYVITA TM or HBOC-201, or any combination thereof.

[0182] The pharmaceutical compositions disclosed herein can be administered by a variety of routes, depending on whether local or systemic treatment is desired and on the area to be treated.

[0183] The pharmaceutical compositions include those suitable for parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary) or intraperitoneal administration, although the most suitable route can depend on, for example, the condition and disorder of the recipient. The parenteral compositions can be formulated for injection or infusion, or for intracranial, e.g., intrathecal or intraventricular administration. The parenteral compositions can be in the form of a single bolus dose or can be in the form of an infusion over a relatively short or relatively long period of time. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. In some embodiments, the compounds can be included in such pharmaceutical compositions in combination with pharmaceutically acceptable diluents, fillers, disintegrating agents, binding agents, lubricants, surface active or wetting agents, hydrophilic or hydrophobic carriers, emulsifiers, buffers, moistening agents, preservatives, humectants, solubilizers, antioxidants, salts, and the like, used in connection with car- rying or delivery of drugs. The skilled artisan can consult various pharmacological references for guidance. For example, reference can be made to Modern Pharmaceutics, 5th Edition, Banker & Rhodes, CRC Press (2009); and Goodman & Gilman, The Pharmaceutical Basis of Therapeutics, 13th Edition, McGraw Hill, New York (2018). The compositions can conveniently be presented in unit dosage form and can be prepared by any methods known in the art of pharmacy. In general, such methods include the step of bringing into association the isolated recombinant RcoM molecule or derivative thereof disclosed herein (“active ingredient”) with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product into the desired composition.

[0184] The recombinant RcoM protein can be formulated for parenteral administration by injection. Injection compositions can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The pharmaceutical compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. The composition can be present in a unit dosage or multi-dose container, for example, sealed ampoules and vials, and can be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, physiological saline or sterile pyrogen-free water upon immediate use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

[0185] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions of the active compound which can contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which can include 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 can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension also can include suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0186] It should be understood that in addition to the ingredients particularly mentioned above, the pharmaceutical compositions described above can include other agents conventional in pharmaceutical preparations, for example those suitable for oral administration can include flavoring agents.

[0187] Unit dosage pharmaceutical compositions are those containing effective dosages or appropriate fractions thereof of the active ingredient as described below. The term "unit dosage form" refers to physically discrete units suitable for unitary dosing to 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.

[0188] The RcoM protein can be effective over a wide dosage range and will typically be administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound being administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0189] In some embodiments, the disclosed recombinant RcoM proteins can be administered at a therapeutically effective dose of about 0.01 g to about 1000 g per day. In some examples, the dose of the 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, about 1 g to about 800, about 1 g to about 700 g, about 1 g to about 600 g, about 1 g to about 500 g, about 1 g to about 400 g, 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 g, 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.

[0190] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. In some embodiments disclosed herein, the pharmaceutical compositions comprise one or more of the disclosed RcoM proteins (as active ingredient) in combination with one or more pharmaceutically acceptable carriers (excipients).

[0191] 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 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%, about 0.05% to about 30%, about 0.05% to about 20%, about 0.05% to about 10%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.5% to about 50%, about 0.5% to about 45%, about 0.5% to about 40%, about 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 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. Particular 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 above represent weight percent of the pharmaceutical composition.

[0192] The amount of recombinant RcoM protein administered to a patient will vary depending on the agent administered, the purpose of the administration, such as prophylaxis or treatment, the state of the patient, the mode of administration, and the like. In therapeutic applications, an amount effective for curing or at least partially arresting the symptoms of a disease and its complications can be administered to a patient already suffering from the disease.

[0193] In some embodiments, the pharmaceutical compositions can be sterilized by conventional sterilization techniques, or can be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation 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. It will be appreciated that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of a pharmaceutically acceptable salt.

[0194] In certain embodiments, the pharmaceutical composition comprises a reducing agent. In some examples, the reducing agent is selected from ascorbic acid, N-acetyl cysteine, sodium dithionite, methylene blue, glutathione, B5 / B5-reductase / NADH, tris(2-carboxyethyl)phosphine, dithiothreitol, or a combination thereof. Other agents with the property of reducing heme-containing molecules can also be used.

[0195] In other particular embodiments, the pharmaceutical composition comprises an oxidizing agent. In 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.

[0196] In certain embodiments, the pharmaceutical composition can be deoxygenated by producing and maintaining the RcoM protein or pharmaceutical composition in an oxygen-free environment.

[0197] VI. Methods of treating CO, H2S, and cyanide poisoning

[0198] The recombinant RcoM proteins disclosed herein (see Section IV) exhibit very high affinity for carbon monoxide. Based on this property, the disclosed RcoM proteins are useful in a variety of in vivo and in vitro methods, including as a detoxifying agent for carbon monoxide poisoning. The use of the disclosed RcoM proteins for treating cyanide and hydrogen sulfide (H2S) poisoning is also described.

[0199] Provided herein are methods of 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 a recombinant RcoM protein disclosed herein. In some embodiments, the method comprises selecting a subject having carboxyhemoglobinemia (carbon monoxide poisoning) prior to administering the RcoM protein or a 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 in its reduced form. In some examples, the reducing agent comprises sodium dithionite, ascorbic acid, N-acetyl cysteine (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.

[0200] Further provided herein is a method of removing carbon monoxide from native hemoglobin, myoglobin, or mitochondria (i.e., from cytochrome c oxidase in mitochondria) in a subject's blood or tissue by contacting the subject's blood or tissue with a recombinant RcoM protein or a pharmaceutical composition disclosed herein. In some embodiments, the method comprises selecting a subject having carboxyhemoglobinemia (carbon monoxide poisoning) prior to contacting the subject's blood or tissue with the disclosed RcoM protein or a 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 in its reduced form. In some examples, the reducing agent comprises sodium dithionite, ascorbic acid, N-acetyl cysteine (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.

[0201] Also provided herein is a method of removing hydrogen sulfide from native hemoglobin, myoglobin, or mitochondria (such as from cytochrome c oxidase in mitochondria) 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 the step of selecting a subject having hydrogen sulfide poisoning prior to contacting the subject's blood or tissue with the RcoM protein or pharmaceutical composition. Further provided is a method of 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 method further comprises the step of selecting a subject having hydrogen sulfide poisoning prior to administering the RcoM protein or pharmaceutical composition. In some embodiments of these methods, the RcoM protein is in its reduced form. Examples of reducing agents included in the pharmaceutical composition include, but are not limited to, sodium dithionite, ascorbic acid, N-acetyl cysteine (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.

[0202] Further provided herein is a method of removing cyanide from native hemoglobin, myoglobin, or mitochondria (such as from cytochrome c oxidase in mitochondria) 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 the step of selecting a subject having cyanide poisoning prior to contacting the subject's blood or tissue with the RcoM protein or pharmaceutical composition. Also provided is a method of 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 the step of selecting a subject having cyanide poisoning prior to administering the RcoM protein or pharmaceutical composition. In some embodiments of these methods, the RcoM protein is in its oxidized form. In some examples, the oxidizing agent includes an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

[0203] In some embodiments of the in vivo methods disclosed herein, the RcoM protein or pharmaceutical composition is administered intravenously or intramuscularly. In some examples, the RcoM protein or pharmaceutical composition is administered by intravenous infusion, intraperitoneal injection, or intramuscular injection.

[0204] In some embodiments, the RcoM protein is administered alone or as part of a pharmaceutical composition at a dose of about 0.1 to about 300 g per day. Other dosage ranges are described in Section V above.

[0205] Also provided herein are in vitro methods of removing carbon monoxide from hemoglobin, myoglobin, or mitochondria (e.g., from cytochrome c oxidase in mitochondria) in blood or animal tissue, 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 in its reduced form.

[0206] Further provided herein are in vitro methods of removing hydrogen sulfide from hemoglobin, myoglobin, or mitochondria (e.g., from cytochrome c oxidase in mitochondria) in blood or animal tissue, 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 in its reduced form.

[0207] Also provided herein are in vitro methods of removing cyanide from hemoglobin, myoglobin, or mitochondria (e.g., from cytochrome c oxidase in mitochondria) in blood or animal tissue, comprising contacting the blood or animal tissue with an effective amount of a recombinant RcoM protein disclosed herein. In some embodiments, the recombinant RcoM protein is in its oxidized form.

[0208] In some embodiments of the disclosed methods, the recombinant RcoM protein is pegylated, polymerized, or cross-linked.

[0209] VII. Recombinant RcoM as a blood substitute

[0210] The recombinant RcoM proteins disclosed herein are capable of binding and carrying oxygen (see Figure 8 and 15A -15D; Examples 3 and 4). Thus, the use of the disclosed RcoM proteins as a blood substitute is contemplated.

[0211] Provided herein is a method of replacing blood and / or increasing oxygen delivery to tissue in a subject. In some embodiments, the method comprises 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.

[0212] For example, the subject to be treated is any subject in need of increasing blood volume or increasing oxygen delivery to tissues. In some embodiments, the subject has or is at risk of having a disease, condition, or injury associated with a deficiency in red blood cells and / or hemoglobin or a decrease in oxygen delivery to tissues. In some examples, the disease, condition, or injury comprises a bleeding condition, a bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, a burn, an ulcer, an ectopic pregnancy, microcytosis, rhabdomyolysis, a hemoglobinopathy, a globinopathy, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever.

[0213] In some embodiments, the bleeding episode in the subject to be treated with the recombinant RcoM protein is caused by an anticoagulant overdose, an aneurysm, a blood vessel rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or infection.

[0214] In some embodiments, the bleeding condition in the subject to be treated with the recombinant RcoM protein comprises hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, a platelet disorder, a coagulopathy, Favism, thrombocytopenia, vitamin K deficiency, or von Willebrand disease.

[0215] In some embodiments, the anemia in the subject to be treated comprises microcytic anemia, iron deficiency anemia, a heme synthesis defect, a globin synthesis defect, a sideroblastic defect, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of prematurity, Fanconi's anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia.

[0216] In some embodiments, the shock in the subject to be treated comprises septic shock, hemorrhagic shock, or hypovolemic shock.

[0217] In some embodiments, the subject to be treated has or is at risk of having a disease or condition associated with reduced blood flow, such that increased oxygen delivery is beneficial to the treatment of the subject. Examples of diseases or conditions that can be treated using the disclosed methods include, but are not limited to, ischemia, myocardial infarction, stroke, ischemia-reperfusion injury, elevated blood pressure, pulmonary arterial hypertension (including neonatal pulmonary arterial hypertension, primary pulmonary arterial hypertension, and secondary pulmonary arterial hypertension), systemic hypertension, skin ulcers, acute renal failure, chronic renal failure, intravascular thrombosis, ischemic central nervous system events, vasospasm (such as cerebral arterial vasospasm), hemolytic conditions, peripheral vascular disease, trauma, cardiac arrest, general surgery, or organ transplantation.

[0218] In some embodiments, the recombinant RcoM protein is administered intravenously to the subject.

[0219] In some embodiments, the method further comprises administering to the subject a second blood substitute, blood product, or whole blood. In some examples, the second blood substitute comprises a hemoglobin-based oxygen carrier, artificial red blood cell, or oxygen-releasing compound. In some examples, the blood product comprises packed red blood cells, plasma, or serum.

[0220] In some examples, the subject is a human. In other examples, the subject is a non-human animal.

[0221] Also provided are compositions comprising the disclosed RcoM protein and an oxygen carrier such as a native or recombinant globin molecule (such as native or recombinant hemoglobin or neuroglobin) or 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 cross-linked.

[0222] VIII. Embodiments

[0223] Embodiment 1. A recombinant carbon monoxide metabolic regulator (RcoM) protein, wherein the recombinant RcoM protein comprises a heme-binding domain (HBD), and wherein the amino acid sequence of the HBD has at least 90% identity to SEQ ID NO: 2 and comprises an amino acid substitution at one or more of H74, C94, M104, M105, C127, and C130.

[0224] Embodiment 2. The recombinant RcoM protein of Embodiment 1, wherein:

[0225] the substitution at H74 is selected from H74S, H74T, H74M, H74W, H74A, H74L, H74I, H74V, and H74G;

[0226] the substitution at C94 is selected from C94S, C94T, C94H, C94W, C94M, C94A, C94L, C94I, C94V, and C94G;

[0227] the substitution at M104 is selected from M104S, M104T, M104H, M104W, M104A, M104L, M104I, M104V, and M104G;

[0228] the substitution at M105 is selected from M105S, M105T, M105H, M105W, M105A, M105L, M105I, M105V, and M105G;

[0229] the substitution at C127 is selected from C127S, C127T, C127M, C127A, C127L, C127I, C127V, and C127G; and / or

[0230] the substitution at C130 is selected from C130S, C130T, C130M, C130A, C130L, C130I, C130V, and C130G.

[0231] Embodiment 3. The recombinant RcoM protein of Embodiment 1 or Embodiment 2, wherein the amino acid sequence of the HBD has at least 95% identity to SEQ ID NO: 2 and comprises an amino acid substitution at one or more of C94, M104, C127, and C130.

[0232] Embodiment 4. The recombinant RcoM protein of any one of Embodiments 1 to 4, wherein the HBD comprises:

[0233] a C94S substitution;

[0234] a C127S substitution and a C130S substitution;

[0235] a C94S substitution, a C127S substitution, and a C130S substitution;

[0236] a M104A substitution, a C127S substitution, and a C130S substitution;

[0237] a M104H substitution, a C127S substitution, and a C130S substitution;

[0238] a M104L substitution, a C127S substitution, and a C130S substitution;

[0239] a C94S substitution, a M104A substitution, a C127S substitution, and a C130S substitution;

[0240] C94S replacement, M104H replacement, C127S replacement, and C130S replacement; or

[0241] C94S, M104L, C127S, and C130S are all replacements.

[0242] Implementation Scheme 5. The recombinant RcoM protein as described in any one of Implementation Schemes 1 to 4, wherein:

[0243] The amino acid sequence of the RcoM protein contains or consists of the following: 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

[0244] The amino acid sequence of the RcoM protein contains SEQ ID NO:1 or SEQ ID NO:2 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.

[0245] Implementation Scheme 6. The recombinant RcoM protein as described in any one of Implementation Schemes 1 to 5, wherein the RcoM protein comprises an N-terminal tag or a C-terminal tag.

[0246] Implementation Scheme 7. The recombinant RcoM protein as described in Implementation Scheme 6, wherein the tag is an affinity tag.

[0247] Implementation Scheme 8. The recombinant RcoM protein as described in Implementation Scheme 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.

[0248] Implementation Scheme 9. The recombinant RcoM protein as described in any one of Implementation Schemes 6 to 8, wherein the tag is cleavable.

[0249] Implementation Scheme 10. An in vitro method for removing carbon monoxide from hemoglobin, myoglobin, or mitochondria in blood or animal tissue, comprising contacting blood or animal tissue with an effective amount of recombinant RcoM protein of any one of Implementation Schemes 1 to 9, thereby removing carbon monoxide from hemoglobin in blood or animal tissue.

[0250] Implementation Scheme 11. A method for treating carboxyhemoglobinemia in a subject, comprising administering to the subject a therapeutically effective amount of RcoM protein of any one of Implementation Schemes 1 to 9.

[0251] Embodiment 12. The method of embodiment 11, further comprising selecting a subject having carboxyhemoglobinemia prior to administering the recombinant RcoM protein.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] Embodiment 17. The method of embodiment 16, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetyl cysteine (NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

[0257] Embodiment 18. A method of treating cyanide poisoning in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant RcoM protein of any one of embodiments 1 to 9, wherein the RcoM protein is in its oxidized form, thereby treating cyanide poisoning in the subject.

[0258] Embodiment 19. The method of embodiment 18, further comprising selecting a subject having cyanide poisoning prior to administering the recombinant RcoM protein.

[0259] 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.

[0260] 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.

[0261] Embodiment 22. A method of treating hydrogen sulfide (H2S) poisoning in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant RcoM protein of any one of embodiments 1 to 9, wherein the RcoM protein is in its reduced form, thereby treating H2S poisoning in the subject.

[0262] Embodiment 23. The method of embodiment 22, further comprising selecting a subject having H2S poisoning prior to administering the recombinant RcoM protein.

[0263] 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.

[0264] Embodiment 25. The method of embodiment 24, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetyl cysteine (NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), trehalose, dithiothreitol (DTT), or any combination thereof.

[0265] Embodiment 26. A method of replacing blood in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant RcoM protein of any one of embodiments 1 to 9, thereby replacing blood in the subject.

[0266] Embodiment 27. The method of embodiment 26, wherein the subject has or is at risk of having a disease, condition, or injury associated with a deficiency in red blood cells and / or hemoglobin or a reduction in oxygen delivery to tissues.

[0267] Embodiment 28. The method of embodiment 27, wherein the disease, condition, or injury comprises a bleeding condition, a bleeding episode, anemia, shock, ischemia, hypoxia, anoxia, hypoxemia, a burn, an ulcer, an ectopic pregnancy, microcytosis, rhabdomyolysis, a hemoglobinopathy, a globinopathy, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever.

[0268] Embodiment 29. The method of embodiment 28, wherein:

[0269] the bleeding episode is caused by an anticoagulant overdose, an aneurysm, a blood vessel rupture, surgery, trauma, gastrointestinal bleeding, pregnancy, hemorrhage, or an infection;

[0270] The bleeding disorder comprises hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, a platelet disorder, a coagulopathy, patelet disease, patelet deficiency, vitamin K deficiency, or von Willebrand's disease;

[0271] The anemia comprises microcytic anemia, iron deficiency anemia, heme synthesis defect, globin synthesis defect, sideroblastic defect, 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

[0272] The shock comprises septic shock, hemorrhagic shock, or hypovolemic shock.

[0273] Embodiment 30. The method of embodiment 26, wherein the subject has or is at risk of having myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary arterial hypertension, or vasospasm.

[0274] Embodiment 31. The method of any one of embodiments 26-30, wherein the recombinant RcoM protein is administered intravenously to the subject.

[0275] Embodiment 32. The embodiment of any one of claims 26-31, wherein the recombinant RcoM protein is pegylated, polymerized, or cross-linked.

[0276] Embodiment 33. The method of any one of embodiments 26-32, further comprising administering to the subject a second blood substitute, blood product, or whole blood.

[0277] Embodiment 34. The method of embodiment 33, wherein the second blood substitute comprises a hemoglobin-based oxygen carrier, artificial red blood cell, or oxygen-releasing compound.

[0278] Embodiment 35. The method of embodiment 33, wherein the blood product comprises packed red blood cells, plasma, or serum.

[0279] Embodiment 36. The method of any one of embodiments 11-35, wherein the subject is a human.

[0280] Embodiment 37. The method of any one of embodiments 11-35, wherein the subject is a non-human animal.

[0281] Embodiment 38. A pharmaceutical composition comprising the recombinant RcoM protein of any one of embodiments 1 to 9 and a pharmaceutically acceptable carrier.

[0282] Embodiment 39. The pharmaceutical composition of embodiment 38, further comprising a reducing agent or an oxidizing agent.

[0283] Embodiment 40. The pharmaceutical composition of embodiment 39, wherein the reducing agent comprises sodium dithionite, ascorbic acid, N-acetyl cysteine (NAC), methylene blue, glutathione, cytochrome b5 / b5-reductase, hydralazine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or any combination thereof.

[0284] 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.

[0285] Embodiment 42. The pharmaceutical composition of any one of embodiments 38 to 41, wherein the recombinant RcoM protein is pegylated, polymerized, or cross-linked. Example

[0286] Example 1: Transfer of CO from HbCO to RcoM-1 in an aerobic environment

[0287] Hemoglobin-CO (Hb-CO) transfer kinetics were evaluated 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 of the different ligand-bound species of RcoM-1 and hemoglobin. The concentrations of Hb-CO and Fe(II)RcoM-1 were 20 μΜ and the experiments were performed in triplicate. The data for hemoglobin-CO loss were fit to a bi-exponential curve, showing a slow phase half-life (t 1 / 2 ) of 1.4 seconds. The data for Fe(II)-CO RcoM increase were fit to a single exponential curve, showing a half-life of 0.93 seconds. The results are shown in Figure 4 . This data demonstrates that RcoM has equally high affinity for CO and allows for the rapid and efficient transfer of CO from Hb to RcoM.

[0288] Example 2: Transfer of CO from Hb-CO to RcoM-1 in an anaerobic environment

[0289] Hemoglobin-CO turnover kinetics in the presence of WT, full-length RcoM-1 (SEQ ID NO: 1) was measured using UV-Vis spectroscopy under anaerobic conditions at 37°C. The concentrations of Hb-CO and Fe(II) RcoM-1 were 15 μΜ and 15.8 μΜ, respectively. The absorbance changes at 530, 562, and 583 nm were tracked for the transition from Fe(II) to Fe(II)-CO RcoM, and a single exponential curve was fit, showing a half-life of 50 seconds. The results are shown in Figure 5 Figure 2. These results indicate that RcoM-1 is able to scavenge CO from Hb-CO species and, therefore, can be used as a CO scavenger in vivo.

[0290] Example 3: Characterization of truncated RcoM with substitution of C94C (HBD C94S)

[0291] This example describes studies to characterize a modified RcoM protein that lacks the PAS domain and has a C94S substitution (SEQ ID NO: 7). The results of these studies indicate that altering the heme binding residues can change the gas binding properties of RcoM.

[0292] Stability

[0293] 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 (~480 mM heme, compared to ~130 mM for WT RcoM). HBD C94S can also be reduced using dithionite in the absence of a stabilizing reductant (e.g., DTT, TCEP). Furthermore, both the oxidized and reduced forms of RcoM are stable to aggregation when stored at 4°C for more than 1 week. Studies indicate that the T m of 90°C (recorded in a septum-sealed cuvette under anaerobic conditions at a RcoM concentration of 7 μΜ). WT RcoM-1 irreversibly unfolded at about 40°C under the same conditions.

[0294] Comparison of UV-Vis spectra

[0295] The spectra of full-length wild-type RcoM-1 and HBD C94S RcoM were evaluated. The spectra of iron (Fe(III), deoxy ferrous (Fe(II)), and ferrous-CO species (Fe(II)-CO) were determined. The maximum peak wavelength (nm) was calculated for each species, as well as the estimated molar absorptivity (mM -1 cm -1 ) for each peak. The results are shown in Figure 7As expected, the Fe(II) and Fe(II)-CO spectra of the WT and HBD C94S RcoM proteins are very similar. However, the Fe(III) spectra look very different, indicating that the Fe(III) heme coordination environment has changed due to C94S substitution.

[0296] Another study provided evidence of stable O2 adducts in HBD C94S. HBD C94S was reduced with excess dithionite to produce ferrous Fe(II) compounds. The reduced HBD C94S was then desalted and UV-Vis samples were prepared under microaerobic conditions. After recording the UV-Vis spectra under microaerobic conditions, the cuvette lid was removed to introduce air, and the spectra were recorded again, showing the oxygen-bound compounds (RcoM concentration = 8 μM). Figure 8 The visible spectra of ferrous (Fe(II)) in the presence of a reducing agent, desalted Fe(II), Fe(II) after exposure to air, and reoxidized Fe(III) are shown.

[0297] HBD C94S RcoM-CO binding and dissociation rates

[0298] The reaction kinetics of the heme-binding domain (HBD) of HBD C94S RcoM with carbon monoxide (CO) were determined using a stop-flow technique. Figure 9 This study was conducted at RcoM concentrations of 10 μM, CO concentrations ranging from 55 to 287 μM, and temperatures of 37 °C. The calculated rate at different CO concentrations was 1.2 × 10⁻⁶. 5 M -1 s -1 The reaction binding rate (k on Similar values ​​were obtained for the wild-type full-length protein. Therefore, the CO binding rate was not affected by C94S substitution.

[0299] The CO dissociation rate of HBD C94S was determined using a concentration of 10 μM RcoM, 2 mM nitric oxide (NO) (generated using 1 mM ProliNONOate), and a temperature of 37 °C. The reaction was monitored by changes in absorbance as the ferrous-CO complex dissociated in the presence of NO. Upon CO dissociation, NO binds to heme, causing a change in the absorption spectrum. Excess NO prevents CO from recombinizing with heme. The time course of absorbance changes allowed for the determination of a dissociation rate of 4.9 × 10⁻⁶. -2 s -1 ( Figure 10 ).

[0300] Thermolytic folding of HBD C94S

[0301] Unfolding was monitored by changes in absorbance at the heme Soret maximum at 420 nm. Figure 11 The samples were allowed to equilibrate for 5 minutes at each temperature prior to recording each UV-Vis spectrum. Small losses in Soret intensity observed between 20 °C and 75 °C can be due to changes in heme coordination number. Losses in Soret intensity between 75 °C and 98 °C are attributed to thermal unfolding leading to loss of heme from the protein. UV-Vis spectra of Fe(III) HBD RcoM-1 carrying the C94S mutation were recorded at each temperature between 20 °C and 98 °C. The T m was determined to be 91 °C.

[0302] Example 4: RcoM heme-binding domain (HBD) variants

[0303] This example describes the generation and characterization of several truncated RcoM HBD variants.

[0304] Eight RcoM variants were generated. The variants listed in Table 4 have been successfully cloned, expressed in E. coli, and purified to homogeneity. These variants encompass the heme-binding domain (HBD) from RcoM-1 from Paraburkholderia xenovorans and have various mutations at one or more of residues C94, M104, C127, and C130 to enhance solubility, stability, and CO scavenging properties. The expressed variants also include a C-terminal 6-His tag. With the 6-His tag, the variants are 17 kDa.

[0305] Table 4. RcoM HDB16 variants

[0306]

[0307]

[0308] Electronic absorption (UV-Vis) spectra of RcoM HBD WT and variants are shown in Figures 12A-12D , 13A-13B, and 14A-14C. Figure 13C A schematic showing the protein-derived ligand-switch mechanism of RcoM highlighting the coordination sphere changes in the M104 variants. Figure 14D A schematic showing the protein-derived ligand-switch mechanism of RcoM highlighting the coordination sphere changes in the CCC M104 variants.

[0309] Quantification of oxygen binding affinity (P 50 ) in RcoM HBD truncates is shown in Figures 15A-15DUsing an intraocular tonometer equipped with optical cuvettes, the fraction of oxygen-bound hemoglobin was measured as a function of oxygen partial pressure using UV-Vis spectroscopy. Representative spectral changes in the UV-Vis characteristics of the CC HBD RcoM variant were used as the oxygen partial pressure (P). O2 The function is shown in Figure 15A The oxygen binding curves of CCHBD, C94S HBD, and CCC HBD are shown in the figure. Figures 15B-15D The second-order rate constants (kJ / kJ) of CO binding with RcoM WT HBD and HBD variants CC HBD, C94S HBD and CCC HBD were determined. on ,CO)( Figures 16A-16D The CO binding rate at each CO concentration was measured using stopped UV-Vis spectroscopy and fitted to a single exponent. Linear regression was applied to each curve, and the second-order rate constant was estimated as the slope. The results are as follows:

[0310] RcoM variants k on,CO ]]> WT HBD 4.0 x 10 4 M -1 s -1 ]]> CC HBD 4.4 x 10 4 M -1 s -1 ]]> C94S HBD 2.8 x 10 4 M -1 s -1 ]]> CCC HBD 4.1 x 10 4 M -1 s -1 ]]>

[0311] The auto-oxidation rate (k) of WT HBD RcoM oxid The value was determined to be 0.87h. -1 . Figure 17A Reference spectra of Fe(III) and Fe(II)-O2 proteins are shown. Spectral variations of the UV-Vis characteristics of the Fe(II)-O2 WT HBD are shown in [the diagram]. Figure 17B In the middle, the spectral changes at 542 nm and 573 nm were fitted to a single exponent to determine k. oxid ( Figure 17C ). Figure 18 A table summarizing the ligand binding parameters and heme stability properties of WT RcoM and RcoM HBD variants C94S, CC HBD, and CCC HBD is provided.

[0312] The unfolding of Fe(III)CCC HBD RcoM in the presence of urea (0M, 4M, and 8M urea) was evaluated. Unfolding was monitored by changes in absorbance at the heme Solvay maximum at 415 nm. Samples were equilibrated for 10 minutes before recording each UV-Vis spectrum. Figure 19A The unfolded data were fitted with an S-shaped curve to determine the denaturant concentration at which half of the protein sample unfolded ([D]). 50 ()( Figure 19B ). CCC HBD's [D] 50 It is 4.6M.

[0313] 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 mM hydrogen peroxide at pH 7.4, 25 °C, and monitored by UV-Vis spectroscopy every 2 minutes for 30 minutes ( Figures 20A-20D ). Minimal spectral changes were observed for each variant, indicating that hydrogen peroxide does not react with the Fe(III) heme center of RcoM HBD truncates to produce highly oxidized species.

[0314] Nitrite reduction was assessed for full-length and HBD-truncated RcoM variants. Diron proteins (10-15 mM) were incubated with 1-5 mM sodium nitrite in the presence of 2.5 mM sodium hydrosulfite at 37 °C. UV-Vis spectroscopy was used to monitor the conversion of Fe(II) heme to Fe(II)-NO ( Figure 21A ). The spectral feature changes at 562 nm and 578 nm were fit to a single exponential curve to determine the observed rate of nitrite reduction. The observed rates were plotted as a function of nitrite concentration, a linear regression was applied to each plot, and the second-order rate constant was estimated as the slope ( Figures 21B-21C ).

[0315] Additional studies were performed to assess the CO scavenging ability of RcoM. Kinetic traces were developed for the transfer of CO from hemoglobin (Hb) to WT RcoM HBD and RcoM HBD variants CC HBD, C94S HBD, and CCC HBD in vitro under aerobic conditions at 37 °C. CO-bound Hb (20 mM) was incubated with an equimolar amount of oxygenated iron RcoM, and CO transfer from Hb to RcoM was monitored using UV-Vis spectroscopy. The fraction of each CO-bound hemoglobin was determined using spectral deconvolution, and the corresponding kinetic traces were fit to a single or double exponential equation. The half-lives of each CO-bound species are shown in Figures 22A-22D , curves fit to a double exponential show a fast species half-life and amplitude. Figures 23A-23B Kinetic traces are shown for the monitoring of CO transfer from RBC-encapsulated HbCO to extracellular RcoM HBD truncates under aerobic conditions at 37 °C. Hemoglobin was incubated at equimolar concentrations (50-100 mM), and RBCs were separated from extracellular RcoM by centrifugation at each time point. CO transfer from Hb to WT HBD RcoM ( Figure 23A ) and C94S HBD RcoM ( Figure 23BCO transfer. The fraction of each CO-bound hemoglobin was determined using spectral deconvolution, and the corresponding kinetic traces were fit to a single exponential equation. The half-life of COHb in the presence of WT HBD and C94S HBD was 24 ± 6 s and 23 ± 5 s, respectively.

[0316] These results show that RcoM HBD variants rapidly clear CO from RBC-encapsulated Hb under aerobic conditions, similar to what can occur in vivo during acute CO poisoning. The RcoM HBD variants are selective for CO over oxygen, as CO is transferred from HbCO under aerobic conditions.

[0317] Example 5: Toxicity screening of RcoM HBD variants in mice

[0318] Recombinantly expressed RcoM truncates were introduced into healthy mice via tail vein catheter at a concentration of 1 mM or 10 mM and an injection volume of 10 μL / g body weight. Behavior, including nest building, was monitored for 48 hours, followed by sacrifice and collection of blood for toxicology evaluation. Results are shown in Table 5. Blood chemistry results for liver function (AST and ALT) and kidney function (BUN and creatinine) for all mice treated with RcoM truncates were comparable to those of control mice given phosphate buffered saline (PBS). These results show that intravenous infusion of RcoM truncates does not cause organ-specific toxicity in mice.

[0319] Table 5. Toxicity screening results

[0320]

[0321]

[0322] Example 6: In vivo RcoM CO clearance

[0323] The ability of C94S and CCC HBD RcoM variants to clear CO from HbCO was evaluated in a lethal CO poisoning mouse model. Anesthetized, mechanically ventilated mice were exposed to 3,000 ppm CO in air for 4.5 minutes, followed by intravenous infusion of Fe(II)-02 CCC HBD RcoM at an injection volume of 10 μL / g body weight (hemoglobin concentration as in Example 4). The mice were then returned to room air and monitored for survival. Figure 24Blood samples (15 μί) were taken immediately before and after infusion, as well as 25 minutes after CO exposure. At each time point, RBC were separated from plasma by centrifugation, and the separated RBC pellet and plasma samples were immediately frozen at -80°C. Subsequently, the fraction of CO-bound hemoglobin from RBC (%HbCO) and the fraction of CO-bound RcoM (%RcoM-CO) were determined using spectral deconvolution. Infusion of RcoM resulted in a greater reduction in the fraction of CO-bound Hb (Δ%HbCO) Figure 24 than infusion of PBS, indicating that RcoM is able to clear CO in vivo.

[0324] In view of the many possible embodiments to which the principles of the disclosed subject matter can be applied, it should be recognized that the examples described herein are merely illustrative of the present disclosure and should not be considered as limiting the scope of the present disclosure. Rather, the scope of the present disclosure is defined by the following claims. Thus, we

Claims

1. A recombinant carbon monoxide metabolism regulatory factor protein, wherein the recombinant carbon monoxide metabolism regulatory factor protein comprises a heme-binding domain, and wherein the amino acid sequence of the recombinant carbon monoxide metabolism regulatory factor protein is composed of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:

14.

2. The recombinant carbon monoxide metabolism regulatory factor protein according to claim 1, wherein the recombinant carbon monoxide metabolism regulatory factor protein further comprises an N-terminal tag or a C-terminal tag.

3. The recombinant carbon monoxide metabolism regulator protein according to claim 2, wherein the tag is an affinity tag.

4. The recombinant carbon monoxide metabolism regulator protein according to claim 3, 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.

5. The recombinant carbon monoxide metabolism regulator protein according to claim 2, wherein the tag is cleavable.

6. 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 carbon monoxide metabolism regulator protein of claim 1, thereby removing carbon monoxide from the hemoglobin in the blood or animal tissue.

7. Use of the recombinant carbon monoxide metabolism regulator protein of claim 1 in the preparation of a medicament for treating carboxyhemoglobinemia in a subject.

8. The use according to claim 7, further comprising selecting a subject with carboxyhemoglobinemia prior to administration of the recombinant carbon monoxide metabolism regulator protein.

9. The use according to claim 7, wherein the object 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 its blood.

10. The use according to claim 7, wherein the recombinant carbon monoxide metabolism regulator protein is administered by intravenous infusion, intraperitoneal injection or intramuscular injection.

11. The use according to claim 7, wherein the recombinant carbon monoxide metabolism regulator protein is administered as a pharmaceutical composition comprising a reducing agent.

12. The use according to claim 11, 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.

13. Use of the recombinant carbon monoxide metabolism regulator protein of claim 1 in the preparation of a medicament for treating cyanide poisoning in a subject, wherein the recombinant carbon monoxide metabolism regulator protein is in its oxidized form, thereby treating cyanide poisoning in the subject.

14. The use according to claim 13, further comprising selecting a subject suffering from cyanide poisoning prior to administering the recombinant carbon monoxide metabolism regulator protein.

15. The use according to claim 13, wherein the recombinant carbon monoxide metabolism regulator protein is administered as a pharmaceutical composition comprising an oxidant.

16. The use according to claim 15, wherein the oxidant comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, or any combination thereof.

17. Use of the recombinant carbon monoxide metabolism regulator protein of claim 1 in the preparation of a medicament for treating hydrogen sulfide (H2S) poisoning in a subject, wherein the recombinant carbon monoxide metabolism regulator protein is in its reduced form, thereby treating H2S poisoning in the subject.

18. The use according to claim 17, further comprising selecting subjects suffering from H2S poisoning prior to administration of the recombinant carbon monoxide metabolism regulator protein.

19. The use according to claim 17, wherein the recombinant carbon monoxide metabolism regulator protein is administered as a pharmaceutical composition comprising a reducing agent.

20. The use according to claim 19, 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.

21. Use of the recombinant carbon monoxide metabolism regulator protein of claim 1 in the preparation of a medicament for replacing the blood of a recipient, wherein: The subject suffers from a disease, condition, or injury associated with a deficiency of red blood cells and / or hemoglobin or with reduced oxygen delivery to tissues, or is at risk of developing a disease, condition, or injury associated with a deficiency of red blood cells and / or hemoglobin or with reduced oxygen delivery to tissues, wherein the disease, condition, or injury includes bleeding disorders, bleeding episodes, anemia, shock, ischemia, hypoxia, hypoxia, hypoxemia, burns, ulcers, ectopic pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathies, spherocytosis, hemolytic uremic syndrome, thalassemia, disseminated intravascular coagulation, stroke, or yellow fever; or The subjects had myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary hypertension, or vasospasm, or were at risk of having myocardial infarction, stroke, ischemia-reperfusion injury, pulmonary hypertension, or vasospasm.

22. The use according to claim 21, wherein: The bleeding episodes were caused by anticoagulant overdose, aneurysm, ruptured blood vessel, surgery, trauma, gastrointestinal bleeding, pregnancy, bleeding, or infection; The bleeding disorders include hemophilia A, hemophilia B, hemophilia C, factor VII deficiency, factor XIII deficiency, platelet disorders, coagulation disorders, favism, thrombocytopenia, vitamin K deficiency, or Villebrand disease. The anemias mentioned include microcytic anemia, iron deficiency anemia, heme synthesis deficiency, globin synthesis deficiency, sideroblast deficiency, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, premature infant anemia, Fanconi anemia, hereditary spherocytosis, sickle cell anemia, warm autoimmune hemolytic anemia, or cold agglutinin hemolytic anemia; or Shock includes septic shock, hemorrhagic shock, or hypovolemic shock.

23. The use according to claim 21, wherein the recombinant carbon monoxide metabolism regulator protein is administered intravenously to the subject.

24. The use according to claim 21, wherein the recombinant carbon monoxide metabolism regulator protein is polyethylene glycol-modified, polymerized, or cross-linked.

25. The use according to claim 21, wherein the drug further comprises a second blood substitute, blood product or whole blood.

26. The use according to claim 25, wherein the second blood substitute comprises a hemoglobin-based oxygen carrier, artificial red blood cells, or an oxygen-releasing compound.

27. The use according to claim 25, wherein the blood product comprises packed red blood cells, plasma or serum.

28. The use according to claim 7, wherein the object is a person.

29. The use according to claim 7, wherein the object is a non-human animal.

30. A pharmaceutical composition comprising the recombinant carbon monoxide metabolism regulator protein of claim 1 and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition according to claim 30, further comprising a reducing agent or an oxidizing agent.

32. The pharmaceutical composition according to claim 31, 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.

33. The pharmaceutical composition according to claim 31, wherein the oxidant comprises an oxygen-containing gas mixture, an oxygen-containing liquid mixture, a ferricyanide salt, a quinone, or any combination thereof.

34. The pharmaceutical composition of claim 30, wherein the recombinant carbon monoxide metabolism regulator protein is polyethylene glycolated, polymerized, or cross-linked.

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