βcysteine 93 residue plays a key role in oxygen (O2)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O2 affinity, cooperativity, and Hb’s sensitivity to changes in pH. In recent years, βCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. βCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe4+), a higher oxidation state of Hb is formed together with its protein radical (.HbFe4+). This radical migrates to βCys93 and interacts with other “hotspot” amino acids that are highly susceptible to oxidative modifications. Oxidized βCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield βCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.
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Dickerson RE and Geis I Hemoglobin: structure, function, evolution, and pathology. New York: Benjamin-Cummings Publishing Company, 1983.
Alayash AI. Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants? Nat Biotech. 1999;17:545–9.
Perutz MF. Stereochemistry of cooperative effects in haemoglobin: haem-haem interaction and the problem of allostery. Nature. 1970;228:726–39.
Kan HI, Chen IY, Zulfajri M, Wang CC. Subunit disassembly pathway of human hemoglobin revealing the site-specific role of its cysteine residues. J Physical Chem. 2013;117:9831–9.
Cheng Y, Shen TJ, Simplaceanu V, Ho C. Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine. Biochemistry. 2002;41:11901–13.
Alayash AI. Mechanisms of toxicity and modulation of hemoglobin-based oxygen carriers. Shock. 2019;52:41–49.
Khan I, Dantsker D, Samuni U, Friedman AJ, Bonaventura C, Manjula B, et al. Beta 93 modified hemoglobin: kinetic and conformational consequences. Biochemistry. 2001;40:7581–92.
Boykins RA, Buehler PW, Jia Y, Venable R, Alayash AI. O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification. Proteins. 2005;59:840–55.
Lancaster, JR. Historical origins of the discovery of mammalian nitric oxide (nitrogen monoxide) production/physiology/pathophysiology. Biochem Pharmacol. 2020;176:113793. https://doi.org/10.1016/j.bcp.2020.113793.
Gardner PR, Gardner AM, Brashear WT, Suzuki T, Hvitved AN, Setchell KD, et al. Hemoglobins dioxygenate nitric oxide with high fidelity. Inorg Biochem. 2006;100:542–50.
Sharma VS, Traylor TG, Gardiner R, Mizukami H. Reaction of nitric oxide with heme proteins and model compounds of hemoglobin. Biochemistry. 1987;26:3837–43.
Alayash AI, Fratantoni JC, Bonaventura C, Bonaventura J, Cashon RE. Nitric oxide binding to human ferrihemoglobins cross-linked between either alpha or beta subunits. Arch Biochem Biophys. 1993;303:332–8.
Alayash AI, Cashon RE. Hemoglobin and free radicals: implications for the development of a safe blood substitute. Mol Med Today. 1995;1:122–7.
Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature. 1996;380:221–6.
Premont RT, Stamler JS. Essential role of hemoglobin βCys93 in cardiovascular physiology. Physiology. 2020;35:234–43.
Minneci PC, Deans KJ, Shiva S, Zhi H, Banks SM, Kern S, et al. Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis. Am J Physiol. 2008;295:H743–H754.
Winslow RM, Intaglietta M. SNO job; red cell age and loss of function: advance or SNO-job? Transfusion. 2008;48:411–4.
Buehler PW, Karnaukhova E, Gelderman MP, Alayash AI. Blood aging, safety, and transfusion: capturing the “radical” menace. Antioxid Redox Signal. 2011;14:1713–28.
Roche CJ, Cassera MB, Dantsker D, Hirsch RE, Friedman JM. Generating S-nitrosothiols from hemoglobin: mechanisms, conformational dependence, and physiological relevance. J Biol Chem. 2013;288:22408–25.
Eich RF, Li T, Lemon DD, Doherty DH, Curry SR, Aitken JF, et al. Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry. 1996;35:6976–83.
Doctor A, Gaston B, Kim-Shapiro DB. Detecting physiologic fluctuations in the S-nitrosohemoglobin micropopulation: Triiodide versus 3C. Blood. 2006;108:3225–7.
Gell DA. Structure and function of haemoglobins. Blood Cells Mol Dis. 2018;70:13–42.
Hrinczenko BW, Schechter AN, Wojtkowski TL, Pannell LK, Cashon RE, Alayash AI. Nitric oxide-mediated heme oxidation and selective β-globin nitrosation of hemoglobin from normal and sickle erythrocytes. Biochem Biophys Res Comm. 2000;275:962–7.
Winterbourn CC, Carrell RW. Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93. Biochem J. 1977;165:141–8.
Balagopalakrishna C, Abugo OO, Horsky J, Manoharan PT, Nagababu E, Rifkind JM. Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical. Biochemistry. 1998;37:13194–202.
Shikama K. A controversy on the mechanism of autoxidation of oxymyoglobin and oxyhaemoglobin: oxidation, dissociation, or displacement? Biochem J. 1984;223:279–80.
Vitturi DA, Sun CW, Harper VM, Thrash-Williams B, Cantu-Medellin N, Chacko BK, et al. Antioxidant functions for the hemoglobin β93 cysteine residue in erythrocytes and in the vascular compartment in Vivo. Free Radic Biol Med. 2013;55:119–29.
Jia Y, Buehler PW, Boykins RA, Venable RM, Alayash AI. Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway. J Biol Chem. 2007;282:4894–907.
Momozono A, Kodera Y, Sasaki S, Nakagawa Y, Konno R, Shichiri M. Oxidised Met147 of human serum albumin is a biomarker of oxidative stress, reflecting glycaemic fluctuations and hypoglycaemia in diabetes. Scientific Reports. 2020;10:268 https://doi.org/10.1038/s41598-019-57095-.
Stadtman ER, Van Remmen H, Richardson A, Wehr NB, Levine RL. Methionine oxidation and aging. Biochim. Biophys. Acta. 2005;1703:135–40.
Khor HK, Fisher MT, Schöneich C. Potential role of methionine sulfoxide in the inactivation of the chaperone GroEL by hypochlorous acid (HOCl) and peroxynitrite (ONOO-). J Biol Chem. 2004;279:19486–93.
Bonaventura C, Henkens R, Alayash AI, Banerjee S, Crumbliss AL. Molecular controls of the oxygenation and redox reactions of hemoglobin. Antioxid Redox Signal. 2013;18:2298–313.
Pimenova T, Pereira CP, Gehrig P, Buehler PW, Schaer DJ, Zenobi RJ. Quantitative mass spectrometry defines an oxidative hotspot in hemoglobin that is specifically protected by haptoglobin. Journal of Proteome Res. 2010;9:4061–70.
Strader MB, Alayash AI. Exploring oxidative reactions in hemoglobin variants using mass spectrometry: lessons for engineering oxidatively stable oxygen therapeutics. Antioxid Redox Signal. 2017;26:777–93.
Kassa T, Strader MB, Nakagawa A, Zapol WM, Alayash AI. Targeting βCys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects. Metallomics. 2017;9:1260–70.
Meng F, Kassa T, Strader MB, Soman J, Olson JS, Alayash AI. Substitutions in the β subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation. J Biol Chem. 2019;294:4145–59.
Kettisen K, Strader MB, Wood F, Alayash AI, Bülow L. Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin. Redox Biol. 2018;19:218–25.
Koch CG, Duncan AI, Figueroa P, Dai L, Sessler DI, Frank SM, et al. Real age: red blood cell aging during storage. Ann Thorac Surg. 2019;107:973–80.
Wither M, Dzieciatkowska M, Nemkov T, Strop P, D’Alessandro A, Hansen KC. Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells. Transfusion. 2016;56:421–6.
Blaken GR, Wang Y, Lopez JA, Fu X Cysteine 93 of hemoglobin beta chain is the major target of oxidation during red blood cell storage. Blood 2009; 114: Poster 4040. 10.1182/blood.V114.22.4040.4040.
Alayash AI. Haptoglobin: old protein with new functions. Clin Chim Acta. 2011;412:493–8.
Cooper CE, Schaer DJ, Buehler PW, Wilson MT, Reeder BJ, Silkstone G, et al. Haptoglobin binding stabilizes hemoglobin ferryl iron and the globin radical on tyrosine β145. Antioxid Redox Signal. 2013;18:2264–73.
Mollan TL, Jia Y, Banerjee S, Wu G, Kreulen RT, Tsai AL, et al. Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin. Free Radic Biol Med. 2014;69:265–77.
CB Andersen, et al. Structure of the haptoglobin-haemoglobin complex. Nature. 2012;489:456–9.
Alayash AI, Andersen CB, Moestrup SK, Bülow L. Haptoglobin: the hemoglobin detoxifier in plasma. Trends Biotechnol. 2013;31:2–3.
RP Hebbel, Ney PA, Foker W. Autoxidation, dehydration, and adhesivity may be related abnormalities of sickle erythrocytes. Am J Physiol. 1989;256:C579–C583.
Kassa T, Jana S, Strader MB, Meng F, Jia Y, Wilson MT, et al. Sickle cell hemoglobin in the ferryl state promotes βCys-93 oxidation and mitochondrial dysfunction in epithelial lung cells (E10). J Biol Chem. 2015;290:27939–58.
Kelman DJ, Mason RP. Characterization of the rat hemoglobin thiyl free radical formed upon reaction with phenylhydrazine. Arch Biochem Biophys. 1993;306:439–42.
Scott MD, van den Berg JJ, Repka T, Rouyer-Fessard P, Hebbel RP, Beuzard Y, et al. Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes. J Clin Invest. 1993;91:1706–12.
Strader MB, Kassa T, Meng F, Wood FB, Hirsch RE, Friedman JM, et al. Oxidative instability of hemoglobin E (β26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/β-thalassemia. Redox Biol. 2016;8:363–74.
Kassa T, Wood F, Strader MB, Alayash AI Antisickling drugs targeting βCys93 reduce iron oxidation and oxidative changes in sickle cell hemoglobin. Front Physiol. 2019; 24; 10:931. 10.3389/fphys.2019.00931.
Jana S, Strader MB, Meng F, Hicks W, Kassa T, Tarandovskiy I, et al. Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles. 20183. pii: 120451. 10.1172/jci.insight.120451
Nakagawa A, Ferrari M, Schleifer G, Cooper MK, Liu C, Yu B, et al. A Triazole disulfide compound increases the affinity of hemoglobin for oxygen and reduces the sickling of human sickle cells. Mol Pharm. 2018;15:1954–63.
Abdulmalik O, Safo MK, Chen Q, Yang J, Brugnara C, Ohene-Frempong K, et al. 5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells. Br J Haematol. 2005;128:552–61.
Niihara Y, Zerez CR, Akiyama DS, Tanaka KR. Increased red cell glutamine availability in sickle cell anemia: Demonstration of increased active transport, affinity, and increased glutamate level in intact red cells. J Lab Clin Med. 1997;130:83–90.
I would like to thank past and present members of my laboratory for their valuable contribution to this work. I would like to thank Drs. Tigist Kassa and Sirsendu Jana for assisting in constructing figures included in this article. I would also like to thank Drs. Michael Heaven and Felice D’Agnillo for reading the manuscript. Funding was provided by internal FDA and NIH/NHLBI grants (HL110900).
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Alayash, A.I. βCysteine 93 in human hemoglobin: a gateway to oxidative stability in health and disease. Lab Invest (2020). https://doi.org/10.1038/s41374-020-00492-3