Heme Redox Properties of S-Nitrosated Hemoglobin A0 and Hemoglobin S

Abstract

S-NitrosatedThe reactivity of heme proteins with nitro and nitroso compounds has been under intense scrutiny since Ignarro et al. (1,2) reported the ability of some of these compounds to activate an ␣␤ heterodimer enzyme containing a b-type heme, sGC, involved in the relaxation of the endothelium (3, 4). Nitric oxide (NO) has been observed to react with Fe-porphyrin complexes in various oxidation states (Fe . Reaction of NO with oxygenated heme groups results in heme oxidation and nitrate formation. This process, like NO binding to deoxy-Hb, occurs rapidly and with a similar rate constant (10 -12). As shown in this report, reactions of NO at the sulfhydryl groups of Hb also promote met-Hb formation. Several lines of study have shown that S-nitrosated Hb (SNO-Hb) can be formed in vivo and in vitro, and although present at low concentration, may be of importance in blood pressure regulation. Hb contains a highly conserved cysteine residue at position ␤93 whose reactions with NO may account for its persistence in hemoglobin's evolutionary history. A dynamic cycle of SNO-Hb formation in the lungs and NO release in the tissues was implicated by finding the presence of greater levels of SNO-Hb in aortic relative to venous blood (13). This cycle has, however, been brought into question. Notably, Gladwin and co-workers (14) did not see higher levels of SNO-Hb in aortic blood, even in patients given low levels of NO in breathing gas (4 M NO delivered to 10 mM heme).It is now well established that the formation of SNO-Hb is under allosteric control (13). Functional and crystallographic studies demonstrate that the ␤Cys 93 residues at which NO is bound in SNO-Hb A 0 are more accessible in the high affinity conformation of oxy (R-state) Hb than in deoxy (T-state) Hb (6,15,16). This conformational sensitivity results in a rate dependence for SNO-Hb formation that mirrors the greater relative exposure of ␤Cys 93 in conditions that favor the R-state. Allosteric considerations were also invoked to explain the decreased stability of the deoxy form of SNO-Hb (13), but allosteric control of NO release from SNO-Hb in vivo is still under debate (14,17,18). In a purified condition, free of red blood cell constituents, SNO-Hb is sufficiently stable to allow oxygen binding studies (7) and oxidation-reduction studies (this report) to be carried out over a period of several hours without significant loss of NO from the SNO-Hb derivative.Stamler and co-workers (19) recently presented evidence showing that interactions of SNO-Hb with the Band 3 protein on the erythrocyte membrane can facilitate NO release from * This work was supported by the National Institutes of Health Grants RO1 HL-58248, 1R43 HL-6586, and GM24417, NIEHS Center Grant ESO-1908, and the North Carolina Biotechnology Center through financial support of the Duke University Mass Spectrometry Facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with ...

Nitrite Reductase Activity of Hemoglobin S (Sickle) Provides Insight into Contributions of Heme Redox Potential Versus Ligand Affinity

“…Human HbS is unique among the heme-globins in that its ligand-binding affinity and kinetics approximate those of HbA (in the unpolymerized state) (38 -41), yet its considerably lower heme redox potential renders is more prone to oxidation (42)(43)(44)(45)(46). However, the solubility of the deoxygenated form of HbS is ϳ17 g/dl at 37°C, whereas it is 70 g/dl for HbA (49).…”

“…This correction now reveals a dampening of allosteric autocatalysis by the redox Bohr effect, such that the normalized maximum bimolecular rate constants actually decrease with decreasing pH, as the T-to-R shift is retarded and allosteric autocatalysis is limited. These normalized maximum constants are 42 7.04, and 25.58 Ϯ 4.81 M Ϫ1 s Ϫ1 at pH 6.73 (n ϭ 4 each; Fig. 5D).…”

“…HbS and HbA, such that these species similarly transition from T-to R-state (41 (42). In an aerobic environment unpolymerized HbS also oxidizes more easily than HbA (43-45) with reported rates of autoxidation nearing 0.05 h Ϫ1 for oxy-HbS versus 0.029 h Ϫ1 for oxy-HbA at 37°C (1.7-fold faster) (46).…”

Molecular Controls of the Oxygenation and Redox Reactions of Hemoglobin

“…Exposure of Hb to NO and formation of HbNO in the absence of O 2 does not trigger SNO-Hb formation in either T-or R-states (39), but transfer of NO + from intraerythrocytic S-nitrosoglutathione to the reactive thiol group of b93 Cys in the R-state of Hb could occur in the lungs, forming a limited amount of SNO-Hb. SNO-Hb could also result from formation of nitrosating agents such as NO x when NO is generated from nitrite in the presence of O 2 (7,21,39). Deoxygenation of SNO-Hb in the microvasculature, accompanied by the reverse R-to-T process, was hypothesized to cause the release of NO equivalents.…”

“…Deoxygenation of SNO-Hb in the microvasculature, accompanied by the reverse R-to-T process, was hypothesized to cause the release of NO equivalents. However, SNO-Hb was subsequently shown to be remarkably stable in both R and T conformations (17,21), and could not be a significant storage form of bioactive NO due to its instability in the reductive environment of red blood cells (21,50). In subsequent revisions of the SNO-Hb proposal, release of bioactive NO was suggested to be facilitated by Hb binding to the anion exchanger 1 (AE1) protein (Band 3) in the red blood cell membrane, with transfer of NO + from Cysb93 of SNO-Hb to thiols in AE1 and subsequent release (89).…”