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

Abstract

Hemoglobin A (HbA) is an allosterically regulated nitrite reductase that reduces nitrite to NO under physiological hypoxia. The efficiency of this reaction is modulated by two intrinsic and opposing properties: availability of unliganded ferrous hemes and R-state character of the hemoglobin tetramer. Nitrite is reduced by deoxygenated ferrous hemes, such that heme deoxygenation increases the rate of NO generation. However, heme reactivity with nitrite, represented by its bimolecular rate constant, is greatest when the tetramer is in the R quaternary state. The mechanism underlying the higher reactivity of R-state hemes remains elusive. It can be due to the lower heme redox potential of R-state ferrous hemes or could reflect the high ligand affinity geometry of R-state tetramers that facilitates nitrite binding. We evaluated the nitrite reductase activity of unpolymerized sickle hemoglobin (HbS), whose oxygen affinity and cooperativity profile are equal to those of HbA, but whose heme iron has a lower redox potential. We now report that HbS exhibits allosteric nitrite reductase activity with competing proton and redox Bohr effects. In addition, we found that solution phase HbS reduces nitrite to NO significantly faster than HbA, supporting the thesis that heme electronics (i.e. redox potential) contributes to the high reactivity of R-state deoxy-hemes with nitrite. From a pathophysiological standpoint, under conditions where HbS polymers form, the rate of nitrite reduction is reduced compared with HbA and solution-phase HbS, indicating that HbS polymers reduce nitrite more slowly.The various redox reactions catalyzed by ferrous hemes of the heme-globins (primarily hemoglobin and myoglobin) have interested scientists for over 150 years (1). In 1901, while studying the mechanism of meat curing, John Haldane described the ability of nitrite to oxidize hemoglobin (Hb) 5 to methemoglobin (met-Hb) and generate iron-nitrosyl-hemoglobin (iron-nitrosyl-Hb) in sections of meat not exposed to air (2), although he was most likely observing the analogous reaction with myoglobin (Mb). The reaction of nitrite with deoxyhemoglobin (deoxy-Hb) was further characterized by Brooks in 1937 (3) and by Doyle and colleagues in 1981 (4). Recent work has demonstrated that deoxy-Hb can effectively catalyze the reduction of nitrite to NO along physiological oxygen and pH gradients (5-9). This reaction has been implicated in nitrite-and deoxy-Hb-dependent vasodilation in vivo (5, 10 -12) and in vitro (5, 13), as well as in nitrite-mediated cytoprotection following ischemia-reperfusion injury (14 -17). An analogous reaction of nitrite with deoxymyoglobin (deoxy-Mb) has been associated with the regulation of cardiomyocyte respiration during physiological and pathological hypoxia (18,19).In an anaerobic environment nitrite is reduced to NO as deoxy-Hb is oxidized to met-Hb (Equation 1) (6,20). NO generated in this reaction can then be bound by other ferrous deoxy-hemes to form iron-nitrosyl-Hb (Equation 2) (21).Importantly, this reaction is a...

Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling

“…This group includes hemoglobin and myoglobin (43)(44)(45)(46)(47). The rate of NO production by Cco increases with decreasing pH and increasing NO 2 Ϫ concentration (21), suggesting that that Cco functions in a nitrite reductase reaction (NO 2 Ϫ ϩ FeII ϩ H ϩ 3 NO ϩ FeIII ϩ OH Ϫ ) similar to that proposed for hemoglobin and myoglobin (48)(49)(50)(51)(52). Unlike the Cco oxidase reaction, which requires four electrons, the Cco NO 2 Ϫ reductase reaction involves a one-electron transfer.…”

The functional nitrite reductase activity of the heme-globins

“…This is clearly illustrated when the instantaneous rate itself is plotted versus time as in Figure 1B. 14,15,22 …”

“…In another study, the rate that deoxygenated normal adult hemoglobin, HbA 0 , reacts with nitrite was compared with the rate that sickle cell hemoglobin, HbS, reacts with nitrite. 22 The ligand affinity of solution phase …”

“…It should be noted that polymerized HbS was found to react more slowly with nitrite than HbA 0 , which could have important implications in the pathology of sickle cell disease. 22 A complete characterization of the reaction rate constants for nitrite reduction by different hemoglobin variants and allosteric states that have been measured is presented in Table 1. These standard measurements for hemoglobin species may provide insight into the physiologic function of the heme-globins in NO signaling.…”

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

“…2, A and B). It is important to note that dithionite does not effectively reduce nitrite (49). Dithionite can compete with Fe(II)-CBS for NO ⅐ because the formation of Fe(II)NO ⅐ -CBS is a relatively slow process (32).…”