Nitrite reductase activity of hemoglobin S (sickle) provides insight into contributions of heme redox potential versus ligand affinity.
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.
Vascular Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892; Howard Hughes Medical Institute, National Institutes of Health Research Scholars Program, Bethesda, Maryland 20814.
Rozalina
R
Grubina
Department of Physics, Wake Forest University, Winston-Salem, North Carolina 27109.
Swati
S
Basu
Vascular Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Mauro
M
Tiso
Department of Physics, Wake Forest University, Winston-Salem, North Carolina 27109. Electronic address: shapiro@wfu.edu.
Daniel B
DB
Kim-Shapiro
Vascular Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892; Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: mgladwin@nih.gov.
Mark T
MT
Gladwin
D000495:Allosteric Site; D004227:Dithionite / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry; D006451:Hemoglobin, Sickle / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008954:Models, Biological; D009569:Nitric Oxide / Q000737:chemistry; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D008024:Ligands; D004227:Dithionite; D009569:Nitric Oxide; D006418:Heme; D006441:Hemoglobin A; D009572:Nitrite Reductases