β93cys residue controls nitrite reductase activity of Hb. (A) Hb(C→A) potentiates nitrite-dependent relaxation. Final concentrations: nitrite (2 μM), Hb(C→A) (25 μM), and IHP (625 μM). *P < .001 relative to nitrite alone. #P < .01 relative to Hb(C→A) + IHP. Data represent mean ± SEM (n = 3-5). (B) Relationship between P₅₀ of HbA (▪) or Hb (C→A) (⬡) and the oxygen tension at the initiation of vessel dilation in the presence of nitrite (2 μM). Data for HbA are taken from Figure 3C. P₅₀ was modulated by the addition of varying amounts of IHP and was determined under identical buffer, pH, temperature, and PCO₂ conditions as for vessel dilation studies. A linear relationship between P₅₀ and oxygen tension at the onset of relaxation was observed for both Hbs (black line: y = 20.7 + 0.67x; r = 0.96; gray line: y = 11.6 + 1.48x; r = 0.94). Data represent mean ± SEM (n = 3-6). (C) Representative reaction traces for metHb formation from the reaction between nitrite and various deoxyHbs (experiments performed under anoxic conditions). (D) Relationship between Hb P₅₀ and rate of nitrite reduction. Shown are the best fit line (R 2 = 0.99) and the average redox potentials (E_1/2) from values in the literature.^37,40,42