Covalent modifications of hemoglobin by nitrite anion: formation kinetics and properties of nitrihemoglobin

The green nitrihemoglobin (α(2)β(2) tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A(0) under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion, and oxygen concentrations: -d[HbNO(2)]/dt = [k(1) + k(2)(K(a)[HNO(2)])[O(2)](1/2)][HbNO(2)], where k(1) = (2.4 ± 0.9) × 10(-4) s(-1), k(2) = (1 ± 0.2) × 10(5) M(-5/2) s(-1), and K(a) is the acid dissociation constant for nitrous acid (4.5 × 10(-4) M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Because nonphotochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A(0) and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.