STUDIES ON THE REACTION-MECHANISM FOR REDUCTIVE NITROSYLATION OF FERRIHEMOPROTEINS IN BUFFER SOLUTIONS

Ferrihemoproteins in buffer solutions bind nitric oxide to yield their nitric oxide adducts. Reversible binding of NO was found for ferricyt ochrome c (Cyt(III)) and metmyoglobin (Mb(III)) at pH values lower tha n ca. 7.0. The equilibrium constants were obtained as (1.6 +/- 0.1) x 10(4) M(-1) for Cyt(III) and (1.3 +/- 0.1) x 10(4) M(-1) for Mb(III). At higher pH, the reversible formation of the NO adducts is no longer observed; the NO adduct of Cyt(III) (Cyt(III)-NO) undergoes reduction to ferrocytochrome c, Cyt(II), and that of Mb(III) (Mb(III)-NO) to the nitrosyl adduct of Mb(II) (Mb(II)-NO). Methemoglobin (Hb(III)) reacts readily with NO even at pH < 6 to give the nitrosyl adduct of hemoglo bin (Hb(II)-NO). The rates for the formation of Cyt(II), Mb(II)-NO, an d Hb(II)-NO were measured as functions of NO and OH- concentrations. K inetic analysis indicates that Cyt(III)-NO and Mb(III)-NO undergo nucl eophilic attack by OH- at higher pH to yield Cyt(II) and Mb(II), respe ctively. Mb(II) thus produced further reacts with NO to give Mb(II)-NO . For Hb(III), the nitrosyl adduct (Hb(III)-NO) was found to react wit h both OH- and H2O to give Hb(II)-NO in the presence of excess NO. The rate constants for the reaction between the nitrosyl ferrihemoprotein s and OH- were determined as (1.5 +/- 0.1) x 10(3) M(-1) s(-1) for Cyt (III)-NO, (3.2 +/- 0.2) x 10(2) M(-1) s(-1) for Mb(III)-NO, and (3.2 /- 0.2) x 10(3) M(-1) s(-1) for Hb(III)-NO. The reductive nitrosylatio n of Hb(III) observed at pH < 6.0 is explained by reaction of H2O with Hb(III)-NO: the rate constant is (1.1 +/- 0.1) x 10(-3) s(-1).