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 ferricytochrome c (Cyt III ) and metmyoglobin (Mb III ) at pH values lower than ca. 7.0. The equilibrium constants were obtained as (1.6 ± 0.1) × 10 4 M -1 for Cyt III and (1.3 ± 0.1) × 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 hemoglobin (Hb II −NO). The rates for the formation of Cyt II, Mb II −NO, and Hb II −NO were measured as functions of NO and OH - concentrations. Kinetic analysis indicates that Cyt III −NO and Mb III −NO undergo nucleophilic attack by OH - at higher pH to yield Cyt II and Mb II, respectively. 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 with both OH - and H 2 O to give Hb II −NO in the presence of excess NO. The rate constants for the reaction between the nitrosyl ferrihemoproteins and OH - were determined as (1.5 ± 0.1) × 10 3 M -1 s -1 for Cyt III −NO, (3.2 ± 0.2) × 10 2 M -1 s -1 for Mb III −NO, and (3.2 ± 0.2) × 10 3 M -1 s -1 for Hb III −NO. The reductive nitrosylation of Hb III observed at pH < 6.0 is explained by reaction of H 2 O with Hb III −NO: the rate constant is (1.1 ± 0.1) × 10 -3 s -1 .