The mechanism of electron donation to molecular oxygen by phagocytic cytochrome b558

Abstract

Phagocytic cytochrome b558 is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction kinetics of cytochrome b558 purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b558 was rapidly reoxidized by O2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b558 with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O2 linearly depended on the O2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pKa of 9.7. Under complete anaerobic conditions, ferrous cytochrome b558 was oxidized by ferricyanide at a rate faster than by O2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b558 are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O2 complex. These findings are most consistent with the electron transfer from the heme to O2 by an outer-sphere mechanism.