THE MECHANISM OF ELECTRON DONATION TO MOLECULAR-OXYGEN BY PHAGOCYTIC CYTOCHROME B(558)

Phagocytic cytochrome b(558) 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 b(558) purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b(558) was rapidly reoxidized by O-2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b(558) with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O-2 linearly depended on the O-2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pK(a) of 9.7. Under complete anaerobic conditions, ferrous cytochrome b(558) was oxidized by ferricyanide at a rate faster than by O-2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b(558) are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O-2 complex. These findings are most consistent with the electron transfer from the heme to O-2 by an outer-sphere mechanism.