MECHANISM OF THE SUPEROXIDE-PRODUCING OXIDASE OF NEUTROPHILS - O-2 IS NECESSARY FOR THE FAST REDUCTION OF CYTOCHROME B-245 BY NADPH

A soluble oxidase from phorbol-stimulated pig neutrophils contained FAD and cytochrome b-245. A typical preparation produced 13.03 mol of superoxide (O2-.) .cntdot. s-1 .cntdot. mol of cytochrome b-1 (348 nmol .cntdot. min-1 .cntdot. mg of protein-1). In the aerobic steady state, cytochrome b was 8.9% reduced. Steady-state cytochrome b reduction was absent from extracts of unstimulated cells; Km values for NADPH, for O2-. production and cytochrome b reduction were similar. The calculated aerobic rate of cytochrome b reduction was equal to the measured rate of O2-. production in a variety of preparations and in the presence of a range of inhibitors. Under anaerobic conditions the rate was slow: O2 is apparently required for rapid electron flow into the oxidase complex. Cytochrome b is shown to be kinetically competent to act as part of the O2-.-generating complex.