Superoxide-producing cytochrome b. Enzymatic and electron paramagnetic resonance properties of cytochrome b558 purified from neutrophils.

Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.