Purified leukocyte cytochrome b(558) incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O-2(-)-generating NADPH oxidase in a cell-free system, It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O-2(-) generation. Both O-2(-) generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mob, Blob, Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b(558)-associated fraction, The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O-2(-) generation. Upon incorporation into liposomes, the cytochrome b(558)-containing fraction demonstrates high O-2(-) and INT reductase activities in the presence of cytosolic factors. Both O-2(-) generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTP gamma S. Phenylarsine oxide inhibits both O-2(-) generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the K-m values (O-2(-) formation) for NADPH and NADH are 27.2 mu M and 810 mu M, and for INT reductase the K-m values are 27.5 mu M and 1017 mu M,, respectively. Under anaerobic conditions and thus in the absence of O-2(-) formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O-2(-) anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b(558) is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O-2(-) generation and INT reduction by cytochrome b(558) incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b(558).