SUPEROXIDE PRODUCTION BY CYTOCHROME B(559) - MECHANISM OF CYTOSOL-INDEPENDENT ACTIVATION

Purified cytochrome b(559) relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O-2(-)) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b(559) binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b(559) relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH-driven O-2(-) producing capacity. A kinetic study of O-2(-) production by cytochrome b(559) reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O-2(-) production by high- and low-activity relipidated and reflavinated cytochrome b(559), at varying substrate concentrations, and the determination of steady-state difference spectra of such preparations, reduced by NADPH, indicated that O-2(-) production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.