Superoxide production by cytochrome b559

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Abstract
Purified cytochrome b 559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2) 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 O2 − producing capacity. A kinetic study of O2 − production by cytochrome b 559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2 production by high‐ and low‐activity relipidated and reflavinated eytochrome b 559, at varying substrate concentrations, and the determination of steady‐state difference spectra of such preparations, reduced by NADPH, indicated that O2 − production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.