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

Purified cytochrome b(559) relipidated with either a mixture of phosph atidylcholine and phosphatidic acid or with phosphatidylcholine only e xhibits high and low superoxide (O-2(-)) producing ability, respective ly, 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, cytochro me b(559) binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity bindin g of FAD to cytochrome b(559) relipidated with phosphatidylcholine com bined with phosphatidic acid is associated with an enhanced NADPH-driv en O-2(-) producing capacity. A kinetic study of O-2(-) production by cytochrome b(559) reflavinated under stoichiometric FAD binding condit ions 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 determ ination of steady-state difference spectra of such preparations, reduc ed by NADPH, indicated that O-2(-) production is activated by facilita tion of electron transfer from NADPH to FAD rather than by an enhancem ent of NADPH binding.