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Superoxide production by cytochrome b 559
Author(s) -
Koshkin Vasilij,
Pick Edgar
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80285-8
Subject(s) - chemistry , phosphatidic acid , phosphatidylcholine , cytochrome , cytochrome c , nadph oxidase , biochemistry , cytochrome p450 reductase , cytochrome c oxidase , phospholipid , superoxide , coenzyme q – cytochrome c reductase , reactive oxygen species , enzyme , mitochondrion , membrane
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 eytochrome 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.