title: NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain.
abstract: Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.
authors.affiliation: Department of Biochemistry, University of Glasgow, UK.
authors.forename: A W
authors.initials: AW
authors.lastname: Munro
authors.forename: J G
authors.initials: JG
authors.lastname: Lindsay
authors.forename: J R
authors.initials: JR
authors.lastname: Coggins
authors.forename: S M
authors.initials: SM
authors.lastname: Kelly
authors.forename: N C
authors.initials: NC
authors.lastname: Price
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins; D001713:Biphenyl Compounds / Q000494:pharmacology; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D008163:Luminescent Measurements; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D017382:Reactive Oxygen Species
chemical_list: D001426:Bacterial Proteins; D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C031291:diphenyliodonium; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D019255:NADPH Oxidases