title: Electron transfer between the FMN and heme domains of cytochrome P450BM-3. Effects of substrate and CO.
abstract: Cytochrome P450BM-3 has the P450 heme domain and FAD/FMN reductase domain linked together in a single polypeptide chain arranged as heme-FMN-FAD. In the accompanying article (Govindaraj, S., and Poulos, T. L. (1997) J. Biol. Chem. 272, 7915-7921, we have described the preparation and characterization of the various domains of cytochrome P450BM-3. One reason for undertaking this study was to provide simpler systems for studying intramolecular electron transfer reactions. In particular, the heme-FMN version of P450BM-3 that is missing the FAD domain should prove useful in studying the FMN-to-heme electron transfer reaction. This version of P450BM-3 has been designated truncated P450BM-3 or BM3t. In this study we have used laser flash photolysis techniques to generate the reduced semiquinone of 5-deazariboflavin which in turn reduces the FMN of BM3t to the semiquinone, FMN-, at a rate constant of 6600 s-1, whereas the heme is not reduced by the 5-deazariboflavin radical. The reduction of the heme by FMN- does not proceed in the absence of carbon monoxide (CO), whereas in the presence of CO the FMN- to heme electron transfer rate constant is 18 s-1. If a fatty acid substrate is present, this rate constant increases to 250 s-1. Somewhat surprisingly, the rate of heme reduction also is dependent on [CO] which indicates that CO causes some change within the heme pocket and/or interaction between the heme and FMN domains that is required for intramolecular electron transfer.
authors.affiliation: Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Hazzard
authors.forename: S
authors.initials: S
authors.lastname: Govindaraj
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
authors.forename: G
authors.initials: G
authors.lastname: Tollin
mesh_terms: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry; D013057:Spectrum Analysis; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C007228:5-deazariboflavin; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D012256:Riboflavin