Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase.

Haoming Zhang, Larry Gruenke, A Sami Saribas, Sang-Choul Im, Anna L Shen, Charles B Kasper, Lucy Waskell

University of Michigan and Veterans Administration Medical Center, 2215 Fuller Road, Ann Arbor, Michigan 48105, USA.

Biochemistry 2003 Jun 10

filter terms:

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.

Citation

Haoming Zhang, Larry Gruenke, A Sami Saribas, Sang-Choul Im, Anna L Shen, Charles B Kasper, Lucy Waskell. Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase. Biochemistry. 2003 Jun 10;42(22):6804-13