Preparation and characterization of a 5'-deazaFAD XXXX @entity1110 -cytochrome P450 reductase.

@entity8156 @entity4674 @entity21890 @entity1110 @entity28094 @entity72565 @entity13508
@entity1110 -cytochrome P450 reductase is a flavoprotein which contains both an FAD and @entity21890 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 @entity72565 mutant of @entity1110 -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 @entity21890 to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total @entity28094 . Addition of 2 electron equivalents to the 5'-deazaFAD @entity72565 reductase from dithionite generated a stoichiometric amount of the @entity21890 @entity8156 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 @entity21890 domain to its redox partners, ferric @entity4674 and @entity13508 (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 @entity1110 -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.
