Investigation of the Rate Limiting Step for Electron Transfer from NADPH:Cytochrome P450 Reductase to Cytochrome b5: A Laser Flash-Photolysis Study

The reduction kinetics of the one-electron-reduced cytochrome P450 reductase:cytochrome b5 complex (P450R1e:b5ox) has been investigated by the laser flash-photolysis technique, using the semiquinone of 5-deaza-riboflavin (5-dRfH ·) as the reductant. Investigation of the kinetic properties of the individual components at 470 nm indicated that P450R1e and b5ox were reduced via second-order kinetics by 5-dRfH · with rate constants of 1 × 108 M−1 s−1 and 4.2 × 108 M−1 s−1, respectively. Intramolecular electron transfer from (laser reduced) FADH · to FMNH · was measured at 585 nm and a first-order rate constant of 36 s−1 was obtained for this process. Reduction of the preformed P450R1e:b5ox complex by 5-dRfH · was biphasic and second-order rate constants of 5.4 × 108 M−1 s−1 and 7.5 × 107 M−1 s−1 were obtained for the fast and slow phases of reduction. The time-resolved flash-induced difference spectrum was consistent with the simultaneous direct reduction (by 5-dRfH ·) of protein-bound flavin and heme, followed by an additional slower first-order intracomplex electron transfer (kLim = 37 s−1 from protein-bound flavin semiquinone to heme. The results indicate that the (laser-generated) two-electron-reduced form of cytochrome P450 reductase is catalytically competent in the transfer of reducing equivalents to oxidized cytochrome b(5) and suggest that formation of FMNH2 as a result of internal electron transfer from FADH · to FMNH · within P450R1e is the rate limiting step in the reduction of cytochrome b5 by cytochrome P450 reductase. The Kd of the cytochrome P450 reductase:cytochrome b5 complex was estimated to be approximately 1.5 × 10−6 M.