INVESTIGATION OF THE RATE-LIMITING STEP FOR ELECTRON-TRANSFER FROM NADPH -CYTOCHROME-P450 REDUCTASE TO CYTOCHROME-B(5) - A LASER FLASH-PHOTOLYSIS STUDY

The reduction kinetics of the one-electron-reduced cytochrome P450 reductase:cytochrome b(5) complex (P450R(le):b(5ox)) 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 P450R(le) and b(5ox) were reduced via second-order kinetics by 5-dRfH. with rate constants of 1 X 10(8) M(-1) s(-1) and 4.2 X 10(8) 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 P450R(le):b(5ox) complex by 5-dRfH. was biphasic and second-order rate constants of 5.4 X 10(8) M(-1) s(-1) and 7.5 X 10(7) 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 (k(Lim) = 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 FMNH(2) as a result of internal electron transfer from FADH. to FMNH within P450R(le) is the rate limiting step in the reduction of cytochrome b(5) by cytochrome P450 reductase. The K-d of the cytochrome P450 reductase:cytochrome b(5) complex was estimated to be approximately 1.5 X 10(-6) M. (C) 1994 Academic Press, Inc.