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-deazariboflavin (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 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 P450R1e:b5ox 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 (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 b5 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 x 10(-6) M.
Bhattacharyya
A K
AK
Department of Anesthesiology, University of California, San Francisco.
Hurley
J K
JK
Tollin
G
G
Waskell
L
L
D000818:Animals; D015786:Cytochromes b5 / Q000378:metabolism*; D003902:Detergents; D007700:Kinetics; D007834:Lasers; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010782:Photolysis; D011485:Protein Binding; D011817:Rabbits
D003902:Detergents; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase