Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase.
The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)
Bhattacharyya
A K
AK
Department of Anesthesia, University of California, San Francisco.
Lipka
J J
JJ
Waskell
L
L
Tollin
G
G
D000818:Animals; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007834:Lasers; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010782:Photolysis; D011817:Rabbits; D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism; D012995:Solubility; D014357:Trypsin
C007228:5-deazariboflavin; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D012256:Riboflavin