Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

Abstract

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)

Mechanism of cytochrome P450 reductase from the house fly: evidence for an FMN semiquinone as electron donor

“…(ii) In early measurements of the stoichiometry of cytochrome c reduction [15] it was found that at least two thirds of the reducing equivalents were transferred from NADPH to cytochrome c with high rates under single turnover conditions, suggesting heterogeneity of the FMN semiquinone enzyme form, a fraction of which is catalytically competent. (iii) As shown by £ash photolysis [16] (Table 1), and is clearly not an intermediate of catalysis, but rather represents a sluggish enzyme form analogous to the inactive three-electron reduced £avoprotein of P450BM3 [5,19]. This`air-stable' semiquinone form of the enzyme is unable to rapidly reduce cytochrome c despite the fact that the reduction is thermodynamically favored by +350 mV.…”

Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

“…In the wild-type protein, there is a rapid (30 -70 s Ϫ1 ), reversible exchange of electrons between the two flavins (20,21). At equilibrium, the distribution of the electrons is governed by the redox potentials of the individual flavin half-reactions (FMN ox/sq ϭ Ϫ110 mV, FMN sq/hq ϭ Ϫ270 mV, FAD ox/sq ϭ Ϫ290 mV, and FAD sq/hq ϭ Ϫ365 mV) (7)(8)(9).…”

“…Reaction 3 decreased 7-fold in ⌬TG (k ϭ 6.5 s Ϫ1 ) and 410-fold in ⌬TGEE (k ϭ 0.11 s Ϫ1 ) (Table 3A) (20,21).…”

“…Based on the juxtaposition of the two flavins, an extrinsic electron transfer rate of ϳ10 10 s Ϫ1 is predicted (19). However, the experimentally observed electron transfer rate between the two flavins is 30 -55 s Ϫ1 (20,21). This modest rate and slowing of electron transfer in a viscous solvent (75% glycerol) suggest that interflavin electron transfer is likely conformationally gated.…”

Mechanistic Studies on the Intramolecular One-electron Transfer between the Two Flavins in the Human Neuronal Nitric-oxide Synthase and Inducible Nitric-oxide Synthase Flavin Domains

“…In the presence of CaM, this lock is removed, facilitating electron transfer between FAD and FMN. Intramolecular electron transfer between the two flavins in CPR has been studied using laser flash photolysis (31) and temperature-jump relaxation spectroscopy (32). Pulse radiolysis was used to study the electron transfer in nNOS (33).…”