Abstract
NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.
Highlights
In the present study, threeaction of NADPH with the reductase wasexaminedunder anaerobic conditionsby stopped flow spectrophotometry
Thispermitted a precise assignment of the NADPH concentration employed, a third phase occurs individual reduction potentials to the specific flavin half-rewith very slow changes leading to an equilibrium mix- actions
The absorption spectrum for the semiquinone form of the FMN-free enzyme shown inFig. 6A was recorded with a spectral bandwidth of 2.5 nm and a scan rate of 5.0 nm/s.Solutions were placed in quartzcuvettes of 1.0-ml sample volume and IO-mm optical path length, and beam masks of 3-mm aperture were used
Summary
Prepared by incubation of the native enzyme with apotlavodoxin ir the presence of 2 M KBr as has been described [15].The specifit activity of the resultingpreparation, assayed in 0.3 M potassiur phosphate buffer, pH 7.7,at 30 "C,with and without prior incubation with excess FMN, was 54 and 0.8 pmol of cytochrome c reduced/min/ mg of protein, respectively. The absorption spectrum for the semiquinone form of the FMN-free enzyme shown inFig. 6A was recorded with a spectral bandwidth of 2.5 nm and a scan rate of 5.0 nm/s.Solutions were placed in quartzcuvettes of 1.0-ml sample volume and IO-mm optical path length, and beam masks of 3-mm aperture were used. The column removed debris that would have contributed to light scattering, and the precision of the absorption spectra was correspondingly improveci Absorption coefficients for both the native enzyme and the FMN-. The observed first order rate constant for the reduction reaction appeared to be invariant with respect to NADPH concentration, lending further credence to the suggestion that the reductase is saturated with reduced pyridine nucleotide.
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