Kinetics of Reduction of Cytochrome P-450Lm4 and Nadph-Cytochrome P-450 Reductase.

The reduction of cytochrome P-450 in liver microsomal suspensions by NADPH is known from the work of others to exhibit biphasic kinetics, but the interpretation of this finding is complicated by the occurrence of multiple forms of the cytochrome. In the present study, the kinetics of reduction of highly purified P-450LM4 (5,6-benzoflavone-inducible form of the cytochrome, isolated in the high spin state from rabbit liver microsomes) was examined in a reconstituted system containing highly purified NADPH-cytochrome P-450 reductase from the same source, phosphatidycholine, NADPH, and carbon monoxide under anaerobic conditions. When a mixture of the two enzymes was rapidly mixed with NADPH in a stopped flow spectrophotometer, the reduction reaction, monitored by the appearance of the ferrous carbonyl complex of P-450LM4 at 448 nm, exhibited biphasic kinetics irrespective of the NADPH concentration or the molar ratio of reductase to cytochrome. The first order rate constants were about 1.0 sec('-1) and 0.2 sec('-1). The presence of a typical substrate, benzphetamine, had no effect on the results, and the omission of the phospholipid, although causing no change in the kinetics, resulted in a decrease in the extent of the fast phase. The relative magnitude of the two phases displayed complex behavior with varying NADPH concentrations. Substitution of the airstable semiquinone form of the reductase (known from earlier studies in this laboratory to contain FMN in the semiquinone state and FAD in the oxidized state) for the fully oxidized form in the reconstituted system caused no change in the kinetics of P-450LM4 reduction. When the reduction reaction was initiated by the rapid mixing of P-450LM4 and reductase, evidence was obtained that association of the two enzymes from their separate sources in bulk solution is a relatively slow process, whereas once the two enzymes have associated into large aggregates the formation of a catalytically functional complex between the two proteins proceeds very rapidly. Our results indicate that the occurrence of biphasic kinetics in the 1-electron transfer to P-450LM4 in the reconstituted system is apparently due not to the cytochrome, but to some property of the reductase. The reduction of the reductase by NADPH was found to exhibit triphasic first order kinetics. The first order rate constants for the three phases were 70 sec('-1), 7 sec('-1), and 0.05 sec('-1). A complete spectral analysis (400 to 640 nm) of the reaction mixture was carried out and the spectrum for each phase of the triphasic reaction was reconstructed from component spectra summed in proportions governed by their relative reduction potentials. For example, the first phase of the reaction with a 10-fold excess of NADPH could be spectrally described by a mixture of 31.5% disemiquinone and 68.5% (FMNH(,2),FAD). The second phase was well represented by an equimolar mixture of 2- and 4- electron-reduced species and , finally, thermodynamic relaxation in the third phase resulted in an equilibrium mixture of all possible states of the reductase. These results suggest that interflavin electron transfer, within a single reductase molecule, is much faster than the transfer of reducing equivalents from pyridine nucleotide to flavin and that the distribution of electrons between the two flavin moieties is governed by the reduction potentials for the pertinent half-cells of each flavin.Ph.D.BiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/157801/1/8017332.pd