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A schematic kinetic model is proposed for the hydroperoxide-mediated substrate reaction and cytochrome P450 inactivation. From the model, the relationships between the product concentration at infinite time (P infinity), the apparent rate constant of P450 inactivation (A), and the substrate concentrations are predicted, and the predictions were experimentally examined. The reciprocal of P infinity is proportional to the reciprocal of the substrate concentration in both CuOOH- and H2O2-supported substrate reactions. The reciprocal of P infinity is proportional to cumene hydroperoxide (CuOOH) concentration, but P infinity is independent of H2O2 concentration, indicating different effects of CuOOH and H2O2 in P450 inactivation. The apparent rate constant (A) is proportional to the reciprocal of the substrate concentrations, suggesting substrate protection of P450 from hydroperoxide inactivation. The model also suggests that a second CuOOH molecule may compete with substrate for binding to the P450 substrate binding site. Simulated kinetics of production formation vs time are quite consistent with the experimental kinetics.

A metastable intermediate was found in the course of the denaturation of purified cytochrome P450 2B1 by increasing concentrations of guanidine hydrochloride (GuHCl). The metastable intermediate has no or low absorbance at 450 nm in the reduced carbon monoxide difference spectrum and has no absorbance at 420 nm. The intermediate is easily converted to P420 by increasing concentrations of GuHCl. Before it becomes P420, the cytochrome can be completely reconverted to native P450 by dilution and incubation at 4 degrees C. Cytochrome P420 resulting from exposure to higher concentrations of GuHCl (> 3 M) failed to be reconverted to P450 by dilution. Denaturation of P450 2B1 by exposure to low concentrations of urea (< 2 M) is also completely reversible but no obvious intermediate is detectable. An intermediate is observed, however, when the urea denaturation is conducted in the presence of 1 M NaCl. As is the case with higher concentrations of GuHCl, cytochrome P450 denatured by exposure to 5 M or higher concentrations of urea is not reversible. The failure of reconversion of P420 denatured by exposure of cytochrome P450 to high concentrations of GuHCl or urea is probably attributable to the extensive unfolding of the apoprotein, which favors aggregation, rather than to heme loss. Our results also suggest that the active site is more sensitive to denaturants than other regions of the protein.

Two regions (amino acid residues 110-130 and 204-218) of NADPH-cytochrome P450 reductase (reductase) have been shown to be the putative binding sites for the interaction with cytochrome P450 or cytochrome c. To obtain further insight into the molecular mechanism of protein-protein interaction between these proteins, three anti-peptide antibodies (1A, 2A, and 3A) were generated against the peptides corresponding to these two regions on rat reductase to study the interaction between the reductase and cytochrome P450 or cytochrome c. All three anti-peptide antibodies have high affinity for their peptide antigens on ELISA (titre > 1 x 10(-6) g/L), and they also bind to rat reductase on ELISA under both denatured and native conditions, suggesting that these regions are on the surface of the protein. 1A and 3A also bind to rabbit and human reductase, though 1A binds to human reductase with lower affinity. Antibody 2A does not bind to rabbit or human reductase. Western blot analysis using these anti-peptide antibodies showed similar results. Antibodies 1A and 3A inhibit both cytochrome P4501A1-dependent ethoxycoumarin hydroxylation activity and P4502B1-dependent pentoxyresorufin dealkylation activity, but the inhibition by 1A and 3A was not additive. Antibodies 1A and 3A also have inhibitory effects on the activity of P4501A1-dependent ethoxycoumarin hydroxylation reconstituted with reductase from rabbit and human. However, none of the three anti-peptide antibodies inhibits cytochrome c reduction by rat reductase. These data suggest that reductases from rat, rabbit, and human share similar structure in at least two regions which appear to be on the surface of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Hexachlorobenzene (HCB) elicits concentration-dependent and saturable type 1 binding spectra when added to oxidized (Fe3+) cytochrome P-450 (CYT P-450) in control, phenobarbital- (PB) induced, and beta-naphthoflavone- (BNF) induced male Sprague-Dawley rat liver microsomes. The spectral binding constants (Ks) for HCB in control and PB-induced microsomes are 180 microM and 83 microM, respectively, and correlate inversely with the specific content of CYT P-450 (0.9 and 2.1 nmol/mg) in the two microsomal preparations. BNF-induced microsomes show type 1 interaction only at low HCB concentration. Overall biotransformation of HCB, monitored by loss of [14C]HCB from the reaction medium, is dependent on NADPH and intact microsomes. Dimethyl sulfoxide (Me2SO), a potent hydroxyl radical scavenger and the solvent used for HCB dissolution, does not affect the biotransformation of HCB in aerobic reactions. Pentachlorobenzene (PCB) appears to be the initial and major isolatable CYT P-450 mediated dechlorination product of HCB with NADPH-fortified rat liver microsomes. Trace levels of pentachlorophenol (PCP) and an unidentified metabolite are also observed. PCB formation is enhanced under anaerobic conditions but is inhibited by metyrapone and carbon monoxide. PCB formation is also inhibited with aerobic reaction conditions, while PCP formation is observed. The data indicate that CYT P-450 in hepatic microsomes supports the reductive dechlorination of HCB to PCB.

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.