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Cytochrome b5 and NADPH‐Cytochrome P450 Reductase Compete for a Binding Site on the Proximal Surface of Cytochrome P450 2B4
Author(s) -
Waskell Lucy,
Zhang Haoming,
Im SangChoul,
Razeghifard Reza
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a670-d
Subject(s) - cytochrome b5 , cytochrome p450 , chemistry , cyclohexanol , reductase , cyclohexane , cytochrome p450 reductase , catalysis , stereochemistry , biochemistry , cytochrome c , enzyme , organic chemistry , coenzyme q – cytochrome c reductase , mitochondrion
To elucidate the mechanism by which cytochrome b 5 ( cyt b 5) affects catalysis by cytochrome P450 (P450) 2B4, the kinetics of product formation by oxyferrous cyt P450 2B4 were compared in the presence of cyt b 5 and cyt P450 reductase (CPR) under single turnover conditions. In the presence of cyt b 5 , P450 catalyzes the oxidation of cyclohexane monophasically (k=29 ± 4.5 s −1 ), forming 58 pmoles of cyclohexanol/nmole P450. In contrast, in the presence of CPR, P450 catalyzes the oxidation of cyclohexane ≅ 10‐fold slower (k= 2.5 ± 0.35 s −1 ) and less efficiently, generating 21 pmoles of cyclohexanol/nmole P450. The ten‐fold different catalytic rate constants of cyt b 5 and CPR enabled us to investigate whether cyt b 5 or CPR catalyzed the oxidation of cyclohexane when both were present. Increasing the cyt b 5 concentration enhanced the amount of product formed by cyt b 5 and diminished the amount of cyclohexanol produced by CPR. Under steady‐state conditions, increasing the concentration of cyt b 5 progressively inhibits NADPH consumption while product formation increases at cyt b 5 : P450 ratios≤ 1 and decreases at cyt b 5 : P450 ratios ≥ 1. These data are consistent with the hypothesis that cyt b 5 binds to P450 and prevents CPR from reducing ferric P450 and initiating catalysis. Present experiments, previous mutagenesis studies, and model complexes suggest that cyt b 5 and CPR compete for a binding site on cyt P450 2B4. Supported by NIGMS 35533 & VA Merit Grant