title: Protonation of the Hydroperoxo Intermediate of Cytochrome P450 2B4 Is Slower in the Presence of Cytochrome P450 Reductase Than in the Presence of Cytochrome b5.
abstract: Microsomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe3+OOH)-and subsequent formation of the active oxidizing species (Fe+4═O POR•+), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within the dead time of the instrument. However, when P450 reductase was the reductant, a hydroperoxo P450 intermediate was observed. The effect of b5 on the enzymatic efficiency in D2O and the kinetic solvent isotope effect under steady-state conditions are both consistent with the ability of b5 to promote rapid protonation of the hydroperoxo species and more efficient catalysis. In summary, by binding to the proximal surface of P450, b5 stimulates the activity of P450 2B4 by enhancing the rate of protonation of the hydroperoxo intermediate and formation of Compound I, the active oxidizing species, which allows less time for side product formation.
authors: Department of Anesthesiology, University of Michigan Medical School , Ann Arbor, Michigan 48109-0112, United States.
authors: Naw May
authors: NM
authors: Pearl
authors: Department of Chemistry, University of California, Davis , Davis, California 95616, United States.
authors: Jarett
authors: J
authors: Wilcoxen
authors: Department of Anesthesiology, University of Michigan Medical School , Ann Arbor, Michigan 48109-0112, United States.
authors: Sangchoul
authors: S
authors: Im
authors: Department of Biological Chemistry, University of Michigan Medical School , Ann Arbor, Michigan 48109-0600, United States.
authors: Ryan
authors: R
authors: Kunz
authors: Department of Biological Chemistry, University of Michigan Medical School , Ann Arbor, Michigan 48109-0600, United States.
authors: Joseph
authors: J
authors: Darty
authors: Department of Chemistry, University of California, Davis , Davis, California 95616, United States.
authors: R David
authors: RD
authors: Britt
authors: Department of Biological Chemistry, University of Michigan Medical School , Ann Arbor, Michigan 48109-0600, United States.
authors: Stephen W
authors: SW
authors: Ragsdale
authors: Department of Anesthesiology, University of Michigan Medical School , Ann Arbor, Michigan 48109-0112, United States.
authors: Lucy
authors: L
authors: Waskell
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D055162:Biocatalysis; D000072467:Cytochrome P450 Family 2 / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D006865:Hydrogenation; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011522:Protons; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D011522:Protons; D009243:NAD; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase