1. 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
- Author
-
R. David Britt, Sang Choul Im, Ryan C. Kunz, Lucy Waskell, Jarett Wilcoxen, Joseph E. Darty, Stephen W. Ragsdale, and Naw May Pearl
- Subjects
0301 basic medicine ,Electrons ,Protonation ,Reductase ,Photochemistry ,Models, Biological ,Biochemistry ,Redox ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Cytochrome b5 ,medicine ,Animals ,Cytochrome P450 Family 2 ,Heme ,NADPH-Ferrihemoprotein Reductase ,030102 biochemistry & molecular biology ,biology ,Electron Spin Resonance Spectroscopy ,Cytochrome P450 reductase ,Cytochrome P450 ,NAD ,Kinetics ,Cytochromes b5 ,chemistry ,Biocatalysis ,biology.protein ,Ferric ,Aryl Hydrocarbon Hydroxylases ,Hydrogenation ,Rabbits ,Protons ,Oxidation-Reduction ,Protein Binding ,medicine.drug - 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...
- Published
- 2016