Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.
Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 × 10(7) per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Jonathan
J
Rittle
Michael T
MT
Green
D055162:Biocatalysis; D020134:Catalytic Domain; D055598:Chemical Phenomena; D002723:Chlorobenzoates / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005615:Freezing; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D015204:Spectroscopy, Mossbauer; D016945:Sulfolobus acidocaldarius / Q000201:enzymology
D002723:Chlorobenzoates; D005227:Fatty Acids; D007850:Lauric Acids; D008024:Ligands; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; C000433:3-chloroperbenzoic acid; D010100:Oxygen