How Does Product Isotope Effect Prove the Operation of a Two-State “Rebound” Mechanism in C−H Hydroxylation by Cytochrome P450?

• Published in: Journal of the American Chemical Society 2003-10, Vol.125 (43), p.13024-13025
• Main Authors: Kumar, Devesh, de Visser, Samuël P, Shaik, Sason
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Chemistry; Cyclopropanes - chemistry; Cyclopropanes - metabolism; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Deuterium - chemistry; Deuterium Exchange Measurement; Exact sciences and technology; Ferric Compounds - chemistry; Fundamental and applied biological sciences. Psychology; Hydrogen Peroxide - chemistry; Hydroxylation; Kinetics; Kinetics and mechanisms; Mechanisms. Catalysis. Electron transfer. Models; Molecular biophysics; Organic chemistry; Physical chemistry in biology; Reactivity and mechanisms; Thermodynamics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja036906x

C−H hydroxylation is a fundamental process. In Nature it is catalyzed by the enzyme cytochrome P450, in a still-debated mechanism that poses a major intellectual challenge for both experiment and theory; currently, the opinions keep swaying between the original single-state rebound mechanism, a two-oxidant mechanism (where ferric peroxide participates as a second oxidant, in addition to the primary active species, the high-valent iron−oxo species), and two-state reactivity (TSR) mechanism (where two spin states are involved). Recent product isotope effect (PIE) measurements for the trans-2-phenyl-methyl cyclopropane probe (1), led Newcomb and co-workers (Newcomb, M.; Aebisher, D.; Shen, R.; Esala, R.; Chandrasena, P.; Hollenberg, P.; Coon, M. J. J. Am. Chem. Soc. 2003 , 125, 6064−6065) to rule out TSR in favor of the two-oxidant scenario, since the direction of the PIE was at odds with the one predicted from calculations on methane hydroxylation. The present report describes a density functional theoretical study of C−H hydroxylation of the Newcomb probe, 1, leading to rearranged (3) and unrearranged (2) products. Our study shows that the reaction occurs via TSR in which the high-spin pathway gives dominant rearranged products, whereas the low-spin pathway favors unrearranged products. The calculated PIE(2/3) values based on TSR are found to be in excellent agreement with the experimental data of Newcomb and co-workers. This match between experiment and theory makes a strong case that the reaction occurs via TSR mechanism.