Quantum Mechanical/Molecular Mechanical Calculated Reactivity Networks Reveal How Cytochrome P450cam and Its T252A Mutant Select Their Oxidation Pathways

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• 作者：Binju Wang ; Chunsen Li ; Kshatresh Dutta Dubey ; Sason Shaik
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：June 17, 2015
• 年：2015
• 卷：137
• 期：23
• 页码：7379-7390
• 全文大小：732K
• ISSN：1520-5126

文摘

Quantum mechanical/molecular mechanical calculations address the longstanding-question of a 鈥渟econd oxidant鈥?in P450 enzymes wherein the proton-shuttle, which leads to formation of the 鈥減rimary-oxidant鈥?Compound I (Cpd I), was severed by mutating the crucial residue (in P450cam: Threonine-252-to-Alanine, hence T252A). Investigating the oxidant candidates Cpd I, ferric hydroperoxide, and ferric hydrogen peroxide (Fe^III(O₂H₂)), and their reactions, generates reactivity networks which enable us to rule out a 鈥渟econd oxidant鈥?and at the same time identify an additional coupling pathway that is responsible for the epoxidation of 5-methylenylcamphor by the T252A mutant. In this 鈥渟econd-coupling pathway鈥? the reaction starts with the Fe^III(O₂H₂) intermediate, which transforms to Cpd I via a O鈥揙 homolysis/H-abstraction mechanism. The persistence of Fe^III(O₂H₂) and its oxidative reactivity are shown to be determined by interplay of substrate and protein. The substrate 5-methylenylcamphor prevents H₂O₂ release, while the protein controls the Fe^III(O₂H₂) conversion to Cpd I by nailing鈥攖hrough hydrogen-bonding interactions鈥攖he conformation of the HO^{鈥?/sup> radical produced during O鈥揙 homolysis. This conformation prevents HO鈥?/sup> attack on the porphyrin鈥檚 meso position, as in heme oxygenase, and prefers H-abstraction from FeIVOH thereby generating H₂O + Cpd I. Cpd I then performs substrate oxidations. Camphor cannot prevent H₂O₂ release and hence the T252A mutant does not oxidize camphor. This 鈥渟econd pathway鈥?transpires also during H₂O₂ shunting of the cycle of wild-type P450cam, where the additional hydrogen-bonding with Thr252 prevents H₂O₂ release, and contributes to a successful Cpd I formation. The present results lead to a revised catalytic cycle of Cytochrome P450cam.}