Quantum Mechanical/Molecular Mechanical Calculated Reactivity Networks Reveal How Cytochrome P450cam and Its T252A Mutant Select Their Oxidation Pathways Binju Wang Chunsen Li Kshatresh Dutta Dubey Sason Shaik 10.1021/jacs.5b02800.s001 https://acs.figshare.com/articles/journal_contribution/Quantum_Mechanical_Molecular_Mechanical_Calculated_Reactivity_Networks_Reveal_How_Cytochrome_P450cam_and_Its_T252A_Mutant_Select_Their_Oxidation_Pathways/2157718 Quantum mechanical/molecular mechanical calculations address the longstanding-question of a “second oxidant” in P450 enzymes wherein the proton-shuttle, which leads to formation of the “primary-oxidant” 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<sup>III</sup>(O<sub>2</sub>H<sub>2</sub>)), and their reactions, generates reactivity networks which enable us to rule out a “second oxidant” and at the same time identify <i>an additional coupling pathway</i> that is responsible for the epoxidation of 5-methylenylcamphor by the T252A mutant. In this “second-coupling pathway”, the reaction starts with the Fe<sup>III</sup>(O<sub>2</sub>H<sub>2</sub>) intermediate, which transforms to Cpd I via a O–O homolysis/H-abstraction mechanism. The persistence of Fe<sup>III</sup>(O<sub>2</sub>H<sub>2</sub>) and its oxidative reactivity are shown to be determined by interplay of substrate and protein. The substrate 5-methylenylcamphor prevents H<sub>2</sub>O<sub>2</sub> release, while the protein controls the Fe<sup>III</sup>(O<sub>2</sub>H<sub>2</sub>) conversion to Cpd I by nailingthrough hydrogen-bonding interactionsthe conformation of the HO<sup>•</sup> radical produced during O–O homolysis. <i>This conformation prevents HO<sup>•</sup> attack on the porphyrin’s meso position, as in heme oxygenase, and prefers H-abstraction from Fe</i><sup><i>IV</i></sup><i>OH thereby generating H</i><sub><i>2</i></sub><i>O + Cpd I</i>. Cpd I then performs substrate oxidations. Camphor cannot prevent H<sub>2</sub>O<sub>2</sub> release and hence the T252A mutant does not oxidize camphor. This “second pathway” transpires also during H<sub>2</sub>O<sub>2</sub> shunting of the cycle of wild-type P450cam, where the additional hydrogen-bonding with Thr252 prevents H<sub>2</sub>O<sub>2</sub> release, and contributes to a successful Cpd I formation. The present results lead to a revised catalytic cycle of Cytochrome P450cam. 2015-06-17 00:00:00 substrate T 252A Mutant Select Cytochrome P 450cam H 2O release H 2O shunting ferric hydrogen peroxide HO oxidant candidates Cpd T 252A pathway P 450 enzymes FeIII Reactivity Networks Reveal