title: Quantum mechanical/molecular mechanical calculated reactivity networks reveal how cytochrome P450cam and Its T252A mutant select their oxidation pathways.
abstract: 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(III)(O2H2)), and their reactions, generates reactivity networks which enable us to rule out a "second oxidant" 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 "second-coupling pathway", the reaction starts with the Fe(III)(O2H2) intermediate, which transforms to Cpd I via a O-O 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-through hydrogen-bonding interactions-the conformation of the HO(•) radical produced during O-O homolysis. This conformation prevents HO(•) attack on the porphyrin's meso position, as in heme oxygenase, and prefers H-abstraction from Fe(IV)OH 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 "second pathway" 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.
authors.affiliation: †Institute of Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Binju
authors.initials: B
authors.lastname: Wang
authors.affiliation: ‡State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China.|§Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, Xiamen, Fujian 361005, China.
authors.forename: Chunsen
authors.initials: C
authors.lastname: Li
authors.affiliation: †Institute of Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Kshatresh Dutta
authors.initials: KD
authors.lastname: Dubey
authors.affiliation: †Institute of Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Sason
authors.initials: S
authors.lastname: Shaik
mesh_terms: D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D011789:Quantum Theory
chemical_list: D050505:Mutant Proteins; D019475:Camphor 5-Monooxygenase