Quantum Mechanical/Molecular Mechanical Calculated Reactivity Networks Reveal How Cytochrome P450cam and Its T252A Mutant Select Their Oxidation Pathways
2015-06-17T00:00:00Z (GMT) by
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 (FeIII(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 FeIII(O2H2) intermediate, which transforms to Cpd I via a O–O homolysis/H-abstraction mechanism. The persistence of FeIII(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 FeIII(O2H2) conversion to Cpd I by nailingthrough hydrogen-bonding interactionsthe 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 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 “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.