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 nailingthrough hydrogen-bonding interactionsthe
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