PubMed · 11830388
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P Gillery. [Looking for unity].. https://pubmed.ncbi.nlm.nih.gov/11830388/
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Cytochrome P450 enzymes catalyze a number of oxidations in nature including the difficult hydroxylations of unactivated positions in an alkyl group. The consensus view of the hydroxylation reaction 10 years ago was that a high valent iron-oxo species abstracts a hydrogen atom from the alkyl group to give a radical that subsequently displaces the hydroxy group from iron in a homolytic substitution reaction (hydrogen abstraction-oxygen rebound). More recent mechanistic studies, as summarized in this review, indicated that the cytochrome P450-catalyzed "hydroxylation reaction" is complex, involving multiple mechanisms and multiple oxidants. In addition to the iron-oxo species, another electrophilic oxidant apparently exists, either the hydroperoxo-iron intermediate that precedes iron-oxo or iron-complexed hydrogen peroxide formed by protonation of the hydroperoxo-iron species on the proximal oxygen. The other electrophilic oxidant appears to react by insertion of OH(+) into a C-H bond to give a protonated alcohol. Computational work has suggested that iron-oxo can react through multiple spin states, a low-spin ensemble that reacts by insertion of oxygen, and a high-spin ensemble that reacts by hydrogen atom abstraction to give a radical.
Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.