Chemical mechanisms for cytochrome P-450 hydroxylation: evidence for acylation of heme-bound dioxygen.

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Abstract
Isotopic tracer methods showed that dihydrolipoic acid (2,3-thioctic acid) acylates the distal O2 of ferrous oxygenated Pseudomonas cytochrome P-450, forming a transient acyl peroxide intermediate that facilitates O-O bond cleavage. Single-turnover studies with 18O2 indicate that one 18O atom is incorporated into the carboxylate group of lipoic acid for each 18O inserted into the substrate, camphor, forming the product, exo-5-hydroxycamphor. Such a branching ratio for label indicates that water is initially released from an unlabeled position and illustrates that the general P-450 mixed-function oxidase stoichiometry generates H218O from 18O2 only after multiple-turnover equilibration with the acylating carboxylate O. Formation of an acyl peroxide state is a natural intermediate in peracid, oxene, or radical mechanisms for methylene C oxygenation.