Chemical mechanisms for cytochrome P-450 hydroxylation: evidence for acylation of heme-bound dioxygen.
Using isotopic tracer methods, we have shown that dihydrolipoic acid (2,3-thioctic acid) acylates the distal oxygen of ferrous oxygenated Pseudomonas cytochrome P-450, forming a transient acyl peroxide intermediate that facilitates oxygen-oxygen bond cleavage. Single-turnover studies with 18O2 indicate one oxygen-18 atom incorporated into the carboxylate group of lipoic acid for each oxygen-18 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 unlageled 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 oxygen. Formation of an acyl peroxide state is a natural intermediate in peracid, "oxene", or radical mechanisms for methylene carbone oxygenation.
D000215:Acylation; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006900:Hydroxylation; D010100:Oxygen / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology
D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen