Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alp ha -hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residue s in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form o f a ferric P450. Further investigation of the Pro243 site revealed that a P 243H mutant also exhibited a nitrogen-bound form, but that the mutants P243 A or P243S did not. On the hydroxylation of myristic acid by the Lys mutant s, we observed a large decrease in activity for R242K and A246K. We therefo re examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic a cid than the wild type. Replacing Ala246 with Ser decreased the catalytic a ctivity, but did not affect affinity for the substrate. An A246V mutant sho wed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The K-m values for H2O2, increased and V-max values decreas ed in the order of wild type, R242K, and R242A when H2O2 was used; furtherm ore, V-max/K-m was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the K-m values wer e not affected much by these substitutions. Together, our results suggest t hat in CYP152A1 the side chain of Pro243 is located close to the iron at th e distal side of a heme molecule; the fatty acid substrate may be positione d near to Ala246 in the catalytic pocket, although Ala246 does not particip ate in hydrophobic interactions with the substrate; and that Arg242 is a cr itical residue for substrate binding and H2O2-specific catalysis. (C) 2001 Academic Press.