Novel heme coordination variants of flavocytochrome P450 BM3
Résumé
Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the heme (P450) domain of the enzyme lead to novel amino acid 6th (distal) coordination ligands to the heme iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and heme domains of P450 BM3. Crystal structures of the mutant heme domains demonstrate axial ligation of P450 heme iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel heme iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the heme, although EPR data indicate Cys264 interactions with heme iron in solution. The A264M heme potential is elevated by comparison to wild-type heme domain, and substrate binding to the A264Q heme domain results in a ~360 mV increase in potential. All mutant heme domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher lauric acid affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at residue A264 is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel heme iron ligand sets unprecedented in nature.
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