Putative helix F contributes to regioselectivity of hydroxylation in mitochondrial cytochrome P450 27A1

On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P 450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe -215 within putative helix F and at Trp-235 and Tyr-238 within putative hel ix G in P450 27A1 and compared wild type and mutants with respect to cataly tic activity, product pattern, substrate binding, formation of hydrogen per oxide, and interaction with redox partner. Results indicate that the mutate d residues are important for delivery of the correctly oriented substrate t o the P450 active site. The I211K and F215K mutations, for example, affecte d the regioselectivity of P450 27A1-dependent hydroxylation reactions and c onferred the P450 capacity to cleave the C-C bond of the substrate during t he catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has function s similar to those of its counterpart in P450 27A1 (Phe-215). We propose th at putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hyd roxylation in mitochondrial P450s.