A highly conserved acid residue is found in the I-helix of most cytochrome P450s and has been suggested to play a critical function in oxygen activation and substrate hydroxylation in these monooxygenases. We have investigated this hypothesis for cytochrome P45O(BM-3) by replacing the naturally occurring glutamate at position 267 with a glutamine residue. In the case of P45O(BM-3), mutation of the glutamate to glutamine as position 267 drastically reduces the catalytic activity of the enzyme when palmitate is used as a substrate for hydroxylation. On the other hand, the activity change toward laurate hydroxylation is relatively small. The much slower catalytic turnover by the mutant enzyme in palmitate hydroxylation compared with wild type allows the observation of a new spectral intermediate in the hemoprotein. This intermediate is similar to that observed in the corresponding active site acid-to-amide replacement in cytochrome P450(cam) (N. C. Gerber and S. G. Sligar (1994) J. Biol. Chem. 269, 4260-4266). Also, in analogy with P450(cam), this mutation does not lead to any side oxidation processes which produce hydrogen peroxide. Interestingly, however, the alteration in the active site structure which is implied by the change in regio specificity may also effect substrate packing thus leading to the uncoupling of the enzyme to produce additional water rather than a commitment to substrate oxidation. In addition, the distribution of hydroxylation products is altered by this mutation, suggesting some perturbation of the recognition property in P450(BM-3).
ASJC Scopus subject areas
- Molecular Biology