Peroxygenase activity of cytochrome c peroxidase and three apolar distal heme pocket mutants: hydroxylation of 1-methoxynaphthalene.

The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the "peroxide shunt" pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific "peroxygenation" catalysts. The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig's blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25°C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 ± 0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150 ± 0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction. Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.