Relatively few studies of naphthalene hydroxylation by the cytochrome P450s have been reported. Rat liver microsomes hydroxylate naphthalene with a monooxygenation turnover rate of 0.32 min-1 and cytochrome P450cam hydroxylates naphthalene with a rate of 0.7 min-1. Protein engineering of cytochrome P450cam produced a Y96F mutant, which increases the rate of naphthalene hydroxylation to 100 min-1, the fastest reported for a cytochrome P450. Joo et al. have been using laboratory evolution techniques to enhance the peroxygenase activity of cytochrome P450cam. Using a fluorescence technique to monitor naphthalene hydroxylation, Joo and colleagues screened approximately 200,000 random mutants of cytochrome P450cam and were able to select a mutant that had 11-fold higher peroxygenase activity than did the wild-type enzyme although absolute rates were not reported.
Cytochrome P450BSβ is a naturally-occurring peroxygenase, which hydroxylates fatty acids predominantly at the β position . It has no monooxygenase activity and does not react with hydrogen peroxide in the absence of the fatty acid substrate. Myristic acid is the preferred substrate and the turnover rate is about 300 min-1. In the presence of a short-chain fatty acid, cytochrome P450BSβ will catalyze the peroxygenation of a variety of nonnatural substrates such as styrene, ethylbenzene, and 1-methoxynaphthalene.
Shoji et al. investigated the rates of peroxygenation of 1-methoxynaphthalene by three heme proteins, cytochrome P450BSβ, HRP, and metmyoglobin. HRP had no detectable activity, while sperm whale metmyoglobin has a turnover number of about 0.03 min-1. The H64A mutation of metmyoglobin increases the rate of Russig’s blue formation almost 800-fold to 23 min-1. Cytochrome P450BSβ had a turnover number of 112 min-1 for 1-methoxynaphthalene hydroxylation.
In this study, we show that CcP has detectable hydroxylation activity with a turnover number of 0.0044 min-1, faster than that of HRP but slower than that of metMb and cytochrome P450BSβ. The three CcP mutants designed to bind non-polar substrates within the heme pocket, CcP(triAla), CcP(triVal), and CcP(triLeu), are about 30-fold more active than rCcP, however, none approached the activities of metMb(H64A) or cytochrome P450BSβ. Two CcP mutants, H175C and H175C/D235L, designed to simulate the heme ligation in cytochrome P450 have peroxygenase activities that are intermediate between those of rCcP and CcP(triAla).
One of the anticipated strengths of CcP as a platform to develop specific peroxygenase catalysts is the stability of the initial oxidized intermediate in the CcP/hydrogen peroxide reaction, CcP Compound I, and the stability with respect to oxidative degradation by excess hydrogen peroxide [25, 26]. The half-life of CcP compound I is about 6 hours at pH 6  and CcP can react with up to 10 equivalents of hydrogen peroxide in the absence of oxidizable substrates before significant reduction in catalytic activity occurs . However, in the Russig’s blue assay, we saw rapid bleaching of the heme, Figure 2, and complete inactivation of CcP(triAla) within four catalytic cycles. In our screening process, we found that CcP(triAla) is one of the most susceptible mutants to heme degradation with a loss of 73% of the Soret absorbance during the Russig’s blue assay, in the Additional file 1: Figure SA.8. Interestingly, there is about a 10-fold variation in heme degradation during the Russig’s blue assay for the twenty CcP mutants screened in this study, Figure A8. CcP(H52N) is the most stable of the screened mutants with only 8% heme degradation while CcP(W51H) is the most susceptible mutant with 74% heme degradation. The degradation does not correlate with either the rate of compound I formation or with the rate of 1-methoxynaphthalene hydroxylation. It may be due to secondary oxidation of the hydroxylated product within the distal heme pocket, Figure 1, forming substrate-based radicals which, in turn, react with the heme causing the observed degradation.