Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101).

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W-V247L (80 min-1 or 2.5 x the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.