Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450(cam) (CYP101)

CYP101 (cytochrome P450(cam)) catalyses the oxidation of camphor but has al so been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biod egradation of these environmental contaminants. The hexachloroethane dehalo genation activity of CYP101 has been investigated by mutagenesis. The effec ts 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 we akened hexachloroethane binding but did not affect the rate of reaction. Th e 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 rat e. The effect of the individual mutations was additive in multiple mutants, acid 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 hexach loroethane binding and dehalogenation, and hexachloroethane binding and deh alogenation places conflicting demands on active-site polarity and compromi ses were necessary to achieve reasonable values for both.