REDUCTIVE DEHALOGENATION BY CYTOCHROME P450(CAM) - SUBSTRATE-BINDING AND CATALYSIS

Biological reductive dehalogenation reactions are important in environ mental detoxification of organohalides. Only scarce information is ava ilable on the enzymology underlying these reactions. Cytochrome P450CA M with a known X-ray structure and well-studied oxygenase reaction cyc le, has been studied for its ability to reduce carbon-halogen bonds un der anaerobic conditions. The reductive reactions functioned with NADH and the physiological electron-transfer proteins or by using artifici al electron donors to reduce cytochrome P450CAM. Halogenated methane a nd ethane substrates were transformed by a two-electron reduction and subsequent protonation, beta-elimination, or alpha-elimination to yiel d alkanes, alkene, or carbene-derived products, respectively. Halogena ted substrates bound to the camphor binding site as indicated by satur able changes in the Fe(III)-heme spin state upon substrate addition. H exachloromethane was bound with a dissociation constant (K(D)) of 0.7 muM and caused >95% shift from low- to high-spin iron. Ethanes bearing fewer chlorine substituents were bound with increasing dissociation c onstants and gave lesser degrees of iron spin-state change. Camphor co mpetitively inhibited hexachloroethane reduction with an inhibitor con stant (K(I)) similar to the dissociation constant for camphor (K(I) = K(D) = 0.9 muM). Rate determinations with pentachloroethane indicated a 100-fold higher enzyme V/K compared to the second-order rate constan t for hematin free in solution. These studies on substrate binding and catalysis will help reveal how biological systems enzymatically reduc e carbon-halogen bonds in the environment.