Reductive Dehalogenation by Cytochrome P450_CAM: Substrate Binding and Catalysis

Biological reductive dehalogenation reactions are important in environmental detoxification of organohalides. Only scarce information is available on the enzymology underlying these reactions. Cytochrome P450_CAM with a known X-ray structure and well-studied oxygenase reaction cycle, has been studied for its ability to reduce carbon-halogen bonds under anaerobic conditions. The reductive reactions functioned with NADH and the physiological electron-transfer proteins or by using artificial electron donors to reduce cytochrome P450_CAM. Halogenated methane and ethane substrates were transformed by a two-electron reduction and subsequent protonation, β-elimination, or α-elimination to yield alkanes, alkene, or carbene-derived products, respectively. Halogenated substrates bound to the camphor binding site as indicated by saturable changes in the Fe(III)-heme spin state upon substrate addition. Hexachloromethane was bound with a dissociation constant (K_D) of 0.7 and caused >95% shift from low- to high-spin iron. Ethanes bearing fewer chlorine substituents were bound with increasing dissociation constants and gave lesser degrees of iron spin-state change. Camphor competitively inhibited hexachloroethane reduction with an inhibitor constant (K_I) similar to the dissociation constant for camphor (K_I = K_D = 0.9 µM). Rate determinations with pentachloroethane indicated a 100-fold higher enzyme V/K compared to the second-order rate constant for hematin free in solution. These studies on substrate binding and catalysis will help reveal how biological systems enzymatically reduce carbon-halogen bonds in the environment.