Reductive dehalogenation by cytochrome P450CAM: 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 P450CAM 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 P450CAM. Halogenated methane and ethane substrates were transformed by a two-electron reduction and subsequent protonation, beta-elimination, or alpha-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 (KD) of 0.7 microM 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 (KI) similar to the dissociation constant for camphor (KI = KD = 0.9 microM). 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.
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Department of Biochemistry, University of Minnesota, St. Paul 55108.
Wackett
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D019475:Camphor 5-Monooxygenase; D002245:Carbon Dioxide / Q000378:metabolism; D002384:Catalysis; D005617:Chlorofluorocarbons, Methane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D006418:Heme / Q000737:chemistry; D006846:Hydrocarbons, Halogenated / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology; D013379:Substrate Specificity
D005617:Chlorofluorocarbons, Methane; D006846:Hydrocarbons, Halogenated; D009243:NAD; D002245:Carbon Dioxide; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C005848:trichlorofluoromethane; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase