Active-site mobility inhibits reductive dehalogenation of 1,1,1-trichloroethane by cytochrome P450cam

SummaryRecent studies by Wackett and co-workers have shown that cytochrome P450cam is capable of reductively dehalogenating hexachloroethane at a significant rate, but that no appreciable dehalogenation of 1,1,1-trichloroethane is observed. A growing body of evidence indicates that differences in intrinsic reactivity can not completely explain this observation. We therefore explored the possible role of differences in preferred binding orientation and in active-site mobility. A detailed analysis of molecular dynamics trajectories with each of these substrates bound at the active site of P450cam is presented. While the dynamics and overall time-average structure calculated for the protein are similar in the two trajectories, the two substrates behave quite differently. The smaller substrate, 1,1,1-trichloroethane, is significantly more mobile than hexachloroethane and has a preferred orientation in which the substituted carbon is generally far from the heme iron. In contrast, for hexachloroethane, one of the chlorine atoms is nearly always in van der Waals contact with the heme iron, which should favor the initial electron transfer step.

[1]  Ortiz de Montellano,et al.  Cytochrome P-450: Structure, Mechanism, and Biochemistry , 1986 .

[2]  Macdonald Tl.  Chemical mechanisms of halocarbon metabolism , 1983 .

[3]  R. Ornstein,et al.  Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3‐hydroxynorcamphor product , 1993, Protein science : a publication of the Protein Society.

[4]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[5]  Mark T. Fisher,et al.  Control of heme protein redox potential and reduction rate: linear free energy relation between potential and ferric spin state equilibrium , 1985 .

[6]  P A Kollman,et al.  Molecular dynamics studies of a DNA‐binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor , 1992, Protein science : a publication of the Protein Society.

[7]  P. R. Montellano.  Oxygen Activation and Transfer , 1986 .

[8]  J. Collins,et al.  THEORETICAL AND EXPERIMENTAL ANALYSIS OF THE ABSOLUTE STEREOCHEMISTRY OF CIS-BETA -METHYLSTYRENE EPOXIDATION BY CYTOCHROME P450CAM , 1991 .

[9]  S. Sligar,et al.  Cytochrome P-450cam and Other Bacterial P-450 Enzymes , 1986 .

[10]  R. Raag,et al.  Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation. , 1991, Biochemistry.

[11]  V. Vilker,et al.  Stability of Cytochrome P‐450 Enzyme and the Dehalogenation Activity in Resting Cultures of Pseudomonas putida PpG 786 , 1989 .

[12]  D. Osguthorpe,et al.  Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase‐trimethoprim, a drug‐receptor system , 1988, Proteins.

[13]  T. Poulos,et al.  High-resolution crystal structure of cytochrome P450cam. , 1987, Journal of molecular biology.

[14]  S. Sligar,et al.  Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation. , 1993, Protein engineering.

[15]  Jeffrey P. Jones,et al.  The binding and regioselectivity of reaction of (R)- and (S)-nicotine with cytochrome P-450cam: parallel experimental and theoretical studies , 1993 .

[16]  R. Raag,et al.  Cytochrome P450cam: crystallography, oxygen activation, and electron transfer1 , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  R. Ornstein,et al.  A 175‐psec molecular dynamics simulation of camphor‐bound cytochrome P‐450cam , 1991, Proteins.

[18]  Jack R. Collins,et al.  Valproic acid metabolism by cytochrome P450 : a theoretical study of stereoelectronic modulators of product distribution , 1991 .

[19]  Bernard R. Brooks,et al.  The effects of environment and hydration on protein dynamics: A simulation study of myoglobin , 1991 .

[20]  D. Lide.  Handbook of Chemistry and Physics , 1992 .

[21]  M. W. Anders.  Bioactivation of foreign compounds , 1985 .

[22]  Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure. , 1993, Protein engineering.

[23]  Rebecca C. Wade,et al.  Solvation of the active site of cytochrome P450-cam , 1990, J. Comput. Aided Mol. Des..

[24]  C. Walsh,et al.  Enzymatic Reaction Mechanisms , 1978 .

[25]  D E Wemmer,et al.  Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box” , 1994, Proteins.

[26]  V. Vilker,et al.  Biodehalogenation of bromotrichloromethane and 1,2‐dibromo‐3‐chloropropane by Pseudomonas putida PpG‐786 , 1987, Biotechnology and bioengineering.

[27]  P. Ortiz de Montellano,et al.  Calculated and experimental absolute stereochemistry of the styrene and .beta.-methylstyrene epoxides formed by cytochrome P 450cam , 1992 .

[28]  C. E. Castro,et al.  Biodehalogenation: Active site versus enzymatic and whole cell rates with cytochromes P‐450 , 1989 .

[29]  F. Guengerich,et al.  Metabolism of trichloroethylene in isolated hepatocytes, microsomes, and reconstituted enzyme systems containing cytochrome P-450. , 1983, Cancer research.

[30]  R. C. Weast.  CRC Handbook of Chemistry and Physics , 1973 .

[31]  S. Sligar,et al.  Metabolic switching in cytochrome P-450cam: Deuterium isotope effects on regiospecificity and the monooxygenase/oxidase ratio , 1987 .

[32]  C. E. Castro,et al.  Biodehalogenation: reactions of cytochrome P-450 with polyhalomethanes. , 1985, Biochemistry.

[33]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[34]  Mark D. Paulsen,et al.  Predicting the product specificity and coupling of cytochrome P450cam , 1992, J. Comput. Aided Mol. Des..

[35]  D W Nebert,et al.  P450 genes: structure, evolution, and regulation. , 1987, Annual review of biochemistry.

[36]  S. Sligar,et al.  Mutagenesis of cytochromes P450cam and b5. , 1991, Methods in enzymology.

[37]  R. Ornstein,et al.  An evaluation of implicit and explicit solvent model systems for the molecular dynamics simulation of bacteriophage T4 lysozyme , 1994, Proteins.

[38]  F. Guengerich.  Reactions and significance of cytochrome P-450 enzymes. , 1991, The Journal of biological chemistry.

[39]  R. Ornstein,et al.  Substrate mobility in a deeply buried active site: Analysis of norcamphor bound to cytochrome P‐450cam as determined by a 201‐psec molecular dynamics simulation , 1992, Proteins.

[40]  R. Ornstein,et al.  Ethylbenzene hydroxylation by cytochrome P450cam. , 1992, Biochemical and biophysical research communications.

[41]  J. M. Zachara,et al.  Chemical contaminants on DOE lands and selection of contaminant mixtures for subsurface science research , 1992 .

[42]  R. Ornstein,et al.  A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding , 1992, Proteins.

[43]  Miller Re,et al.  Oxidation of trichloroethylene by liver microsomal cytochrome P-450: evidence for chlorine migration in a transition state not involving trichloroethylene oxide. , 1982 .

[44]  A. D. McLean,et al.  Theoretical investigation of the anaerobic reduction of halogenated alkanes by cytochrome P-450. 2. Vertical electron affinities of chlorofluoromethanes as a measure of their activity , 1988 .

[45]  C. Di Primo,et al.  Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P-450cam induced by hydrostatic pressure. , 1990, European journal of biochemistry.

[46]  L. Wackett,et al.  Reductive dechlorination catalyzed by bacterial transition-metal coenzymes , 1991 .

[47]  R. Raag,et al.  The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM. , 1991, Biochemistry.

[48]  Analysis of active site motions from a 175 picosecond molecular dynamics simulation of camphor-bound cytochrome P450cam. , 1991, Journal of biomolecular structure & dynamics.