Electrochemical characterisation of the human cytochrome P450 CYP2C9.

The electrochemistry of human cytochrome P4502C9 (CYP2C9) was characterised using purified His-tagged enzyme. The His-tagged enzyme was shown to have similar functional characteristics to native CYP2C9 heterologously expressed in Escherichia coli and to the CYP2C9 activity of human liver microsomes. Evidence was observed for a reversible one-electron transfer between the P450 heme and the electrode. Both pH and ionic strength influenced the electrochemical behaviour of CYP2C9. A range of substrates was investigated to determine the effect of the heme-substrate interaction on CYP2C9 redox potential. In the absence of oxygen, tolbutamide, diclofenac, warfarin and sulfaphenazole did not alter the redox potential of the iron heme. In contrast, torsemide, carbon monoxide and oxygen led to an anodic shift in redox potential. These results suggest alternative mechanisms by which CYP2C9 (and by inference other P450 enzymes) may alter redox potential to facilitate electron delivery from physiological donors.

[1]  James F Rusling,et al.  Optimization of Electrochemical and Peroxide‐Driven Oxidation of Styrene with Ultrathin Polyion Films Containing Cytochrome P450cam and Myoglobin , 2003, Chembiochem : a European journal of chemical biology.

[2]  J. Rusling.  Enzyme Bioelectrochemistry in Cast Biomembrane-Like Films , 1998 .

[3]  Claudio Nicolini,et al.  P450scc Engineering and Nanostructuring for Cholesterol Sensing , 2001 .

[4]  L. Wong,et al.  The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system , 1999, FEBS letters.

[5]  Y. Ueno,et al.  Conformation between the substrate-binding site and heme of cytochrome P-450 studied by excitation energy transfer. , 1987, Biochimica et biophysica acta.

[6]  S. Imaoka,et al.  Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity. , 1992, Biochemistry.

[7]  J. Schenkman,et al.  Differences in the mechanism of functional interaction between NADPH-cytochrome P-450 reductase and its redox partners. , 1986, Molecular pharmacology.

[8]  Robert L. Haining,et al.  Enzymatic Determinants of the Substrate Specificity of CYP2C9: Role of B‘−C Loop Residues in Providing the π-Stacking Anchor Site for Warfarin Binding† , 1999 .

[9]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. , 1964, The Journal of biological chemistry.

[10]  Jose Cosme,et al.  Crystal structure of human cytochrome P450 2C9 with bound warfarin , 2003, Nature.

[11]  F. Guengerich,et al.  Conformational change and activation of cytochrome P450 2B1 induced by salt and phospholipid. , 1998, Archives of biochemistry and biophysics.

[12]  S. Sligar,et al.  A thermodynamic model of regulation: modulation of redox equilibria in camphor monoxygenase. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[13]  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 .

[14]  N. Orme-Johnson,et al.  Beef adrenal cortical cytochrome P-450 which catalyzes the conversion of cholesterol to pregnenolone. Oxidation-reduction potentials of the free, steroid-complexed, and adrenodoxin-complexed P-450. , 1981, The Journal of biological chemistry.

[15]  J. Schenkman,et al.  Quantitative analyses of electrostatic interactions between NADPH-cytochrome P450 reductase and cytochrome P450 enzymes. , 1994, The Journal of biological chemistry.

[16]  G. P. Kuznetsova,et al.  Optical biosensor investigation of interactions of biomembrane and water‐soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers , 2001, Journal of molecular recognition : JMR.

[17]  V. Urlacher,et al.  Redox properties of cytochrome p450BM3 measured by direct methods. , 2003, European journal of biochemistry.

[18]  Kondo‐François Aguey‐Zinsou,et al.  Electrochemistry of P450cin: new insights into P450 electron transfer. , 2003, Chemical communications.

[19]  D. Mansuy,et al.  Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid. , 2003, Archives of biochemistry and biophysics.

[20]  R. Tuckey,et al.  Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode. , 2003, Bioelectrochemistry.

[21]  Timothy S Tracy,et al.  Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations. , 2003, Archives of biochemistry and biophysics.

[22]  D. Mansuy,et al.  The substrate binding site of human liver cytochrome P450 2C9: an NMR study. , 1997, Biochemistry.

[23]  J. Miners,et al.  Human hepatic cytochrome P450 2C9 catalyzes the rate-limiting pathway of torsemide metabolism. , 1995, The Journal of pharmacology and experimental therapeutics.

[24]  D. Lewis,et al.  Allosteric phenomena in cytochrome P450-catalyzed monooxygenations. , 2001, European journal of biochemistry.

[25]  M. J. Coon,et al.  Interactions of cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and substrate in the reconstituted liver microsomal enzyme system. , 1980, The Journal of biological chemistry.

[26]  F. Guengerich,et al.  Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. , 2001, Chemical research in toxicology.

[27]  James F Rusling,et al.  Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions. , 2003, Biophysical chemistry.

[28]  T. Omura,et al.  Forty years of cytochrome P450. , 1999, Biochemical and biophysical research communications.

[29]  J. Miners,et al.  Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and relationship to other cytochrome P-450 dependent xenobiotic oxidations. , 1988, Biochemical pharmacology.

[30]  J. Miners,et al.  Cytochrome P4502C9: an enzyme of major importance in human drug metabolism. , 1998, British journal of clinical pharmacology.

[31]  A Boobis,et al.  Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways. , 1999, Biochemical pharmacology.

[32]  F W Scheller,et al.  Clay-bridged electron transfer between cytochrome p450(cam) and electrode. , 2000, Biochemical and biophysical research communications.

[33]  M. Sutcliffe,et al.  Role of conserved Asp293 of cytochrome P450 2C9 in substrate recognition and catalytic activity. , 2003, The Biochemical journal.

[34]  J. Schenkman,et al.  Direct electron injection from electrodes to cytochromeP450cam in biomembrane-like films , 1997 .

[35]  C. Amatore,et al.  Trends in Molecular Electrochemistry , 2004 .

[36]  J. Miners,et al.  Use of tolbutamide as a substrate probe for human hepatic cytochrome P450 2C9. , 1996, Methods in enzymology.

[37]  E. Laviron.  General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems , 1979 .

[38]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. , 1964, The Journal of biological chemistry.

[39]  C Masimirembwa,et al.  Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function. , 2000, Biochemical and biophysical research communications.

[40]  Israel Hanukoglu.  Electron Transfer Proteins of Cytochrome P450 Systems , 1996 .

[41]  L. S. Wong,et al.  Direct electrochemistry of putidaredoxin at a modified gold electrode , 1995 .

[42]  S. Boye,et al.  Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9 , 2004, Xenobiotica; the fate of foreign compounds in biological systems.

[43]  V. Reipa,et al.  Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate. , 2002, Chemical communications.

[44]  A. Y. Lu,et al.  Studies on the stimulation of cytochrome P-450-dependent monooxygenase activity by dilauroylphosphatidylcholine. , 1981, Archives of biochemistry and biophysics.

[45]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[46]  J. Liu,et al.  Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase activity and alpha-naphthoflavone stimulation. , 1998, Archives of biochemistry and biophysics.

[47]  S. Sligar,et al.  Coupling of spin, substrate, and redox equilibria in cytochrome P450. , 1976, Biochemistry.

[48]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[49]  T. Poulos,et al.  Redox control of the catalytic cycle of flavocytochrome P-450 BM3. , 1997, Biochemistry.

[50]  James F Rusling,et al.  An amperometric biosensor with human CYP3A4 as a novel drug screening tool. , 2003, Biochemical pharmacology.