Surface‐modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine‐free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys‐58, Cys‐85, Cys‐136, Cys‐148 and Cys‐334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin‐state equilibria, NADH turnover rates and camphor‐hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox‐active label, N‐ferrocenylmaleimide, to the single surface cysteine mutant SCF‐K344C, and the electrochemistry of this modified mutant are also described.

[1]  I. C. Gunsalus,et al.  Cytochrome P-450cam. I. Crystallization and properties. , 1974, The Journal of biological chemistry.

[2]  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 .

[3]  H. Hill,et al.  N‐(2‐Ferrocene‐ethyl)maleimide : a new electroactive sulphydryl‐specific reagent for cysteine‐containing peptides and proteins , 1996, FEBS letters.

[4]  J. Peterson.  Camphor binding by Pseudomonas putida cytochrome P-450 , 1971 .

[5]  James F. Rusling,et al.  Direct Electrochemistry of Myoglobin and Cytochrome P450cam in Alternate Layer-by-Layer Films with DNA and Other Polyions , 1998 .

[6]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

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

[8]  L. Wong,et al.  The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme. , 1997, Protein engineering.

[9]  L. Wong,et al.  Covalent Attachment of an Electroactive Sulphydryl Reagent in the Active Site of Cytochrome P450Cam as Revealed by the Crystal Structure of the Modified Protein , 1998 .

[10]  Alan M. Bond,et al.  The transient nature of the diffusion controlled component of the electrochemistry of cytochrome c at ‘bare’ gold electrodes: an explanation based on a self-blocking mechanism , 1997 .

[11]  L. Wong,et al.  The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site‐specific mutagenesis , 1997, FEBS letters.

[12]  R. Brizzolara,et al.  Evidence for covalent attachment of purple membrane to a gold surface via genetic modification of bacteriorhodopsin , 1997 .

[13]  S. Sligar,et al.  Spin state control of the hepatic cytochrome P450 redox potential. , 1979, Biochemical and biophysical research communications.

[14]  H. Shimada,et al.  Role of Arg112 of Cytochrome P450cam in the Electron Transfer from Reduced Putidaredoxin , 1996, The Journal of Biological Chemistry.

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

[16]  K. Nakamura,et al.  Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin. , 1994, Biochimica et biophysica acta.

[17]  J. Peterson,et al.  Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins. , 1990, The Journal of biological chemistry.

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

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

[20]  W. Pryor.  Cytochrome P450: Structure, mechanism, and biochemistry , 1996 .

[21]  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.

[22]  G. Ratnaswamy,et al.  A structure-based model for cytochrome P450cam-putidaredoxin interactions. , 1996, Biochimie.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  S. Sligar,et al.  The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling. , 1990, Biochemistry.

[25]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[26]  T. Poulos,et al.  Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex. , 1989, Biochemistry.

[27]  K. Yura,et al.  Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin , 1993, FEBS letters.

[28]  Fumio Mizutani,et al.  Site-specific immobilization of molecularly engineered dihydrofolate reductase to gold surfaces , 1994 .

[29]  K. Sekimizu,et al.  Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam. , 1994, Biochimica et biophysica acta.

[30]  S. Sligar,et al.  Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. , 1986, The Journal of biological chemistry.

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