Structure of a cytochrome P450-redox partner electron-transfer complex.

The crystal structure of the complex between the heme- and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 A resolution. The flavodoxin-like flavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 A from the peptide that precedes the heme-binding loop and the heme iron, respectively. The heme-binding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the flavin-binding sites, are responsible for different redox properties of the FMN, which, in turn, control electron flow to the P450.

[1]  M. Waterman,et al.  Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding. , 1992, The Journal of biological chemistry.

[2]  G. Sheldrick,et al.  SHELXL: high-resolution refinement. , 1997, Methods in enzymology.

[3]  G. Tollin,et al.  Electrostatic interactions during electron transfer reactions between c-type cytochromes and flavodoxin. , 1985, The Journal of biological chemistry.

[4]  T. Poulos,et al.  Modeling protein-substrate interactions in the heme domain of cytochrome P450(BM-3). , 1994, Acta crystallographica. Section D, Biological crystallography.

[5]  M. Waterman,et al.  Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity* , 1997, The Journal of Biological Chemistry.

[6]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[7]  J Deisenhofer,et al.  Structure and function of cytochromes P450: a comparative analysis of three crystal structures. , 1995, Structure.

[8]  A. Fulco,et al.  Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. , 1989, The Journal of biological chemistry.

[9]  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.

[10]  Jones Ta,et al.  Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. , 1985, Methods in enzymology.

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

[12]  Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains. , 1997, Archives of biochemistry and biophysics.

[13]  A. Makower,et al.  Selective chemical modification of a functionally linked lysine in cytochrome P-450 LM2. , 1984, Biochimica et biophysica acta.

[14]  J Deisenhofer,et al.  Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. , 1993, Science.

[15]  J. Peterson,et al.  The kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin. , 1981, The Journal of biological chemistry.

[16]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

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

[18]  M. J. Coon,et al.  Cytochrome P-450 : multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms , 1991 .

[19]  C. Kasper,et al.  Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Z. Zhou,et al.  The cumulative electrostatic effect of aromatic stacking interactions and the negative electrostatic environment of the flavin mononucleotide binding site is a major determinant of the reduction potential for the flavodoxin from Desulfovibrio vulgaris [Hildenborough]. , 1996, Biochemistry.

[21]  J. Schenkman,et al.  The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing. , 1992, The Journal of biological chemistry.

[22]  G. Ratnaswamy,et al.  An NMR-derived model for the solution structure of oxidized putidaredoxin, a 2-Fe, 2-S ferredoxin from Pseudomonas. , 1994, Biochemistry.

[23]  L. Narhi,et al.  Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. , 1986, The Journal of biological chemistry.

[24]  J. Peterson,et al.  Domain-domain interaction in cytochrome P450BM-3. , 1996, Biochimie.

[25]  I. Vakser,et al.  Identification of the Binding Site on Cytochrome P450 2B4 for Cytochrome b 5 and Cytochrome P450 Reductase* , 1998, The Journal of Biological Chemistry.

[26]  M. J. Coon,et al.  The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. , 1993, DNA and cell biology.

[27]  T. Poulos,et al.  The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid , 1997, Nature Structural Biology.

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

[29]  Electron Transfer between the FMN and Heme Domains of Cytochrome P450BM-3 , 1997, The Journal of Biological Chemistry.

[30]  U. Heinemann,et al.  New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4-108). , 1998, Structure.

[31]  J Deisenhofer,et al.  Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution. , 1994, Journal of molecular biology.

[32]  T. Poulos,et al.  Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains. , 1991, The Journal of biological chemistry.

[33]  R. Swenson,et al.  Regulation of oxidation-reduction potentials through redox-linked ionization in the Y98H mutant of the Desulfovibrio vulgaris [Hildenborough] flavodoxin: direct proton nuclear magnetic resonance spectroscopic evidence for the redox-dependent shift in the pKa of Histidine-98. , 1997, Biochemistry.

[34]  M. J. Coon,et al.  Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase. , 1981, The Journal of biological chemistry.

[35]  F. Richards,et al.  Electrostatic orientation during electron transfer between flavodoxin and cytochrome c , 1983, Nature.

[36]  M. Klein,et al.  Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3. , 1993, The Journal of biological chemistry.

[37]  H. Strobel,et al.  Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450. , 1988, Archives of biochemistry and biophysics.

[38]  Z. Zhou,et al.  Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough). , 1995, Biochemistry.

[39]  Y. Feng,et al.  Evaluation of the role of specific acidic amino acid residues in electron transfer between the flavodoxin and cytochrome c3 from Desulfovibrio vulgaris. , 1997, Biochemistry.

[40]  T. Porter,et al.  An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals. , 1991, Trends in biochemical sciences.

[41]  S. Boddupalli,et al.  Reconstitution of the fatty acid hydroxylation function of cytochrome P-450BM-3 utilizing its individual recombinant hemo- and flavoprotein domains. , 1992, The Journal of biological chemistry.

[42]  Y. Fujii‐Kuriyama,et al.  Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase. , 1991, The Journal of biological chemistry.

[43]  G. Krey,et al.  Site-directed mutagenesis of tyrosine-98 in the flavodoxin from Desulfovibrio vulgaris (Hildenborough): regulation of oxidation-reduction properties of the bound FMN cofactor by aromatic, solvent, and electrostatic interactions. , 1994, Biochemistry.

[44]  B C Finzel,et al.  The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. , 1985, The Journal of biological chemistry.

[45]  S. Boddupalli,et al.  Expression, purification, and properties of the flavoprotein domain of cytochrome P-450BM-3. Evidence for the importance of the amino-terminal region for FMN binding. , 1991, The Journal of biological chemistry.

[46]  J. Peterson,et al.  Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. , 1996, Biochemistry.

[47]  K D Watenpaugh,et al.  Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures. , 1993, Journal of molecular biology.

[48]  B. Masters,et al.  Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[49]  J. Peterson,et al.  The flavoprotein domain of P450BM-3: expression, purification, and properties of the flavin adenine dinucleotide- and flavin mononucleotide-binding subdomains. , 1996, Biochemistry.

[50]  M. Eren,et al.  Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes. , 1997, Biochemistry.

[51]  S. Sligar,et al.  Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochromeb5. , 1988, The Journal of biological chemistry.

[52]  H. Strobel,et al.  Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase. , 1993, Archives of biochemistry and biophysics.

[53]  T. Poulos,et al.  The Domain Architecture of Cytochrome P450BM-3* , 1997, The Journal of Biological Chemistry.

[54]  T. Poulos,et al.  Structure of cytochrome P450eryF involved in erythromycin biosynthesis , 1995, Nature Structural Biology.