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Crystallization and structural analysis of binary complex of P450 electron donor and its electron transfer mechanism

Research Project

Project/Area Number 09680651
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionOsaka University

Principal Investigator

HORI Hiroshi  Graduate School of Engineering Science, Osaka University, Res. Assoc., 大学院・基礎工学研究科, 助手 (20127294)

Co-Investigator(Kenkyū-buntansha) 堀 洋  大阪大学, 大学院・基礎工学研究科, 助手 (20127294)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
KeywordsCytochrome P450cam / substrate / electron donor / electron transfer / X-ray crystallography / EPR / ENDOR / Putidaredoxin / 電子・核二重共鳴(ENDOR) / 単結晶顕微分光
Research Abstract

Probing molecular structure of Pdx-P450cam binary complex by EPR and ENDOR spectroscopy : During the monooxygenation reaction by cytochrome P450cam, a complex of P450cam and reduced putidaredoxin (Pdx) is formed as an obligatory intermediate of the reaction. We found that EPR spectrum of the reduced Pdx in its binary complex with the camphor-bound ferrous P450cam was altered significantly upon ligation of OィイD22ィエD2, CO and NO to the ferrous heme of P450cam and that Arg-112 and 109 at the putative Pdx-binding site of P450cam played important roles associated with this phenomenon. X-band ィイD11ィエD1H-ENDOR results of the Pdx-P450cam binary complex demonstrated that the interactions of protons of cystein ligands with the unpaired electrons on the iron atoms in the reduced Pdx were altered upon CO binding to the ferrous heme of P450cam. We also found that the EPR spectrum of the reduced adrenodoxin (Adx) in its binary complex with cytochrome P450scc was altered upon CO binding to the ferrous heme of P450scc in the presence of 20S-hydroxy cholesterol, substrate analogue. Precise analyses are in progress.
Crystallization of Pdx-P450cam binary complex : A substrate-free P450cam was crystallized having different crystalline form from that had been reported previously. Single crystal microspectrophotometry indicated that substrate-camphor never bound to P450cam in this crystal. X-ray crystallographic analysis of this substrate-free P450cam indicated that the positions of the side chains of the amino acid residues near the substrate-binding site were significantly altered as compared with the previously reported molecular structure. We have also made every effort to crystallize the binary complex of Pdx-P450cam. The crystallization of this binary complex is not succeeded until now. The efforts of the crystallization are in progress.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] Hideo Shimada: "Putidaredoxin-Cytochrome P450cam Interaction : Spin State of the Heme Ion Modulates Putidaredoxin Structure"J. Biol. Chem.. 274. 9363-9369 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Ryu Makino: "EPR Characterization of Axial Bond in Metal Centerof Native and Cobalt-substituted Guanylate Cyclase"J. Biol. Chem.. 274. 7714-7723 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Shiro Kominami: "Electron Paramagnetic Resonance Studies on the Distal Region of the Ferric Heme in Rat Neurnal Nitric Oxide Synthase"J. Biochem.. 126. 756-761 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Motonari Tsubaki: "Furier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coil bo-type ubiquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Motonari Tsubaki: "20β-Hydroxy-C21-Steroid 20β-Oxidase Activity of Cytochrome P450c21 Purified from Bovine Adrenocortical Microsomes"Biochim. Biophys. Acta. 1390. 197-206 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sam-Yong Park: "Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor"FEBS Lett.. 412. 346-350 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hideo Shimada et al.: "Putidaredoxin-Cytochrome P450cam Interaction : Spin State of the Heme Iron Modulates Putidaredoxin Structure"J. Biol. Chem.. 274. 9363-9369 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Ryu Makino et al.: "EPR Characterization of Axial Bond in Metal Center of Native and Cobalt-substituted Guanylate Cyclase"J. Biol. Chem.. 274. 7714-7723 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Shiro Kominami et al.: "Electron Paramagnetic Resonance Studies on the Distal Region of the Ferric Heme in Rat Neuronal Nitric Oxide Synthase"J. Biochem.. 126. 756-761 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Motonari Tsubaki et al.: "Furier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Motonari Tsubaki et al.: "20β-Hydroxy-C21-Steroid 20β-Oxidase Activity of Cytochrome P450c21 Purified from Bovine Adrenocortical Microsomes"Biochim. Biophys. Acta. 1390. 197-206 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sam-Yong Park et al.: "Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor"FEBS Lett.. 412. 346-350 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hideo Shimada: "Putidaredoxin-Cytochrome P450cam Interaction: Spin State of the Heme Iron Modulates Putidaredoxin Structure"J.Biol.Chem.. 274. 9363-9369 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Ryu Makino: "EPR Characterization of Axial Bond in Metal Center of Native and Cobalt-substituted Guanylate Cyclase"J.Biol.Chem.. 274. 7714-7723 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Shiro Kominami: "Electron Paramagnetic Resonance Studies on the Distal Region of the Ferric Heme in Rat Neuronal Nitric Oxide Synthase"J.Biochem.. 126. 756-761 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Motonari Tsubaki: "Furier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Motonari Tsubaki: "Azide-and Cyanide-binding to the Escherichia coli bd-type Ubiquinol oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies"J.Biochem.. 126. 510-519 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Motonari Tsubaki: "Fluoride-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption and EPR Spectroscopies"J.Biochem.. 126. 98-103 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tatsushi Mogi: "Substitutions of Conserved Aromatic Amino Acid Residues in Subunit I Perturb the Metal Centers of the Escherichia coli bo-type Ubiquinol Oxidase" Biochemistry. 37. 1632-1639 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Atsushi Morimoto: "Detection of a Tryptophan Radical as an Intermediate Species in the Reaction of Horseradish Peroxidase Mutant(Phe221→Trp)with Hydrogen Peroxide" J.Biol.Chem. 273. 14753-14760 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Tatsushi Mogi: "Two Terminal Quinol Oxidase Families in Escherichia coli:Variations on Molecular Machinery for Dioxygen Reduction" J.Biochem.Mol.Biol.& Biophys.2. 79-110 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Masaya Suzuki: "Synthesis and Characterization of Mixed μ-Alkoxo-diiron(II,III)Complexes with an Unsymmetric Dinucleating Ligand" Inorg.Chim.Acta. 283. 124-135 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Ryu Makino: "EPR Characterization of Axial Bond in Metal Center of Native and Cobalt-substituted Guanylate Cyclase" J.Biol.Chem.(印刷中). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hideo Shimada: "Putidaredoxin-Cytochrome P450cam Interaction:Spin State of the Heme Iron Modulates Putidaredoxin Structure" J.Biol.Chem. (印刷中). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hirohito Yoneyama: "Optical and electron paramagnetic resonance absorption spectra of complexes of nitric oxide synthase I with isocyanides" Biochim. Biophys. Acta. 1335. 253-264 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Motonari Tsubaki: "Existence of Two Heme B Centers in Cytochrome b_<561> from bovine adrenal chromaffin vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy" J. Biol. Chem.272. 23206-23210 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sam-Yong Park: "Crystallization,preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450ner" FEBS Lett.412. 346-350 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Motonari Tsubaki: "Resonance Raman,infrared and EPR investigation on the binuclear site structure of the heme-copper ubiquinol oxidases from Acetobacter aceti" Biochemistry. 36. 13034-13042 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Motonari Tsubaki: "20β-Hydroxy-C_<21>-Steroid 20β-Oxidase Activity of Cytochrome P450c_<21> Purified from Borine Adrenocortical Microsomes" Biochim. Biophys. Acta. (印刷中). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Hideo Shimada: "Oxygen Binding to P450cam Induces Conformational Changes of Putidaredoxin in the Ferrous P450cam-Reduced Putidaredoxin Complex" Oxygen Homeostasis and Its Dynamics : keio University Symposia for Life Science and Medicine ed.Y.Ishimura. Vol.1. 139-146 (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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