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1999 Fiscal Year Final Research Report Summary

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
KeywordsCytochrome P450cam / substrate / electron donor / electron transfer / X-ray crystallography / EPR / ENDOR / Putidaredoxin
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.

  • Research Products

    (12 results)

All Other

All Publications (12 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 2001-10-23  

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