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Study of Functional Mechanism of Nitricoxide Synthase as FAD containing Flavo Protein

Research Project

Project/Area Number 06680586
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Structural biochemistry
Research InstitutionScience University of Tokyo

Principal Investigator

MIURA Shigetoshi  Science Univrsity of Tokyo, Department of Biological Associate Professor, Science and Technology, 基礎工学部, 助教授 (70190585)

Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
KeywordsNOS / NADPH / FAD / electron transfer / P450 / flavin / adrenodoxin reductase / 一酸化窒素
Research Abstract

NADPH-adrenoferredoxin reductase, a flavoprotein from bovine adrenocortical mitochondria, has been investigated to elucidate the equilibrium and dynamic properties of the interaction with NADP^+ and adrenoferredoxin (adrenodoxin) using proton NMR spectroscopy. The line width of the signals from NADP^+ depends on the presence of the reductase. The off rate constant of NADP^+ from the reductase is estimated to be about 15-20 s^<-1> on the basis of line width measurements. No appreciable difference in off rate is detected between adenine and nicotinamide moieties of NADP^+. Transferred nuclear Overhauser effect (transferred NOE) experiments for NADP^+ indicate the time dependent magnetization transfer profiles with a long lag phase. The proton NMR spectra during the titration of the reductase with adrenodoxin reveal that the reductase possesses distinct binding sites for both NADP^+ and adrenodoxin. The sharp resonances in the aromatic region due to his-10 and his-62 of adrenodoxin were utilized as a probe to explore the interaction with the reductase. In the mixure of adrenodoxin and the reductase at the mole ratio of 6 : 1, T1 values of the histidine residue in adrenodoxin were measured by inversion recovery method. At low ionic strength, T1 values of the resonances are not affected in the presence or absence of the reductase. In the presence of the reductase, T1 values of resonances due to the histidine residues become shorter as the concentration of KC1 increases because of rapid exchange between bound and free states. At low ionic strength (10mM phosphate buffer), the off rate from the reductase is estimated to be less than about 4s^<-1>. The off rate of adrenodoxin from the reductase could be the rate limiting step in cytochrome c reductase activity at low ionic strength.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] Shigetoshi Miura: "Interaction of NADPH-Adrenoferredoxin Reductase with NADP+and Adrenoferredoxin : Equilibrium and Dynamic Properties Investigated by Proton Nuclear Magnetic Resonance" The Journal of Biological Chemistry.269,. 8001-8006 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Motonari Tsubaki: "Electron Paramagnetic Resonance Investigation of CYtochrome P450c21 from Bovine Adrenocortical Microsomes : A New Enzymatic Activity" Biochimica et Biophysica Acta. 1259,. 89-98 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kazuo Kobayashi: "Interaction of NADPH-Adrenodoxin Reductase with NADP+ as Studied by Pulse Radiolysis" Biochemistry. 34,. 12932-12936 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Shigetoshi Miura and yoshiyuki Ichikawa: "Interaction of NADPH-Adrenoferredoxin Reductase with NADP+ and Adrenoferredoxin : Equilibrium and Dynamic Properties Investigated by Proton Nuclear Magnetic Resonance" The Journal of Biological Chemistry. 269. 8001-8006 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Motonari Tsubaki, Kohji Morimoto, Shuhei Tomita, Shigetoshi Miura, Yoshiyuki Ichikawa, Akira Miyatake Futoshi Masuya, and Hiroshi Hori: "Electron Paramagnetic Resonance Investigation of Cytochrome P450c21 from Bovine Adrenocortical Microsomes : A New Enzymatic Activity" Biochimica et Biophysica Acta. 1259. 89-98 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kazuo Kobayashi, Shigetoshi Miura, Miyako Miki, Yoshiyuki Ichikawa, and Seiichi Tagawa: "Interaction of NADPH-Adrenodoxin Reductase with NADP+ as Studied by Pulse Radiolysis" Biochemistry. 34. 12932-12936 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Shigetoshi Miura and Yoshiyuki Ichikawa: Flavins and Flavoproteins (Yagi, K.ed.) (1H-NMR Investigation of NADPH-Adrenoferredoxin Reductase with NADP+ and Adrenoferredoxin). Walter de Gruyer & Co., 459-462 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Maki Tsujita, Shuhei Tomita, Yoshinori Matsuo, Shigetoshi Miura, and Yoshiyuki Ichikawa: Flavins and Flavoproteins (Yagi, K.ed.) Physicochemical Properties of Retinal Oxidase Purified from Rabbit Hepatic Cytosol. Walter de Gruyer & Co., 463-466 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Miyako Miki, Kazuo Kobayashi, Shigetoshi Miura, and Yoshiyuki Ichikawa: Flavins and Flavoproteins (Yagi, K.ed.) One Electron Reduction of Adrenodoxin Reductase as Studied by Pulse Radiolysis. Walter de Gruyer & Co., 463-466 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Shigetoshi Miura: "Interaction of NADPH-Adrenoferredoxin Reductase with NADP_+ and Adrenoferredoxin: Equilibrium and Dynamic Properties" The Journal of Biological Chemistry. 269,. 8001-8006 ((1994))

    • Related Report
      1995 Annual Research Report
  • [Publications] Motonari Tsubaki: "Electron Paramagnetic Resonance Investigation of Cytochrome P450c21 from Bovine Adrenocortical Microsomes: A New Enzymatic Activity" Biochimica et Biophysica Acta. 1259,. 89-98 ((1995))

    • Related Report
      1995 Annual Research Report
  • [Publications] Kazuo Kobayashi: "Interaction of NADPH-Adrenodoxin Reductase with NADP_+ as Studied Pulse Radiolysis" Biochemistry. 34,. 12932-12936 ((1995))

    • Related Report
      1995 Annual Research Report
  • [Publications] Miura Shigetoshi: "Interaction of NADPH-Adrenoferredoxin Reductase with NADP+and Adrenoferredoxin" The Journal of Biological Chemistry. 269. 8001-8006 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Miura Shigetoshi: "Flarins and Flavoproteins" Walter de Gruyter, 4 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Miki Miyako: "Flarins and Flavoproteins" Walter de Gruyter, 4 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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