1995 Fiscal Year Final Research Report Summary
Study of Functional Mechanism of Nitricoxide Synthase as FAD containing Flavo Protein
| Project/Area Number |
06680586
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | Science University of Tokyo |
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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)
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| Project Period (FY) |
1994 – 1995
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| Keywords | NOS / NADPH / FAD / electron transfer / P450 / flavin / adrenodoxin reductase |
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| 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.
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