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Related Experiment Videos

Viscosity effects on eukaryotic nitrate reductase activity.

Guillaume G Barbier1, Wilbur H Campbell

  • 1The Nitrate Elimination Company, Inc., Lake Linden, Michigan 49945, USA.

The Journal of Biological Chemistry
|May 18, 2005
PubMed
Summary

Catalysis by nitrate reductase (NaR) involves conformational changes. Viscosity studies reveal that "hinge" regions in NaR likely gate internal electron-proton transfers, controlling enzyme activity.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Eukaryotic molybdenum-containing nitrate reductase (NaR) catalyzes nitrate reduction.
  • NaR contains internal electron carriers separated by hinge regions.
  • Understanding rate-limiting steps is crucial for enzyme mechanism elucidation.

Purpose of the Study:

  • Investigate rate-limiting processes in NaR catalysis.
  • Determine the role of hinge regions in regulating internal electron-proton transfer (IET).
  • Assess the impact of conformational changes on NaR activity.

Main Methods:

  • Utilized viscosogens (glycerol, sucrose) to alter solution viscosity.
  • Assayed NAD(P)H:NaR activity in corn, Arabidopsis, and yeast NaR.
  • Measured activities of recombinant NaR modules (cytochrome b reductase - CbR) and holo-NaR.

Main Results:

  • Increased solution viscosity negatively affected holo-NaR activity, indicating rate limitation by conformational changes.
  • Viscosity had minimal impact on CbR module activity, suggesting no large conformational changes.
  • Viscosity hindered partial activities involving Hinge 2, implicating it in gating IET.

Conclusions:

  • Both Hinge 1 and Hinge 2 in NaR restrict cytochrome b domain movement.
  • These hinges act as gates for internal electron-proton transfer steps.
  • Conformational flexibility is critical for NaR catalytic efficiency.

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