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A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase.
Hein J Wijma1, Lars J C Jeuken, Martin P Verbeet
1Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
The Journal of Biological Chemistry
|April 15, 2006
Summary
Nitrite reductase (NiR) uses a random-sequential mechanism for nitrite reduction. Electron transfer between copper sites is rate-limiting and pH-dependent, influencing catalytic activity.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Bioinorganic chemistry
Background:
- Nitrite reductase (NiR) is a homotrimeric enzyme containing type-1 and type-2 copper centers.
- It catalyzes the reduction of nitrite to nitric oxide, a crucial step in the nitrogen cycle.
Purpose of the Study:
- To elucidate the catalytic mechanism of NiR.
- To investigate the influence of pH and nitrite concentration on NiR activity.
- To determine the rate-limiting step in the enzymatic reaction.
Main Methods:
- Steady-state kinetic measurements with varying pH and nitrite concentrations.
- Electrochemical analysis of NiR immobilized on a graphite electrode.
- Site-directed mutagenesis (NiR M150T) to probe electron transfer rates.
Main Results:
- NiR follows a random-sequential mechanism with rate-limiting intramolecular electron transfer.
- Enzyme activity is pH-dependent, with different substrate binding orders at low and high nitrite concentrations.
- Mutant NiR (M150T) exhibited significantly slower electron transfer, confirming its role in the catalytic cycle.
Conclusions:
- The study confirms NiR employs a random-sequential mechanism.
- pH and nitrite concentration modulate the catalytic efficiency by affecting electron transfer and substrate binding.
- Intramolecular electron transfer between copper centers is a critical determinant of NiR activity.