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The copper site in nitrous oxide reductase.

P M Kroneck¹, J Riester, W G Zumft

¹Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.

Biology of Metals

|January 1, 1990

Summary

Researchers characterized nitrous oxide reductase from Pseudomonas stutzeri using multifrequency electron paramagnetic resonance spectroscopy. Findings reveal a mixed-valence copper site crucial for enzyme activity and structural organization.

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Area of Science:

Biochemistry
Enzymology
Spectroscopy

Background:

Nitrous oxide reductase (N2OR) is a key enzyme in the denitrification pathway, catalyzing the reduction of N2O to N2.
Understanding the structure and function of N2OR is crucial for microbial ecology and environmental science.
Pseudomonas stutzeri is a well-studied denitrifying bacterium, making its N2OR a relevant subject for biochemical investigation.

Purpose of the Study:

To characterize the novel copper enzyme nitrous oxide reductase from Pseudomonas stutzeri.
To elucidate the electronic and structural properties of the active site using multifrequency electron paramagnetic resonance (EPR) spectroscopy.
To investigate the role of redox-active residues in the enzyme's structure and function.

Main Methods:

Multifrequency electron paramagnetic resonance (EPR) spectroscopy (2.4, 3.4, 4.5, 9.31, and 35 GHz).
Enzyme characterization of various forms of nitrous oxide reductase.
Oxidation/reduction experiments.
Quantitative determination of sulfhydryl and disulfide residues.

Main Results:

A mixed-valence [Cu(1.5)...Cu(1.5)] S = 1/2 species with a delocalized unpaired electron was identified.
This mixed-valence copper site is present in both active and catalytically inactive N2OR derivatives.
EPR spectral similarities between N2OR and the CuA site of cytochrome c oxidase suggest analogous structural and electronic arrangements.
Cysteine/cystine redox couple involvement in the active site's structural organization was proposed.

Conclusions:

The study identifies a unique mixed-valence copper site in nitrous oxide reductase.
Electron paramagnetic resonance spectroscopy provides critical insights into the enzyme's active site structure.
The findings suggest a structural and functional link between the copper site in N2OR and the CuA site in cytochrome c oxidase.
The cysteine/cystine redox couple plays a role in maintaining the active site's structural integrity.

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