A model of the copper centres of nitrous oxide reductase ( Pseudomonas stutzeri) : Evidence from optical, EPR and MCD spectroscopy

• Published in: FEBS letters 1991-12, Vol.294 (1), p.11-15
• Main Authors: Farrar, Jaqui A., Thomson, Andrew J., Cheesman, Myles R., Dooley, David M., Zumft, Walter G.
• Format: Article
• Language: English
• Subjects: Circular Dichroism; Copper - analysis; Copper protein; Electron Spin Resonance Spectroscopy - methods; EPR; Macromolecular Substances; MCD; Nitrous oxide reductase; Oxidoreductases - chemistry; Protein Conformation; Pseudomonas - enzymology; Pseudomonas stutzeri; Spectrophotometry - methods
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)81331-2

Nitrous oxide reductase (N 2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di-nitrogen. The enzyme has 2 identical subunits ( M 1 ∼ 70 000) of known amino acid sequence and contains ∼ 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi-reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron-transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of Cu A in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.