Anaerobic purification, characterization and preliminary mechanistic study of recombinant nitrous oxide reductase from Achromobacter cycloclastes

• Published in: Journal of inorganic biochemistry Vol. 101; no. 11; pp. 1836 - 1844
• Main Authors: Fujita, Koyu, Chan, Jeannine M., Bollinger, John A., Alvarez, Marcela L., Dooley, David M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2007
• Subjects: Achromobacter cycloclastes; Achromobacter cycloclastes - genetics; Anaerobic purification; Anaerobiosis; Catalysis; Circular Dichroism; Copper; Copper - chemistry; Denitrification; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Nitrous Oxide - metabolism; Nitrous oxide reductase; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Denitrification; Nitrous oxide reductase; Anaerobic purification; Copper; Achromobacter cycloclastes
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2007.06.029

An overexpression system for nitrous oxide reductase (N 2OR), an enzyme that catalyzes the conversion of N 2O to N 2 and H 2O, has been developed in Achromobacter cycloclastes. Anaerobically purified A. cycloclastes recombinant N 2OR ( AcN 2OR) has on average 4.5 Cu and 1.2 S per monomer. Upon reduction by methyl viologen, AcN 2OR displays a high specific activity: 124 U/mg at 25 °C. Anaerobically purified AcN 2OR displays a unique absorption spectrum. UV–visible and EPR spectra, combined with kinetics studies, indicate that the as-purified form of the enzyme is predominately a mixture of the fully-reduced Cu Z = [4Cu(I)] state and the Cu Z = [3Cu(I) · Cu(II)] state, with the latter readily reducible by reduced forms of viologens. CD spectra of the as-purified AcN 2OR over a range of pH values reveal perturbations of the protein conformation induced by pH variations, although the principal secondary structure elements are largely unaltered. Further, the activity of AcN 2OR in D 2O is significantly decreased compared with that in H 2O, indicative of a significant solvent isotope effect on N 2O reduction. These data are in good agreement with conclusions reached in recent studies on the effect of pH on catalysis by N 2OR [K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613–615].