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

Abstract

An overexpression system for nitrous oxide reductase (N2OR), an enzyme that catalyzes the conversion of N2O to N2 and H2O, has been developed in Achromobacter cycloclastes. Anaerobically purified A. cycloclastes recombinant N2OR (AcN2OR) has on average 4.5 Cu and 1.2 S per monomer. Upon reduction by methyl viologen, AcN2OR displays a high specific activity: 124U/mg at 25°C. Anaerobically purified AcN2OR 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 CuZ=[4Cu(I)] state and the CuZ=[3Cu(I)·Cu(II)] state, with the latter readily reducible by reduced forms of viologens. CD spectra of the as-purified AcN2OR 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 AcN2OR in D2O is significantly decreased compared with that in H2O, indicative of a significant solvent isotope effect on N2O reduction. These data are in good agreement with conclusions reached in recent studies on the effect of pH on catalysis by N2OR [K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613–615].