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

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 reduc...

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Published inJournal 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
LanguageEnglish
Published United States Elsevier Inc 01.11.2007
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Summary: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].
ISSN:0162-0134
1873-3344
DOI:10.1016/j.jinorgbio.2007.06.029