Purification and some characteristics of a cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.
Nitrous oxide reductase from Wolinella succinogenes was purified very nearly to homogeneity. The enzyme was found to be dimeric, with Mr = 162,000 and subunit Mr = 88,000, and to contain three copper atoms and one iron atom (as cytochrome c) per subunit. The oxidized enzyme exhibited absorption bands at 410 and 528 nm, and the dithionite-reduced enzyme, at 416, 520, and 550 nm. The isoelectric point was 8.6; specific activity was at 25 degrees C and pH 7.1, 160 mumol x min-1 x mg-1; and Km was 7.5 microM N2O under the same conditions. alpha-Chymotrypsin cleaved the enzyme into cytochrome c-depleted dimers with an average Mr = 134,000 and cytochrome c-enriched fragments with an average Mr = 13,000. The enzyme was stable at 4 degrees C for at least 100 h under air and 3 h in the presence of 5 mM EDTA. It exhibited a dithionite-N2O oxidoreductase as well as a BV+-N2O oxidoreductase activity. During turnover with BV+ at 25 mM N2O, the enzyme was observed to undergo an initial activation and a subsequent inactivation. The kinetics of inactivation were approximately first-order in remaining activity, and the first-order rate constant was essentially independent of the initial enzyme concentration. These characteristics are consistent with the occurrence of turnover-dependent inactivation. Acetylene was a relatively weak inhibitor, but cyanide and azide were rather strong inhibitors. The nitrous oxide reductase of W. succinogenes is quite different from that of denitrifying bacteria. The amount of activity in cell extracts and the absence of O2-labile nitrous oxide reductase suggested that the cytochrome c containing enzyme may be the only one produced by W. succinogenes.
Teraguchi
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Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Hollocher
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TC
D001438:Bacteroidaceae / Q000201:enzymology*; D002918:Chymotrypsin; D003574:Cytochrome c Group / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D002918:Chymotrypsin