Sulphide-linked Nitrite Reductase from Thiobacillus denitrificans with Cytochrome Oxidase Activity: Purification and Properties

Abstract

A membrane-bound, sulfide-linked nitrite reductase from T. denitrificans was solubilized and after further purification its properties were examined. The purified enzyme, MW 120,000, contained cytochromes c and d in the ratio of 1:1. Both cytochromes were reduced by sulfide and re-oxidized with nitrite or air. Oxidation by nitrite resulted in the appearance of an absorption peak at 572 nm. The kinetics of the reduction of the enzyme with sulfide indicated that cytochrome c was reduced before cytochrome d. The redox potential of cytochrome d was 22 mV more positive than that of cytochrome c. Cytochromes c and d were dissociated from the purfied enzyme by treatment with sodium dodecyl sulfate. The purified nitrite reductase also had cytochrome oxidase activity and both the activities were stimulated by cytochrome c-551 isolated from T. denitrificans. Reduced cytochrome c-551 was an effective electron donor for the purified enzyme with either nitrite or air as the terminal electron acceptor. Neither cytochrome c-554 (also isolated from T. denitrificans) nor mammalian cytochrome c was effective as reductant for the enzyme. NO and N2O were identified as the products of nitrite reduction by the purified sulfide-linked nitrite reductase.