Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase.
Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006418:Heme / Q000302:isolation & purification*; D007526:Isoelectric Point; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009572:Nitrite Reductases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D000596:Amino Acids; D003574:Cytochrome c Group; C083951:cytochrome c553; D006418:Heme; C022302:cytochrome C-552; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases