Open AccessPurification of a hexaheme cytochrome C 552 from Escherichia coli K 12 and its properties as a nitrite reductase
Author(s) -
KAJIE Shinichi,
ANRAKU Yasuhiro
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09419.x
Subject(s) - hydroxylamine , chemistry , cytochrome , nitrite , heme , cytochrome c , nitrite reductase , polyacrylamide gel electrophoresis , chromatography , nuclear chemistry , biochemistry , organic chemistry , enzyme , nitrate , mitochondrion
Anaerobic cytochrome c 552 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 69000 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 c 552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c 552 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 μM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c 552 was inhibited effectively by cupric ion and cyanide.