Anaerobic cytochrome c 5 5 2 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichiu 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 5 5 2 indicated its similarity to Desulfbvibrio desulfuricans hexaheme cytochrome. The cytochrome ~5 5 2 showed nitrite and hydroxylamine reductase activities with benzyl viologen a s 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 pM and 18 mM, respectively. The nitrite reductase activity of the cytochrome cSs2 was inhibited effectively by cupric ion and cyanide.A soluble c-type cytochrome, cytochrome ( ' 5 5 2 is synthesized in Escherichiu coli and some other Enterobacteriacede when they are grown anaerobically [I, 21. Biosynthesis of this cytochrome is repressed by molecular oxygen and stimulated anaerobically by the presence of nitrate or nitrite in the medium [ 3 , 41. In E. coli K12, this synthesis is under the control of an activator protein encoded by the j n r gene, which is necessary for the induced synthesis of a number of components in the anaerobic respiratory systems, including nitrate reductase, nitrite reductase, fumarate reductase, and hydrogenase reported that reduced cytochrome c S s 2 from E. coli Yamaguchi is rapidly reoxidized by addition of nitrite. They found that cytochrome ~5 5 2 is located in the periplasinic space [8] and concluded that its in vivo function is reduction of toxic nitrite produced by nitrate respiration to ammonia at the cell surface 191. However, the protein-chemical properties of the cytochrome as a nitrite reductase have not been studied extensively.Abou-Jaoudt et al. [lo] proposed the possible involvement of cytochrome ~5 5 2 in the formate/nitrite reductase system in whole cells of E. coli K12. Energy conservation during formate-dependent reduction of nitrite by E. coli was demonstrated [I 1, 121. Moreover, from reconstitution experiments, Liu et al. 1131 suggested the role of cytochrome ~5 5 2 as a terminal nitrite reductase in NADH-dependent nitrite reduction. However, the exact physiological function ofcytochrome has not yet been elucidated.

A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c552 which was ten times higher than that found in wild‐type cells. The level of membrane‐bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l‐lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b, nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c552 was the result of a separate mutation, the location of which is yet to be determined.

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