Purification of a hexaheme cytochrome C552 from Escherichia coli K 12 and its properties as a nitrite reductase

Abstract

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.

Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine–lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli

“…Subsequently, similar cytochromes were isolated from a variety of anaerobic bacteria and, on the basis of their similar sizes, specific activities, spectra and epr properties were classified as a family of hexahaem nitrite reductases (Liu and Peck, 1981;Blackmore et al, 1986;Kajie and Anraku, 1986;Sadana et al, 1986;Liu et al, 1988;Costa et al, 1990;Schumacher and Kroneck, 1991). This view became untenable when the sequence of the E. coli enzyme became available and revealed the presence of only 10 cysteine residues, eight of which were arranged as conventional CXXCH haem-binding motifs (Darwin et al, 1993).…”

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli

“…Attempts to express cytochrome C3 in functional form in Escherichia coli under aerobic or anaerobic growth conditions were unsuccessful because of the inability of E. coli to insert the hemes (19). E. coli does express multiheme c-type cytochromes (e.g., a hexahemoprotein [nitrite reductase] is expressed when nitrate is the terminal oxidant [14]), and its inability to produce holo-cytochrome C3 under these same conditions points to a possible specificity of the heme insertion system. It is therefore appropriate to test more closely related hosts (e.g., D. desulfuricans G200 [8]) for their potential to produce holo-cytochrome C3.…”

“…The objectives of the characterization of the three cytochromes C3 were (i) to define the properties of the G200 cytochrome C3, which has not been purified before, and (ii) to determine whether the Hildenborough cytochrome C3 produced by the G200 strain (the recombinant protein) was the same as that synthesized by D. vulgaris Hildenborough (the native protein). All three isolated proteins migrated as single polypeptides with apparent molecular weights of 14 kilodaltons during sodium dodecyl sulfate-gel electrophoresis (not shown). Isoelectric focusing using a PHAST GEL apparatus from Pharmacia LKB Biotechnology (11) with Phast Gel IEF 3-9 and a Pharmacia Broad Range pl Calibration Kit, containing proteins with isoelectric points ranging from 3 to 10, indicated a pl of 5.8 for the G200 protein ( Table 2).…”

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

“…Nitrite, NO⅐, and hydroxylamine are each reduced stoichiometrically to ammonium such that no intermediates are detectable (15)(16)(17)(18)(19). Because all three substrates form the same product it has been suggested that NO⅐ and hydroxylamine are intermediates during nitrite reduction.…”

“…Quinol oxidation is catalyzed by NrfD and electrons are transferred, most likely via NrfC and NrfB, to NrfA, a homodimeric, deca-heme-containing cytochrome c nitrite reductase (15). NrfA homologs are present in a wide range of bacteria, and in vitro they have been shown to reduce not only nitrite but also NO⅐ and hydroxylamine to ammonium without releasing detectable intermediates (15)(16)(17)(18)(19). As a consequence, it was proposed that NO⅐ reduction by NrfA, rather than nonspecific reduction by another Nrf component, was responsible for providing E. coli with resistance toward NO⅐ under anaerobic conditions (6).…”