Modification of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex

Abstract

Modi¢cation of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex IntroductionWolinella succinogenes can grow by respiratory nitrite ammoni¢cation using formate as electron donor [1,2]. The electron transport chain catalyzing this reaction consists of two membrane-bound enzyme complexes and menaquinone. The formate dehydrogenase complex catalyzes the reduction of menaquinone by formate, and menaquinol oxidation by nitrite is catalyzed by the cytochrome c nitrite reductase complex. The former reaction is known to be coupled to the generation of an electrochemical proton potential [3^5], whereas menaquinol oxidation by nitrite appears to be an electroneutral process [2,6,7]. The cytochrome c nitrite reductase complex consists of two c-type cytochromes, the pentaheme catalytic subunit NrfA and the tetraheme subunit NrfH [6^9]. NrfH anchors the complex in the bacterial membrane and mediates electron transport from menaquinol to NrfA [6,7].NrfH and NrfA are encoded by the ¢rst two genes of the nrfHAIJ operon [6]. The nrfI gene product was shown to be required for the attachment of the active site heme group to NrfA [10] while the function of the nrfJ gene is unknown. A vnrfJ deletion mutant of W. succinogenes had wild-type properties with respect to nitrite respiration [6]. Recently, a genetic system has been described that allows expression of mutated nrfH alleles in W. succinogenes [7]. The procedure is based on the integration of a nrfH-containing plasmid into the genome of the W. succinogenes vnrfH deletion mutant thus restoring the nrfHAIJ operon.NrfH belongs to the NapC/NirT family of multiheme c-type cytochromes [2,7,11]. Members of this protein family are commonly found in bacteria and are generally thought to mediate electron transport from a respiratory quinone to a periplasmic oxidoreductase. To date, no high-resolution structure of any member of the NapC/NirT family is available although crystals of the W. succinogenes NrfHA complex have been obtained recently [12]. Furthermore, no data on site-directed modi¢cation of any NapC/NirT-like cytochrome have yet been reported. NrfH contains four heme c binding motifs (CXXCH) to which heme is covalently attached by forming two thioether bonds [7]. The detailed function of the four heme groups is unclear. It is not known how they are arranged in the protein or whether all four hemes are involved in electron transfer from menaquinol to NrfA. In this communication, the role of the NrfH heme groups was investigated by replacing the cysteine residues of the heme c binding motifs. Materials and methods2.1. Growth of W. succinogenes, cell fractionation and puri¢cation of the nitrite reductase complex Strains of W. succinogenes were grown with formate and nitrate as described [13] but (NH 4 ) 2 SO 4 (5 mM) was present in the medium instead of K 2 SO 4 . The medium was supplemented with brain-heartinfusion broth (0.5%, w/v) which was only left out when the capability of growth by nitrite respiration wa...

Multiple haem lyase genes indicate substrate specificity in cytochrome c biogenesis

“…Richardson and J. Simon, unpublished work). Such evidence might also explain the fact that W. succinogenes has previously been shown to be able to covalently attach haem to a modified SX 2 CH motif of the tetrahaem c-type cytochrome NrfH [19]. In contrast, E. coli is apparently unable to attach haem covalently to a monocysteine motif, which is in line with the fact that its genome encodes only two CCHLs that are apparently dedicated to the respective CX n CH and CXXCK motifs [15,20].…”

The cymA Gene, Encoding a Tetraheme c -Type Cytochrome, Is Required for Arsenate Respiration in Shewanella Species

“…However, the physiological outcomes of single cysteine mutations in CXXCH in multiheme cytochromes are not well studied. In the Wolinella succinogenes nitrite reductase, NrfH, seven out of eight individual cysteine replacements resulted in elimination of nitrite respiration (33). Interestingly, one of the heme mutants (C66S) contained a heme covalently linked to the apocytochrome.…”

What is the substrate specificity of the System I cytochrome c biogenesis apparatus?

“…in the cytochromes c 3 of Desulfovibrio species. System II can also attach haem to at least one XXXCH haem-binding motif in a variant cytochrome [14]. Overall, the results suggest that like System I, System II recognizes short stretches of linear polypeptide for haem attachment and that the attachment occurs before substantial folding of the protein.…”