The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes

Abstract

The electron-transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c-type cytochromes. NrfA is the catalytic subunit; its crystal structure is known. NrfH belongs to the NapC/NirT family of membrane-bound c-type cytochromes and mediates electron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A DnrfH deletion mutant of W. succinogenes was constructed by replacing the nrfH gene with a kanamycin-resistance gene cartridge. This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression. The nrfHAIJ gene cluster was restored by integration of an nrfH-containing plasmid into the genome of the DnrfH mutant. The resulting strain had wild-type properties with respect to growth by nitrite respiration and nitrite reductase activity. A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5 0 end produced wild-type amounts of NrfA in the absence of the NrfH protein. NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacteria. The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH. The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.Keywords: cytochrome c nitrite reductase; NapC/NirT family; nitrite respiration; Wolinella succinogenes.The epsilon proteobacterium Wolinella succinogenes can grow by anaerobic respiration using formate as electron donor and nitrite as electron acceptor [1]. The electron transport chain that catalyzes nitrite reduction by formate is integrated into the bacterial membrane and consists of formate dehydrogenase, menaquinone and the heterodimeric cytochrome c nitrite reductase complex (NrfHA) [2][3][4]. The purified complex of NrfH and NrfA catalyzed nitrite reduction to ammonia by the soluble menaquinol analog 2,3-dimethyl-1,4-naphthoquinol, whereas, on its own, the catalytic subunit NrfA reacted only with artificial electron donors such as reduced viologen dyes [4,5]. Proteoliposomes containing the NrfHA complex together with menaquinone and formate dehydrogenase catalyzed nitrite reduction by formate which was coupled to the generation of an electrochemical proton potential across the liposomal membrane [4]. The crystal structure of NrfA, a pentaheme cytochrome c, was recently determined [6].NrfH apparently mediates the electron transfer from menaquinol located in the membrane to NrfA. The NrfH protein is a c-type cytochrome with four CXXCH heme c binding motifs, which is encoded by the first gene of the nrfHAIJ locu...

Periplasmic nitrate reduction in Wolinella succinogenes: cytoplasmic NapF facilitates NapA maturation and requires the menaquinol dehydrogenase NapH for membrane attachment

“…This mode of respiration depends on two terminal periplasmic reductases, nitrate reductase (NapA) and cytochrome c nitrite reductase (NrfA), and mutants deficient in either napA or nrfA did not grow by nitrate or nitrite respiration, respectively (Simon et al, 2000(Simon et al, , 2003. While the NapCtype tetrahaem cytochrome c NrfH protein was shown to form a membrane-bound complex with NrfA that catalyses nitrite reduction using menaquinone as electron donor (Simon et al, 2000(Simon et al, , 2001Gross et al, 2005), the electrontransfer route from menaquinol to NapA was less obvious until the essential role of the putative menaquinol dehydrogenase complex NapGH in nitrate respiration was demonstrated (Kern et al, 2007;. Fig.…”

Role of individual nap gene cluster products in NapC-independent nitrate respiration of Wolinella succinogenes

“…In most cases NapB is thought to be reduced by NapC, a membrane-bound tetrahaem cytochrome c that is thought to catalyse quinol oxidation. NapC is prototypic for members of the NapC/NirT/NrfH family, which mediate electron transfer in various anaerobic respiratory chains (Roldán et al, 1998;Simon et al, 2000Simon et al, , 2001Gross et al, 2005;Rodrigues et al, 2006). More recently, a putative membrane-bound complex consisting of the two predicted iron-sulfur proteins, NapG and NapH, was proposed to be functionally equivalent to NapC Simon et al, 2003Simon et al, , 2004.…”

Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA

“…In both types, nitrite reductase (NrfA) functions as the terminal reductase. The NrfH protein is a tetraheme c-type cytochrome of the NapC/NirT family that is responsible for passing electrons from menaquinol to NrfA and functionally equivalent to NrfBCD of the NrfABCD type system (Simon et al, 2000(Simon et al, , 2001Simon, 2002).…”