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

The electron-transport chain that catalyzes nitrite respiration with format e in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction b y 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 t he NapC/NirT family of membrane-bound c-type cytochromes and mediates elect ron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A Delta nrfH deletion mutan t of W. succinogenes was constructed by replacing the nrfH gene with a kana mycin-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 pla smid into the genome of the Delta nrfH mutant. The resulting strain had wil d-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' end produced wild- type amounts of NrfA in the absence of the NrfH protein. NrfA was located e xclusively in the soluble cell fraction of the stopH mutant, indicating tha t NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacter ia. The stopH mutant did not grow by nitrite respiration and did not cataly ze 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.