Variants of the tetrahaem cytochrome c quinol dehydrogenase NrfH characterize the menaquinol-binding site, the haem c-binding motifs and the transmembrane segment

Members of the NapC/NrfH family are multihaem c-type cytochromes that exchange electrons with oxidoreductases situated at the outside of the cytoplasmic membrane or in the periplasmic space of many proteobacteria. They form a group of membrane-bound quinol dehydrogenases that are essential components of several electron transport chains, for example those of periplasmic nitrate respiration and respiratory nitrite ammonification. Knowledge of the structure-function relationships of NapC/NrfH proteins is scarce and only one high-resolution structure (Desulfovibrio vulgaris NrfH) is available. In the present study, several Wolinella succinogenes mutants that produce variants of NrfH, the membrane anchor of the cytochrome c nitrite reductase complex, were constructed and characterized in order to improve the understanding of the putative menaquinol-binding site, the maturation and function of the four covalently bound haem c groups and the importance of the N-terminal transmembrane segment. Based on amino acid sequence alignments, a homology model for W. succinogenes NrfH was constructed that underlines the overall conservation of tertiary structure in spite of a low sequence homology. The results support the proposed architecture of the menaquinol-binding site in D. vulgaris NrfH, demonstrate that each histidine residue arranged in one of the four CX(2)CH haem c-binding motifs is essential for NrfH maturation in W. succinogenes, and indicate a limited flexibility towards the length and structure of the transmembrane region.