1887

Abstract

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periplasmic nitrate reductase NapA. The composition of the corresponding gene clusters varies but, in addition to , genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some loci predict accessory proteins such as the iron–sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron–sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CXCP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

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2009-08-01
2020-01-23
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