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

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 nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap 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 Wolinella succinogenes 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 napH 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 CX3CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF 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.

The Mononuclear Molybdenum Enzymes

“…The classical respiratory nitrate reductase, Nar, couples nitrate respiration to proton translocation across the inner membrane of bacteria, thereby contributing to the transmembrane proton gradient that is subsequently used to drive ATP synthesis. 442 The periplasmically localized complexes, that is, Nap and pNar, are more complex and functionally diverse. Neither by itself can contribute to the proton gradient across the inner membrane, although when nitrate reduction is coupled with formate oxidation, they can contribute to the transmembrane proton gradient.…”

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria

“…In W. succinogenes, Nap encoded by the napAGHBFLD gene cluster. The role of individual nap genes in W. succinogenes has been assessed by characterizing nonpolar gene inactivation mutants (Kern, Mager, & Simon, 2007;Kern & Simon, 2008, 2009b. NapB and NapD were shown to be essential for growth by nitrate respiration, with NapD being required for the production of mature NapA.…”

Nitrate and periplasmic nitrate reductases

“…101,102 Similar to Nar, Nap has the ability to function in nitrate respiration. 103 In some organisms, nitrate respiration is dependent on the presence of the nap operon 104 e.g. , Epsilonproteobacteria.…”