Melanie Kern

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd(1) nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.

Characterization of the NapGH quinol dehydrogenase complex involved in Wolinella succinogenes nitrate respiration

Nitrate respiration catalysed by the ε‐proteobacterium Wolinella succinogenes relies on the NapAGHBFLD system that comprises periplasmic nitrate reductase (NapA) and various other Nap proteins required for electron transport from menaquinol to NapA or maturation of Nap components. The W. succinogenes Nap system is unusual as electron transfer to NapA was shown previously to depend on both subunits of the predicted menaquinol dehydrogenase complex NapGH but did not require a cytochrome c of the NapC/NrfH family. Nonetheless, minor residual growth by nitrate respiration was observed in napG and napH gene inactivation mutants. Here, the question is addressed whether alternative membrane‐bound menaquinol dehydrogenases, like NrfH and NosGH, involved in nitrite or N2O reduction systems, are able to functionally replace NapGH. The phenotypes of various gene deletion mutants as well as strains expressing chimeric nap/nos operons demonstrate that NosH is able to donate electrons to the respiratory chain of nitrate respiration at a physiologically relevant rate, whereas NrfH and NosG are not. The iron‐sulphur protein NapG was shown to form a complex with NapH in the membrane but was detected in the periplasmic cell fraction in the absence of NapH. Likewise, NosH is able to bind NapG. Each of the eight poly‐cysteine motifs present in either NapG or NapH was shown to be essential for nitrate respiration. The NapG homologue NosG could not substitute for NapG, even after adjusting the cysteine spacing to that of NapG, implying that NapG and NosG are specific adapter proteins that channel electrons into either the Nap or Nos system. The current model on the structure and function of the NapGH menaquinol dehydrogenase complex is presented and the composition of the electron transport chains that deliver electrons to periplasmic reductases for either nitrate, nitrite or N2O is discussed.

Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration

Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding

In bacterial c‐type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c‐binding motif. Here, a novel octa‐haem c protein, MccA, is described that contains only seven conventional haem c‐binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high‐potential haem c with a red‐shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase‐encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer‐based prediction of putative haem c‐binding motifs in bacterial proteomes.

The oxidative and nitrosative stress defence network of Wolinella succinogenes: cytochrome c nitrite reductase mediates the stress response to nitrite, nitric oxide, hydroxylamine and hydrogen peroxide

Microorganisms employ diverse mechanisms to withstand physiological stress conditions exerted by reactive or toxic oxygen and nitrogen species such as hydrogen peroxide, organic hydroperoxides, superoxide anions, nitrite, hydroxylamine, nitric oxide or NO-generating compounds. This study identified components of the oxidative and nitrosative stress defence network of Wolinella succinogenes, an exceptional Epsilonproteobacterium that lacks both catalase and haemoglobins. Various gene deletion-insertion mutants were constructed, grown by either fumarate respiration or respiratory nitrate ammonification and subjected to disc diffusion, growth and viability assays under stress conditions. It was demonstrated that mainly two periplasmic multihaem c-type cytochromes, namely cytochrome c peroxidase and cytochrome c nitrite reductase (NrfA), mediated resistance to hydrogen peroxide. Two AhpC-type peroxiredoxin isoenzymes were shown to be involved in protection against different organic hydroperoxides. The phenotypes of two superoxide dismutase mutants lacking either SodB or SodB2 implied that both isoenzymes play important roles in oxygen and superoxide stress defence although they are predicted to reside in the cytoplasm and periplasm respectively. NrfA and a cytoplasmic flavodiiron protein (Fdp) were identified as key components of nitric oxide detoxification. In addition, NrfA (but not the hybrid cluster protein Hcp) was found to mediate resistance to hydroxylamine stress. The results indicate the presence of a robust oxidative and nitrosative stress defence network and identify NrfA as a multifunctional cytochrome c involved in both anaerobic respiration and stress protection.

Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

Bacterial c‐type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c‐type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active‐site HBMs (CX2CK or CX2CH). W. succinogenes CcsA2 was found to attach haem to standard CX2CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active‐site CX2CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo‐cytochrome variants carrying the CX15CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non‐standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c‐type cytochromes.

The Wolinella succinogenes mcc gene cluster encodes an unconventional respiratory sulphite reduction system

Assimilatory and dissimilatory sulphite reductions are key reactions in the biogeochemical sulphur cycle and several distinct sirohaem‐containing sulphite reductases have been characterized. Here, we describe that the Epsilonproteobacterium Wolinella succinogenes is able to grow by sulphite respiration (yielding sulphide) with formate as electron donor. Sulphite is reduced by MccA, a prototypical member of an emerging new class of periplasmic cytochrome c sulphite reductases that, phylogenetically, belongs to a multihaem cytochrome c superfamily whose members play crucial roles in the global sulphur and nitrogen cycles. Within this family, MccA represents an unconventional octahaem cytochrome c containing a special haem c group that is bound via two cysteine residues arranged in a unique CX15CH haem c binding motif. The phenotypes of numerous W. succinogenes mutants producing MccA variants underlined the structural importance of this motif. Several open reading frames of the mcc gene cluster were individually inactivated and characterization of the corresponding mutants indicated that the predicted iron‐sulphur protein MccC, the putative quinol dehydrogenase MccD (a member of the NrfD/PsrC family) as well as a peptidyl‐prolyl cis‐trans isomerase, MccB, are essential for sulphite respiration. MccA synthesis in W. succinogenes was found to be induced by sulphite (but not by thiosulphate or sulphide) and repressed in the presence of fumarate or nitrate. Based on the results, a sophisticated model of respiratory sulphite reduction by the Mcc system is presented.

Physiological function and catalytic versatility of bacterial multihaem cytochromes c involved in nitrogen and sulfur cycling

Bacterial MCCs (multihaem cytochromes c) represent widespread respiratory electron-transfer proteins. In addition, some of them convert substrates such as nitrite, hydroxylamine, nitric oxide, hydrazine, sulfite, thiosulfate or hydrogen peroxide. In many cases, only a single function is assigned to a specific MCC in database entries despite the fact that an MCC may accept various substrates, thus making it a multifunctional catalyst that can play diverse physiological roles in bacterial respiration, detoxification and stress defence mechanisms. The present article briefly reviews the structure, function and biogenesis of selected MCCs that catalyse key reactions in the biogeochemical nitrogen and sulfur cycles.

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

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 CX(3)CP 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.

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.