Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, B.; Lenz, Oliver

doi:10.1074/jbc.m113.544668

Cited by 10 publications

(7 citation statements)

References 65 publications

(103 reference statements)

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“…In this regard, other roles are also possible and compatible with the available evidence, for example as specific assembly factors and/or structural scaffolds for the hydrogenases. For example, it has been demonstrated that a rubredoxin-related protein is important for aerobic maturation of the group 1d [NiFe]-hydrogenase in R. eutropha (Fritsch et al, 2014). Furthermore, it is possible that other hypothetical proteins downstream of HucE and HhyE may also serve as electron acceptor candidates, in particular MSMEG_2717 that shares homology to PHG067, the proposed electron acceptor of R. eutropha (Schäfer et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

2019

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Aerobic soil bacteria persist by scavenging molecular hydrogen (H2) from the atmosphere. This key process is the primary sink in the biogeochemical hydrogen cycle and supports the productivity of oligotrophic ecosystems. In Mycobacterium smegmatis, atmospheric H2 oxidation is catalyzed by two phylogenetically distinct [NiFe]-hydrogenases, Huc (group 2a) and Hhy (group 1h). However, it is currently unresolved how these enzymes transfer electrons derived from H2 oxidation into the aerobic respiratory chain. In this work, we used genetic approaches to confirm that two putative iron-sulfur cluster proteins encoded on the hydrogenase structural operons, HucE and HhyE, are required for H2 consumption in M. smegmatis. Sequence analysis show that these proteins, while homologous, fall into distinct phylogenetic clades and have distinct metal-binding motifs. H2 oxidation was reduced when the genes encoding these proteins were deleted individually and was eliminated when they were deleted in combination. In turn, the growth yield and long-term survival of these deletion strains was modestly but significantly reduced compared to the parent strain. In both biochemical and phenotypic assays, the mutant strains lacking the putative iron-sulfur proteins phenocopied those of hydrogenase structural subunit mutants. We hypothesize that these proteins mediate electron transfer between the catalytic subunits of the hydrogenases and the menaquinone pool of the M. smegmatis respiratory chain; however, other roles (e.g., in maturation) are also plausible and further work is required to resolve their role. The conserved nature of these proteins within most Hhy- or Huc-encoding organisms suggests that these proteins are important determinants of atmospheric H2 oxidation.

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Section: Discussionmentioning

confidence: 99%

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

2019

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“…Recruitment of auxiliary proteins is not uncommon in the biogenesis of unique Fe/S proteins. An example of such an auxiliary component is the rubredoxin-related HoxR protein, which prevents oxidative damage to metallocenters during their biogenesis in the [NiFe] hydrogenase of Ralstonia eutropha H16 (38). In this context, NqrM may represent an additional, Na ϩ -NQR-specific protein that directly functions in Na ϩ -NQR maturation in the respective bacteria.…”

Section: Discussionmentioning

confidence: 99%

NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na ⁺ -Translocating NADH:Quinone Oxidoreductase

et al. 2016

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Na؉ -translocating NADH:quinone oxidoreductase (Na ؉ -NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na ؉ translocation across the membrane. Na ؉ -NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na ؉ -NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na ؉ -NQR, resulted in an enzyme incapable of Na ؉ -dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na ؉ -NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na ؉ -NQR, which could be recovered by an nqrM-containing plasmid. The Na ؉ -NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na ؉ -NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane ␣-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na ؉ -NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na ؉ -NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys) 4 [Fe] center between subunits NqrD and NqrE. IMPORTANCE Na؉ -translocating NADH:quinone oxidoreductase complex (Na ؉ -NQR) is a unique primary Na ؉ pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis. Production of Na؉ -NQR in bacteria requires Na ؉ -NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na ؉ -NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na ؉ -NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys 4 [Fe] center during Na ؉ -NQR assembly. Besides highlighting Na ؉ -NQR biogenesis, these findings suggest a novel drug target to combat Na ؉ -NQR-containing bacteria.N a ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR) is a redox-driven sodium pump operating in the respiratory chains of various bacteria, including several pathogens (1). This enzyme is a functional analog of H ϩ -translocating NADH: quinone oxidoreductase, known as mitochondrial complex I or bacterial NDH-1...

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“…Rx. gelatinosus CBS contains HupI (HoxR homolog in R. eutropha H16), a rubredoxin-type FeS protein deemed essential in assembling the O 2 -tolerant MBH in R. eutropha H16 when cultured in aerobic environment [54] , [56] . Future studies may reveal whether the supernumerary cysteines contribute to the O 2 -tolerance of the MBH in Rx.…”

Section: Resultsmentioning

confidence: 99%

Genome Annotation Provides Insight into Carbon Monoxide and Hydrogen Metabolism in Rubrivivax gelatinosus

et al. 2014

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We report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.

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Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

Cited by 10 publications

References 65 publications

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na ⁺ -Translocating NADH:Quinone Oxidoreductase

Genome Annotation Provides Insight into Carbon Monoxide and Hydrogen Metabolism in Rubrivivax gelatinosus

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Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

Cited by 10 publications

References 65 publications

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

Putative Iron-Sulfur Proteins Are Required for Hydrogen Consumption and Enhance Survival of Mycobacteria

NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na + -Translocating NADH:Quinone Oxidoreductase

Genome Annotation Provides Insight into Carbon Monoxide and Hydrogen Metabolism in Rubrivivax gelatinosus

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NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na ⁺ -Translocating NADH:Quinone Oxidoreductase