Identification and characterization of 2‐naphthoyl‐coenzyme A reductase,the prototype of a novel class of dearomatizing reductases

The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl‐coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl‐CoA by ATP‐dependent or ‐independent benzoyl‐CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2‐naphthoyl‐CoA reductase was purified from extracts of the naphthalene‐degrading, sulphidogenic enrichment culture N47. The oxygen‐tolerant enzyme dearomatized the non‐activated ring of 2‐naphthoyl‐CoA by a four‐electron reduction to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin‐containing ‘old yellow enzyme’ family. NCR contained FAD, FMN, and an iron‐sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2‐naphthoyl‐CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl‐CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl‐CoA reductases.     

Two distinct old yellow enzymes are involved in naphthyl ring reduction during anaerobic naphthalene degradation

The 2‐naphthoyl‐coenzyme A (NCoA) reductase (NCR) is so far the only characterized enzyme involved in the anaerobic degradation of the environmentally relevant polycyclic aromatic hydrocarbons. The old yellow enzyme (OYE) family member apparently reduced the nonactivated naphthyl ring to 5,6,7,8‐tetrahydro‐2‐napthoyl‐CoA (THNCoA). In this work, the candidate genes of three NCRs from the sulphate‐reducing, naphthalene‐degrading N47 and NaphS2 cultures were expressed in Escherichia coli. The isolated products contained flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) a [4Fe‐4S] cluster and catalyzed only the two‐electron reduction of NCoA to 5,6‐dihydro‐2‐naphthoyl‐CoA (5,6‐DHNCoA) at a very negative E°′ = ?493 mV. All NCRs exhibited high NCoA‐forming DHNCoA oxidase activities that are proposed to be involved in oxygen‐detoxification during naphthalene degradation. Extracts of N47 and NaphS2 catalyzed the reduction of 5,6‐DHNCoA to THNCoA. Genes putatively coding for 5,6‐DHNCR from N47 and NaphS2 were heterologously expressed in E. coli. The enriched enzyme products specifically catalyzed the reduction of 5,6‐DHNCoA to THNCoA at E°′ = ?375 mV. With the three NCRs and two 5,6‐DHNCRs, five OYEs have been characterized that are involved in the reduction of the nonsubstituted naphthyl‐ring system; these unprecedented enzymatic reactions expand our knowledge of the functional diversity of OYE.     

Of mothballs and old yellow enzymes

Polycyclic aromatic hydrocarbons (PAHs) are persistent and toxic environmental pollutants that accumulate in anoxic habitats. With the exception of naphthalene, nothing is known about the microbial degradation of PAH in these environments. The challenge that must be met in anaerobic PAH degradation is the destabilization of the resonance energy of the aromatic ring system, which requires electrons with very negative redox potentials. Estelmann et al. (2014) identify two enzymes from sulphate‐reducing bacteria that perform successive 2‐electron reductions of a coenzyme A thioester derivative of naphthalene. The first reduces 2‐naphthoyl‐CoA to 5,6‐dihydro‐2‐naphthoyl‐CoA and the second generates 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. Surprisingly, both enzymes are members of the ‘old yellow enzyme’ (OYE) family of flavoproteins. Neither uses adenosine triphosphate to achieve reduction of the aromatic ring. Typically, OYEs have flavin mononucleotide as cofactor and use nicotinamide adenine dinucleotide (phosphate) as reductant. Both ring reductases have flavin adenine dinucleotide and an iron‐sulphur cluster as additional cofactors. Evidence also suggests that in the sulphate‐reducing bacteria, these enzymes form a complex, allowing substrate channeling. The findings of this superb study represent unprecedented biochemistry. This work sheds light on how microbes meet the thermodynamic challenges of life at the redox limit.     

Conversion of cis‐2‐carboxycyclohexylacetyl‐CoA in the downstream pathway of anaerobic naphthalene degradation

The cyclohexane derivative cis‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid [(1R,2R)‐/(1S,2S)‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate‐reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis‐isomer, verifying that this is a true intermediate and not a dead‐end product. Mass‐spectrometric analyses confirmed that conversion is performed by an acyl‐CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl‐group. We propose that a novel kind of ring‐opening lyase is involved in the further catabolic pathway proceeding via pimeloyl‐CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring‐cleavage is achieved via hydratases, this lyase might represent a new ring‐opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl‐CoA and glutaryl‐CoA was proved in cell free extracts, yielding 2,3‐dehydropimeloyl‐CoA, 3‐hydroxypimeloyl‐CoA, 3‐oxopimeloyl‐CoA, glutaconyl‐CoA, crotonyl‐CoA, 3‐hydroxybutyryl‐CoA and acetyl‐CoA as observable intermediates. This indicates a link to central metabolism via β‐oxidation, a non‐decarboxylating glutaryl‐CoA dehydrogenase and a subsequent glutaconyl‐CoA decarboxylase.     

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.     

Genomic insights into the metabolic potential of the polycyclic aromatic hydrocarbon degrading sulfate‐reducing Deltaproteobacterium N47

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) is an important process during natural attenuation of aromatic hydrocarbon spills. However, knowledge about metabolic potential and physiology of organisms involved in anaerobic degradation of PAHs is scarce. Therefore, we introduce the first genome of the sulfate‐reducing Deltaproteobacterium N47 able to catabolize naphthalene, 2‐methylnaphthalene, or 2‐naphthoic acid as sole carbon source. Based on proteomics, we analysed metabolic pathways during growth on PAHs to gain physiological insights on anaerobic PAH degradation. The genomic assembly and taxonomic binning resulted in 17 contigs covering most of the sulfate reducer N47 genome according to general cluster of orthologous groups (COGs) analyses. According to the genes present, the Deltaproteobacterium N47 can potentially grow with the following sugars including d ‐mannose, d ‐fructose, d ‐galactose, α‐d ‐glucose‐1P, starch, glycogen, peptidoglycan and possesses the prerequisites for butanoic acid fermentation. Despite the inability for culture N47 to utilize NO₃^‐ as terminal electron acceptor, genes for nitrate ammonification are present. Furthermore, it is the first sequenced genome containing a complete TCA cycle along with the carbon monoxide dehydrogenase pathway. The genome contained a significant percentage of repetitive sequences and transposase‐related protein domains enhancing the ability of genome evolution. Likewise, the sulfate reducer N47 genome contained many unique putative genes with unknown function, which are candidates for yet‐unknown metabolic pathways.     

The genome of the Gram‐positive metal‐ and sulfate‐reducing bacterium Desulfotomaculum reducens strain MI‐1

Spore‐forming, Gram‐positive sulfate‐reducing bacteria (SRB) represent a group of SRB that dominates the deep subsurface as well as niches in which resistance to oxygen and dessication is an advantage. Desulfotomaculum reducens strain MI‐1 is one of the few cultured representatives of that group with a complete genome sequence available. The metabolic versatility of this organism is reflected in the presence of genes encoding for the oxidation of various electron donors, including three‐ and four‐carbon fatty acids and alcohols. Synteny in genes involved in sulfate reduction across all four sequenced Gram‐positive SRB suggests a distinct sulfate‐reduction mechanism for this group of bacteria. Based on the genomic information obtained for sulfate reduction in D. reducens, the transfer of electrons to the sulfite and APS reductases is proposed to take place via the quinone pool and heterodisulfide reductases respectively. In addition, both H₂‐evolving and H₂‐consuming cytoplasmic hydrogenases were identified in the genome, pointing to potential cytoplasmic H₂ cycling in the bacterium. The mechanism of metal reduction remains unknown.     

Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin‐, K+‐ and Fe2+‐dependent key enzyme of anaerobic phthalate degradation

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2‐dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report on the first purification and characterization of a phthaloyl‐CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD‐family of (de)carboxylases and contains prenylated FMN (prFMN), K⁺ and, unlike other UbiD‐like enzymes, Fe²⁺ as cofactors. The latter is suggested to be involved in oxygen‐independent electron‐transfer during oxidative prFMN maturation. Either oxidation to the Fe³⁺‐state in air or removal of K⁺ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe²⁺/K⁺‐complex in PCD. The PCD‐catalysed reaction was essentially irreversible: neither carboxylation of benzoyl‐CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl‐CoA with ¹³C‐bicarbonate was observed. PCD differs in many aspects from prFMN‐containing UbiD‐like decarboxylases and serves as a biochemically accessible model for the large number of UbiD‐like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.     

Not as easy as π: An insertional residue does not explain the π‐helix gain‐of‐function in two‐component FMN reductases

The π‐helix located at the tetramer interface of two‐component FMN‐dependent reductases contributes to the structural divergence from canonical FMN‐bound reductases within the NADPH:FMN reductase family. The π‐helix in the SsuE FMN‐dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4‐helix. Variants of Tyr118 were generated, and their X‐ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π‐helix. The structure of the Y118A SsuE π‐helix was converted to an α‐helix, similar to the FMN‐bound members of the NADPH:FMN reductase family. Although the π‐helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π‐helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π‐helix contains a His insertional residue. Exchanging the π‐helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure‐based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN‐bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π‐helix. Results from the structural and functional studies of the FMN‐dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π‐helix, and additional structural adaptions occur to provide the altered gain of function.     

Enzymes involved in the anaerobic degradation of meta-substituted halobenzoates

Organohalides are environmentally relevant compounds that can be degraded by aerobic and anaerobic microorganisms. The denitrifying Thauera chlorobenzoica is capable of degrading halobenzoates as sole carbon and energy source under anaerobic conditions. LC‐MS/MS‐based coenzyme A (CoA) thioester analysis revealed that 3‐chloro‐ or 3‐bromobenzoate were preferentially metabolized via non‐halogenated CoA‐ester intermediates of the benzoyl‐CoA degradation pathway. In contrast, 3‐fluorobenzoate, which does not support growth, was converted to dearomatized fluorinated CoA ester dead‐end products. Extracts from cells grown on 3‐chloro‐/3‐bromobenzoate catalysed the Ti(III)‐citrate‐ and ATP‐dependent reductive dehalogenation of 3‐chloro/3‐bromobenzoyl‐CoA to benzoyl‐CoA, whereas 3‐fluorobenzoyl‐CoA was converted to a fluorinated cyclic dienoyl‐CoA compound. The reductive dehalogenation reactions were identified as previously unknown activities of ATP‐dependent class I benzoyl‐CoA reductases (BCR) present in all facultatively anaerobic, aromatic compound degrading bacteria. A two‐step dearomatization/H‐halide elimination mechanism is proposed. A halobenzoate‐specific carboxylic acid CoA ligase was characterized in T. chlorobenzoica; however, no such enzyme is present in Thauera aromatica, which cannot grow on halobenzoates. In conclusion, it appears that the presence of a halobenzoate‐specific carboxylic acid CoA ligase rather than a specific reductive dehalogenase governs whether an aromatic compound degrading anaerobe is capable of metabolizing halobenzoates.     

On nitroaryl reductase activities in several Clostridia

Crude extracts of Clostridium kluyveri, Clostridium spec. La 1, Clostridium sporogenes and Clostridium pasteurianum catalyse the NADH-dependent reduction of the nitro group of p-nitrobenzoate. The former three Clostridia also use pyruvate as electron donor for this reduction. The NADH-dependent reductases have been partially purified and characterized from Clostridium kluyveri. Nitroalkyl compounds as well as nitrite, sulfite, sulfate and hydroxylamine are no substrates. Based on chromatographic behavior, separation pattern, yields, stability, pH optima, molecular masses and EPR studies the three NADH-dependent nitroaryl group reducing enzymes in Clostridium kluyveri (three activities in Clostridium spec. La 1 and two activities in Clostridium sporogenes) are different from alcohol dehydrogenase, aldehyde dehydrogenase, 3-hydroxy-butyryl-CoA dehydrogenase, butyryrl-CoA dehydrogenase, 2-enoate reductase, ferredoxin-NAD and ferredoxin-NADP reductase. The physiological roles of the nitroaryl reductases are not known. The reductase activities show losses of 80-90% during classical protein purification procedures. One of the three nitroaryl reductases exhibits a pH optimum of 10.5. The crude extract reveals a pH optimum at 11.5. The first step of the reduction reaction leads to the nitroradical anion (1 electron transfer). The electron transfer to p-nitrobenzoate is also catalysed by ferrodoxin-NAD reductase from NADH and by ferredoxin-NADP reductase from NADP. Partially purified 2-oxo-acid synthases from Clostridium sporogenes catalyse with low rates the reduction of p-nitrobenzoate as well as 2-nitroethanol in the presence and absence of ferredoxin using pyruvate or 2-oxo-4-methylpentanoate as electron donors, respectively. The NADH-dependent reduction of p-nitro-benzoate accounts for at least 70% and the 2-oxo acid-dependent reduction for about 5% of the total nitroaryl reductase activity in the Clostridia. It seems that the pyridine nucleotide-dependent nitroaryl reductases are enzymes so far unknown in Clostridia.     

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.     

Combining reverse genetics and nuclear magnetic resonance‐based metabolomics unravels trypanosome‐specific metabolic pathways

Numerous eukaryotes have developed specific metabolic traits that are not present in extensively studied model organisms. For instance, the procyclic insect form of Trypanosoma brucei, a parasite responsible for sleeping sickness in its mammalian‐specific bloodstream form, metabolizes glucose into excreted succinate and acetate through pathways with unique features. Succinate is primarily produced from glucose‐derived phosphoenolpyruvate in peroxisome‐like organelles, also known as glycosomes, by a soluble NADH‐dependent fumarate reductase only described in trypanosomes so far. Acetate is produced in the mitochondrion of the parasite from acetyl‐CoA by a CoA‐transferase, which forms an ATP‐producing cycle with succinyl‐CoA synthetase. The role of this cycle in ATP production was recently demonstrated in procyclic trypanosomes and has only been proposed so far for anaerobic organisms, in addition to trypanosomatids. We review how nuclear magnetic resonance spectrometry can be used to analyze the metabolic network perturbed by deletion (knockout) or downregulation (RNAi) of the candidate genes involved in these two particular metabolic pathways of procyclic trypanosomes. The role of succinate and acetate production in trypanosomes is discussed, as well as the connections between the succinate and acetate branches, which increase the metabolic flexibility probably required by the parasite to deal with environmental changes such as oxidative stress.     

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi DSS‐3

Ruegeria pomeroyi DSS‐3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl‐CoA by propionate‐CoA ligase (SPO2934) and other unidentified acyl‐CoA ligases. Acryloyl‐CoA is then reduced to propionyl‐CoA by AcuI or SPO1914. Acryloyl‐CoA is also rapidly hydrated to 3‐hydroxypropionyl‐CoA by acryloyl‐CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl‐CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl‐CoA pathway. Amino acids and nucleosides from cells grown on ¹³C‐enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl‐CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron‐rich substrates. Additionally, label from [¹³C‐methyl] DMSP only appeared in carbons derived from methyl‐tetrahydrofolate, and there was no evidence for a serine cycle of C‐1 assimilation.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.     

Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Sulfate‐reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS‐reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone‐interacting membrane‐bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native‐PAGE complexome profiling and 2D BN‐/SDS‐PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN‐/SDS‐PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na⁺‐based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese‐like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.