Ni-containing Carbon Monoxide Dehydrogenase Research Articles

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO2-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A–G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.

Structural and Phylogenetic Diversity of Anaerobic Carbon-Monoxide Dehydrogenases.

Anaerobic Ni-containing carbon-monoxide dehydrogenases (Ni-CODHs) catalyze the reversible conversion between carbon monoxide and carbon dioxide as multi-enzyme complexes responsible for carbon fixation and energy conservation in anaerobic microbes. However, few biochemically characterized model enzymes exist, with most Ni-CODHs remaining functionally unknown. Here, we performed phylogenetic and structure-based Ni-CODH classification using an expanded dataset comprised of 1942 non-redundant Ni-CODHs from 1375 Ni-CODH-encoding genomes across 36 phyla. Ni-CODHs were divided into seven clades, including a novel clade. Further classification into 24 structural groups based on sequence analysis combined with structural prediction revealed diverse structural motifs for metal cluster formation and catalysis, including novel structural motifs potentially capable of forming metal clusters or binding metal ions, indicating Ni-CODH diversity and plasticity. Phylogenetic analysis illustrated that the metal clusters responsible for intermolecular electron transfer were drastically altered during evolution. Additionally, we identified novel putative Ni-CODH-associated proteins from genomic contexts other than the Wood–Ljungdahl pathway and energy converting hydrogenase system proteins. Network analysis among the structural groups of Ni-CODHs, their associated proteins and taxonomies revealed previously unrecognized gene clusters for Ni-CODHs, including uncharacterized structural groups with putative metal transporters, oxidoreductases, or transcription factors. These results suggested diversification of Ni-CODH structures adapting to their associated proteins across microbial genomes.

The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris

Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO2 and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe–4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooSC, respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooSC is Ni-loaded, reversibly converts CO and CO2, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooSC reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site.

Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster

A unique [Ni–Fe–S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His261, which coordinates one of the Fe atoms with Cys295, is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys295, we constructed CODH-II variants. Ala substitution for the Cys295 substitution resulted in the decrease of Ni content and didn’t result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe–S] clusters. This strongly suggests Cys295 indirectly and His261 together affect Ni-coordination in the C-cluster.

How Light-Harvesting Semiconductors Can Alter the Bias of Reversible Electrocatalysts in Favor of H2 Production and CO2 Reduction

The most efficient catalysts for solar fuel production should operate close to reversible potentials, yet possess a bias for the fuel-forming direction. Protein film electrochemical studies of Ni-containing carbon monoxide dehydrogenase and [NiFeSe]-hydrogenase, each a reversible electrocatalyst, show that the electronic state of the electrode strongly biases the direction of electrocatalysis of CO2/CO and H+/H2 interconversions. Attached to graphite electrodes, these enzymes show high activities for both oxidation and reduction, but there is a marked shift in bias, in favor of CO2 or H+ reduction, when the respective enzymes are attached instead to n-type semiconductor electrodes constructed from CdS and TiO2 nanoparticles. This catalytic rectification effect can arise for a reversible electrocatalyst attached to a semiconductor electrode if the electrode transforms between semiconductor- and metallic-like behavior across the same narrow potential range (<0.25 V) that the electrocatalytic current switches between oxidation and reduction.

A unified electrocatalytic description of the action of inhibitors of nickel carbon monoxide dehydrogenase.

Several small molecules and ions, notably carbon monoxide, cyanide, cyanate, and hydrogen sulfide, are potent inhibitors of Ni-containing carbon monoxide dehydrogenases (Ni-CODH) that catalyze very rapid, efficient redox interconversions of CO(2) and CO. Protein film electrochemistry, which probes the dependence of steady-state catalytic rate over a wide potential range, reveals how these inhibitors target particular oxidation levels of Ni-CODH relating to intermediates (C(ox), C(red1), and C(red2)) that have been established for the active site. The following properties are thus established: (1) CO suppresses CO(2) reduction (CO is a product inhibitor), but its binding affinity decreases as the potential becomes more negative. (2) Cyanide totally inhibits CO oxidation, but its effect on CO(2) reduction is limited to a narrow potential region (between -0.5 and -0.6 V), below which CO(2) reduction activity is restored. (3) Cyanate is a strong inhibitor of CO(2) reduction but inhibits CO oxidation only within a narrow potential range just above the CO(2)/CO thermodynamic potential--EPR spectra confirm that cyanate binds selectively to C(red2). (4) Hydrogen sulfide (H(2)S/HS(-)) inhibits CO oxidation but not CO(2) reduction--the complex on/off characteristics are consistent with it binding at the same oxidation level as C(ox) and forming a modified version of this inactive state rather than reacting directly with C(red1). The results provide a new perspective on the properties of different catalytic intermediates of Ni-CODH--uniting and clarifying many previous investigations.

Carbon Monoxide Dehydrogenase Reaction Mechanism: A Likely Case of Abnormal CO2 Insertion to a Ni−H− Bond

Ni-containing carbon monoxide dehydrogenases (CODH), present in many anaerobic microorganisms, catalyze the reversible oxidation of CO to CO(2) at the so-called C-cluster. This atypical active site is composed of a [NiFe(3)S(4)] cluster and a single unusual iron ion called ferrous component II or Fe(u) that is bridged to the cluster via one sulfide ion. After additional refinement of recently published high-resolution structures of COOH(x)-, OH(x)-, and CN-bound CODH from Carboxydothermus hydrogenoformans (Jeoung and Dobbek Science 2007, 318, 1461-1464; J. Am. Chem. Soc. 2009, 131, 9922-9923), we have used computational methods on the predominant resulting structures to investigate the spectroscopically well-characterized catalytic intermediates, C(red1) and the two-electron more-reduced C(red2). Several models were geometry-optimized for both states using hybrid quantum mechanical/molecular mechanical potentials. The comparison of calculated Mössbauer parameters of these active site models with experimental data allows us to propose that the C(red1) state has a Fe(u)-Ni(2+) bridging hydroxide ligand and the C(red2) state has a hydride terminally bound to Ni(2+). Using our combined structural and theoretical data, we put forward a revised version of an earlier proposal for the catalytic cycle of Ni-containing CODH (Volbeda and Fontecilla-Camps Dalton Trans. 2005, 21, 3443-3450) that agrees with available spectroscopic and structural data. This mechanism involves an abnormal CO(2) insertion into the Ni(2+)-H(-) bond.

Rapid and Efficient Electrocatalytic CO2/CO Interconversions by Carboxydothermus hydrogenoformans CO Dehydrogenase I on an Electrode

The Ni-containing carbon monoxide dehydrogenase I from Carboxydothermus hydrogenoformans adsorbed on a pyrolytic graphite “edge” electrode catalyzes rapid CO2/CO interconversions at the thermodynamic potential.

Structural bases for the catalytic mechanism of Ni-containing carbon monoxide dehydrogenases

Significant progress has been made recently in our understanding of the structure/function relationships of the catalytic C-cluster of carbon monoxide dehydrogenases. Several structures of this enzyme have been reported, some of them at very high resolution. One recurrent problem, however, is the high degree of heterogeneity within each structure, as well as between the different X-ray models. Here, we have tried to relate the structural data with the wealth of spectroscopic and biochemical information gathered over many years. As a result, we propose a catalytic cycle that is consistent with both observations and stereochemistry. We also give alternatives to one of the most difficult aspects of the cycle, namely, the location of the two electrons in the most reduced state of the C-cluster.

Effect of sodium sulfide on Ni-containing carbon monoxide dehydrogenases.

The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans includes a mu(2)-sulfide ion bridged to the Ni and unique Fe, whereas the same cluster in enzymes from Rhodospirillum rubrum (CODH(Rr)) and Moorella thermoacetica (CODH(Mt)) lack this ion. This difference was investigated by exploring the effects of sodium sulfide on activity and spectral properties. Sulfide partially inhibited the CO oxidation activity of CODH(Rr) and generated a lag prior to steady-state. CODH(Mt) was inhibited similarly but without a lag. Adding sulfide to CODH(Mt) in the C(red1) state caused the g(av) = 1.82 EPR signal to decline and new features to appear, including one with g = 1.95, 1.85 and (1.70 or 1.62). Removing sulfide caused the g(av) = 1.82 signal to reappear and activity to recover. Sulfide did not affect the g(av) = 1.86 signal from the C(red2) state. A model was developed in which sulfide binds reversibly to C(red1), inhibiting catalysis. Reducing this adduct causes sulfide to dissociate, C(red2) to develop, and activity to recover. Using this model, apparent K(I) values are 40 +/- 10 nM for CODH(Rr) and 60 +/- 30 microM for CODH(Mt). Effects of sulfide are analogous to those of other anions, including the substrate hydroxyl group, suggesting that these ions also bridge the Ni and unique Fe. This proposed arrangement raises the possibility that CO binding labilizes the bridging hydroxyl and increases its nucleophilic tendency toward attacking Ni-bound carbonyl.

Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans.

From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe-4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different iron-sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.

Carbon monoxide dehydrogenase from Rhodospirillum rubrum produces formate.

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum reversibly catalyzes the oxidation of CO to CO(2) at the active site C-cluster. In this article, the reduction of CO(2) to formate is reported as a slow side reaction catalyzed by both Ni-containing CODH and Ni-deficient CODH. Recently, the structures of R. rubrum CODH and its active site NiFeS cluster (the C-cluster) have been solved. The data in this manuscript describe the formate-producing capability of CODH with or without Ni in the active site.

The Ni-containing carbon monoxide dehydrogenase family: light at the end of the tunnel?

OAK-B135 Metalloenzymes seem to ''come of age'' when their structures are known at atomic resolution, spectroscopic and catalytic properties are basically understood, and genetic expression systems are available. Such foundations allow detailed mechanistic and spectroscopic properties to be probed and correlated to structure. The objective of this article is to summarize what is known about the title group of enzymes, and show that, to a large degree, they have come of age.

Nature of the C-Cluster in Ni-Containing Carbon Monoxide Dehydrogenases

The C-cluster of carbon monoxide dehydrogenase (CODH) appears to be the active site for the oxidation of CO to CO2. We have studied with EPR and Mossbauer spectroscopy the enzymes from Rhodospirill...

Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system

A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.