Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.
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ABSTRACT:
SUBMITTER: Cohen SE
PROVIDER: S-EPMC7796957 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ni-Fe-S-dependent carbon monoxide dehydrogenases (CODHs) are enzymes that interconvert CO and CO2 by using their catalytic Ni-Fe-S C-cluster and their Fe-S B- and D-clusters for electron transfer. CODHs are important in the microbiota of animals such as humans, ruminants, and termites because they can facilitate the use of CO and CO2 as carbon sources and serve to maintain redox homeostasis. The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is responsible for acetate production via the Wood-Ljungdahl pathway, where acetyl-CoA is assembled from two CO2-derived one-carbon units. A Ni-Fe-S A-cluster is key to this chemistry. Whereas acetogens use the A- and C-clusters of CODH/ACS to produce acetate from CO2, methanogens use A- and C-clusters of an acetyl-CoA decarbonylase/synthase complex (ACDS) to break down acetate en route to CO2 and methane production. Here we review some of the recent advances in understanding the structure and mechanism of CODHs, CODH/ACSs, and ACDSs, their unusual metallocofactors, and their unique metabolic roles in the human gut and elsewhere.
| S-EPMC9782325 | biostudies-literature
Project description:A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-A resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe(3)S(4)] and not [Fe(4)S(4)]. The mononuclear site and the four-metal cluster are bridged by means of Cys(531) and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe(4)S(4)] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an alpha/beta (Rossmann-like) domain in the middle, and an alpha/beta (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe(4)S(4)] cluster and another [Fe(4)S(4)] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.
| S-EPMC59822 | biostudies-literature
Project description:The Wood-Ljungdahl pathway allows for autotrophic bacterial growth on carbon dioxide, with the last step in acetyl-CoA synthesis catalyzed by the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). ACS uses a complex Ni-Fe-S metallocluster termed the A-cluster to assemble acetyl-CoA from carbon monoxide, a methyl moiety and coenzyme A. Here, we report the crystal structure of CODH/ACS from Moorella thermoacetica with substrate carbon monoxide bound at the A-cluster, a state previously uncharacterized by crystallography. Direct structural characterization of this state highlights the role of second sphere residues and conformational dynamics in acetyl-CoA assembly, the biological equivalent of the Monsanto process.
| S-EPMC7819276 | biostudies-literature
Project description:Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.
| S-EPMC5819426 | biostudies-literature
Project description:Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60-70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.
| S-EPMC2820341 | biostudies-literature
Project description:Nickel-containing carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidation of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallographically characterized as either a NiFe(4)S(4) or a NiFe(4)S(5) metal center, the latter containing a fifth, additional sulfide that bridges Ni and a unique Fe site. To determine whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H(2)O/OH(-) molecule and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a solution containing potassium cyanide. In both structures, the substrate H(2)O/OH(-) molecule exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H(2)O/OH(-) molecule. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidation by the C-cluster, the catalytic center of an environmentally important enzyme.
| S-EPMC2721637 | biostudies-literature
Project description:A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a methyl group, coenzyme A, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermolecular CO transport. Here, we report a 2.5 A resolution structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid composition surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.
| S-EPMC3040099 | biostudies-literature
Project description:Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO2 using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners. These enzymes tend to be extremely O2-sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from Desulfovibrio vulgaris (Dv) appears unique: exposure to micromolar concentrations of O2 on the minutes-time scale only reversibly inhibits the enzyme, and full activity is recovered after reduction. Here, we examine whether this unusual property of Dv CODH results from the nature of its D-cluster, which is a [2Fe-2S] cluster, instead of the [4Fe-4S] cluster observed in all other characterized CODHs. To this aim, we produced and characterized a Dv CODH variant where the [2Fe-2S] D-cluster is replaced with a [4Fe-4S] D-cluster through mutagenesis of the D-cluster-binding sequence motif. We determined the crystal structure of this CODH variant to 1.83-Å resolution and confirmed the incorporation of a [4Fe-4S] D-cluster. We show that upon long-term O2-exposure, the [4Fe-4S] D-cluster degrades, whereas the [2Fe-2S] D-cluster remains intact. Crystal structures of the Dv CODH variant exposed to O2 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under aerobic conditions retains 30% activity relative to a fully anaerobic purification, compared to 10% for the variant, and the WT enzyme loses activity more slowly than the variant upon prolonged aerobic storage. The D-cluster is therefore a key site of irreversible oxidative damage in Dv CODH, and the presence of a [2Fe-2S] D-cluster contributes to the O2-tolerance of this enzyme. Together, these results relate O2-sensitivity with the details of the protein structure in this family of enzymes.
| S-EPMC7343238 | biostudies-literature
Project description:Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO(2) oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO(2) interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the alpha(2)epsilon(2) component from the alpha(8)beta(8)gamma(8)delta(8)epsilon(8) CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H(2)O/OH(-) bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the epsilon-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
| S-EPMC2474488 | biostudies-literature
Project description:Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of "Candidatus Galacturonibacter soehngenii," from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of "Ca. G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the "Ca. G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of "Ca. G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of "Ca. G. soehngenii."
| S-EPMC7000372 | biostudies-literature