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AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase.

ABSTRACT: Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO, CoA, and a methyl-cation to form acetyl-CoA at a unique Ni,Ni-[4Fe4S] cluster (the A-cluster). However, it was unknown which proteins support the assembly of the A-cluster. We analyzed the product of a gene from the cluster containing the ACS gene, cooC2 from Carboxydothermus hydrogenoformans, named AcsFCh, and showed that it acts as a maturation factor of ACS. AcsFCh and inactive ACS form a stable 2:1 complex that binds two nickel ions with higher affinity than the individual components. The nickel-bound ACS-AcsFCh complex remains inactive until MgATP is added, thereby converting inactive to active ACS. AcsFCh is a MinD-type ATPase and belongs to the CooC protein family, which can be divided into homologous subgroups. We propose that proteins of one subgroup are responsible for assembling the Ni,Ni-[4Fe4S] cluster of ACS, whereas proteins of a second subgroup mature the [Ni4Fe4S] cluster of carbon monoxide dehydrogenases.

SUBMITTER: Gregg CM

PROVIDER: S-EPMC5000062 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans.

Project description:In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS(Mt)/CODH(Mt) from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS(Mt), and a zink-nickel site in the closed form. The ACS(Ch) of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII(Ch). Homogeneous ACS(Ch) and ACS(Ch)/CODHIII(Ch) catalyzed the exchange between [1-(14)C]acetyl-CoA and (12)CO with specific activities of 2.4 or 5.9 micromol of CO per min per mg, respectively, at 70 degrees C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 micromol of acetyl-CoA formed per min per mg, respectively, at 70 degrees C and pH 7.3. The functional cluster A of ACS(Ch) contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS(Ch), a 2.2-A crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy.

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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.

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Project description: Not available

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Dinuclear nickel complexes modeling the structure and function of the acetyl CoA synthase active site.

Project description:A dinuclear nickel complex with methyl and thiolate ligands, Ni(dadt(Et))Ni(Me)(SDmp) (2), has been synthesized as a dinuclear Ni(d)-Ni(p)-site model of acetyl-CoA synthase (ACS) (dadt(Et) is N,N'-diethyl-3,7-diazanonane-1,9-dithiolate; Dmp is 2,6-dimesitylphenyl). Complex 2 was prepared via 2 methods: (i) ligand substitution of a dinuclear Ni(II)-Ni(II) cation complex [Ni(dadt(Et)) Ni(tmtu)2] (OTf)2 (1) with MeMgBr and KSDmp (tmtu is tetramethylthiourea), (ii) methyl transfer from methylcobaloxime Co(dmgBF2)2(Me)(Py) (5) to a Ni(II)-Ni(0) complex such as [Ni(dadt(Et))Ni(cod)] (3), generated in situ from Ni(dadt(Et)) and Ni(cod)(2), followed by addition of KSDmp (cod is 1,5-cyclooctadiene; dmgBF2 is difluoroboryl-dimethylglyoximate). Method ii models the formation of Nip-Me species proposed as a plausible intermediate in ACS catalysis. The reaction of 2 with excess CO affords the acetylthioester CH3C(O)SDmp (8) with concomitant formation of Ni(dadt(Et))Ni(CO)2 (9) and Ni(CO)4 plus Ni(dadt(Et)). When complex 2 is treated with 1 equiv of CO in the presence of excess 1,5-cyclooctadiene, the formation of 9 and Ni(CO)4 is considerably suppressed, and instead the dinuclear Ni(II)-Ni(0) complex is generated in situ, which further affords 2 upon successive treatment with Co(dmgBF2)2(Me)(Py) (5) and KSDmp. These results suggest that (i) ACS catalysis could include the Nid(II)-Nip(0) state as the active species, (ii) The Nid(II)-Nip(0) species could first react with methylcobalamin to afford Nid(II)-Nip(II)-Me, and (iii) CO insertion into the Nip-Me bond and the successive reductive elimination of acetyl-CoA occurs immediately when CoA is coordinated to the Nip site to form the active Nid(II)-Nip(0) species.

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Crystallographic Characterization of the Carbonylated A-Cluster in Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.

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.

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Project description:The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

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Reply to: Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase.

Project description: Not available

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X-ray Absorption Spectroscopy Reveals an Organometallic Ni-C Bond in the CO-Treated Form of Acetyl-CoA Synthase.

Project description:Acetyl-CoA synthase (ACS) is a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO2 fixation, which has long been proposed to operate by a novel mechanism involving a series of protein-bound organometallic (Ni-CO, methyl-Ni, and acetyl-Ni) intermediates. Here we report the first direct structural evidence of the proposed metal-carbon bond. We describe the preparation of the highly active metal-replete enzyme and near-quantitative generation of the kinetically competent carbonylated intermediate. This advance has allowed a combination of Ni and Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure experiments along with density functional theory calculations. The data reveal that CO binds to the proximal Ni of the six-metal metallocenter at the active site and undergoes dramatic structural and electronic perturbation in forming this organometallic Ni-CO intermediate. This direct identification of a Ni-carbon bond in the catalytically competent CO-bound form of the A cluster of ACS provides definitive experimental structural evidence supporting the proposed organometallic mechanism of anaerobic acetyl-CoA synthesis.

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ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma.

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