Corrinoid Iron-sulfur Protein Research Articles

Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per- and polyfluoroalkyl substance) biodegradation

Despite the success of AlphaFold2 (AF2), it is unclear how AF2 models accommodate for ligand binding. Here, we start with a protein sequence from Acidimicrobiaceae TMED77 (T7RdhA) with potential for catalyzing the degradation of per- and polyfluoroalkyl substances (PFASs). AF2 models and experiments identified T7RdhA as a corrinoid iron-sulfur protein (CoFeSP) which uses a norpseudo-cobalamin (BVQ) cofactor and two Fe4S4 iron-sulfur clusters for catalysis. Docking and molecular dynamics simulations suggest that T7RdhA uses perfluorooctanoic acetate (PFOA) as a substrate, supporting the reported defluorination activity of its homolog, A6RdhA. We showed that AF2 provides processual (dynamic) predictions for the binding pockets of ligands (cofactors and/or substrates). Because the pLDDT scores provided by AF2 reflect the protein native states in complex with ligands as the evolutionary constraints, the Evoformer network of AF2 predicts protein structures and residue flexibility in complex with the ligands, i.e., in their native states. Therefore, an apo-protein predicted by AF2 is actually a holo-protein awaiting ligands.

Photosynthesis of Acetate by Sporomusa ovata-CdS Biohybrid System.

Sporomusa ovata, a typical electroautotrophic microorganism, has been utilized in bioelectrosynthesis for carbon dioxide fixation to multicarbon organic chemicals. However, additional photovoltaic devices are normally needed to convert photo energy to electric energy to power the carbon dioxide fixation, which restricts the overall energy conversion efficiency. Herein, we report Sporomusa ovata-CdS biohybrids for artificial photosynthesis driven by light without any other power source. The quantum yield can reach 16.8 ± 9%, and the active duration time of the system can last for 5 days. During the artificial photosynthesis, carbon dioxide is first reduced to formate and finally converted to acetate via the Wood-Ljungdahl pathway. The carbon dioxide fixation, electron transfer, energy metabolism, and reactive oxygen species damage repair processes in the biohybrid system were characterized by proteomic analysis. Key enzymes, e.g., flavoprotein, ferredoxin, formate-tetrahydrofolate ligase, 5-methyltetrahydrofolate:corrinoid iron-sulfur protein methyltransferase, thioredoxin, and rubrerythrin, were found up-regulated in the biohybrid system. The findings are helpful in understanding the mechanism of the artificial photosynthesis and useful for the development of new biohybrid systems using genetically engineered microbes in the future. The study is expected to boost the development of bioabiotic hybrid system in solar energy harvest.

Insights into the interactions between corrinoid iron-sulfur protein and methyl transferase from human pathogen Clostridium difficile

The human pathogen Clostridium difficile infection(CDI) is one of the most important healthcare-associated infections. Methyltransferase(MeTrCd) and corrinoid iron-sulfur protein(CoFeSPCd) are two key proteins in the acetyl-coenzyme A synthesis pathway of Clostridium difficile, which is essential for the survival of the pathogen and is absent in humans. Hence, the interaction between MeTrCd and CoFeSPCd can become innovative targets for the treatment of human CDI. In this study, the interaction between MeTrCd and CoFeSPCd was verified by fluorescence resonance energy transfer measurements. The influence of the interaction on the tertiary structure of MeTrCd and CoFeSPCd was studied by ANS-labeled fluorescence measurements. Molecular docking was also performed to understand the mechanism of the protein interactions. These results provide a molecular basis for innovative drug design and development to treat human CDI.

Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile

The human pathogen Clostridium difficile infection (CDI) is one of the most important healthcare-associated infections. The Wood-Ljungdahl pathway, which is responsible for Acetyl-CoA biosynthesis, is essential for the survival of the pathogen and is absent in humans. The key proteins and enzymes involved in the pathway are attractive targets for the treatment of CDI. Corrinoid iron-sulfur protein (CoFeSP) is a key protein and acts as a methyl transformer in the Wood-Ljungdahl pathway. In this study, CoFeSP from Clostridium difficile (CoFeSPCd) was cloned, expressed in E. coli and characterized for the first time. The structure and function of CoFeSPCd were investigated using homology structure modeling, spectroscopy, electrochemistry, steady state/pre-steady state kinetics and molecular docking. The two metal centers of CoFeSPCd, corrinoid cofactor and [4Fe-4S] cluster, were characterized using metal analysis, structural modeling, UV–Vis, EPR and direct electrochemistry. The methyl transfer activity between CH3-H4folate (CH3-THF) and CoFeSPCd catalyzed by methyl transferase (MeTrCd) was determined by kinetic studies. These results provide a molecular basis for innovative drug design and development to treat human CDI.

Axial Ligation and Redox Changes at the Cobalt Ion in Cobalamin Bound to Corrinoid Iron-Sulfur Protein (CoFeSP) or in Solution Characterized by XAS and DFT.

A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS) at the Co K-edge in combination with density functional theory (DFT) calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb) base of the cofactor is bound in Cbl in solution. As main species, (dmb)CoIII(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II). The absence of an α-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation.

Dramatic Conformational Flexibility of Carbon Monoxide Dehydrogenase/Acetyl‐CoA Synthase Revealed by Electron Microscopy

Carbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS) is a 310‐kDa bifunctional enzyme involved in anaerobic fixation of atmospheric carbon (CO2) by acetogenic bacteria. The CODH subunit reduces CO2 to CO, which passes through a hydrophobic tunnel to the active site of ACS. ACS contains a dinickel iron‐sulfur site where CO combines with a methyl group supplied by corrinoid iron‐sulfur protein (CFeSP) and transfers the acetyl product to coenzyme A (CoA). Structures of CODH/ACS previously observed by X‐ray crystallography indicate that ACS must open to accommodate methyl transfer from CFeSP, but the full extent of that motion was not visualized. To examine the conformational flexibility of CODH/ACS, we performed single‐particle electron microscopy (EM) on anaerobically prepared samples. The resulting structures reveal never before seen conformations of the ACS subunit, suggesting that dramatic movements are involved in the methyl transfer from CFeSP to the ACS subunit of CODH/ACS.

Conformational changes of the carbon‐fixing enzyme CODH/ACS revealed by electron microscopy

Carbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS) is a 310 kDa bifunctional enzyme involved in the anaerobic fixation of atmospheric carbon (CO2) by the acetogenic bacterium Moorella thermoacetica. The CODH subunit reduces CO2 to CO, which transfers through a central hydrophobic tunnel to the active site of ACS. ACS contains a dinickel iron‐sulfur site where CO combines with a methyl group supplied by corrinoid iron‐sulfur protein (CFeSP) and coenzyme A (CoA). Conformational changes of ACS have been hypothesized as the structural mechanism regulating access of methylated‐CFeSP and CoA to the proximal nickel in the active site. However, the conformations of CODH/ACS that have been observed crystallographically are insufficient to accommodate methyltransfer from CFeSP to ACS. To examine the conformations of CODH/ACS we used single‐particle electron microscopy (EM). Because CODH/ACS contains O2 sensitive metallocofactors, we developed a method for preparing negatively stained EM specimens in an anaerobic chamber. The resulting structures of CODH/ACS reveal a dramatic hinge‐like opening of the ACS subunit that can accommodate methyl transfer from CFeSP to CODH/ACS and gives new insight into the catalytic mechanism of this environmentally relevant enzyme.This work is supported by funding from the NIH. Samuel Thompson is supported by the MIT Undergraduate Research Opportunities Program.

Transient B12-Dependent Methyltransferase Complexes Revealed by Small-Angle X-ray Scattering

In the Wood–Ljungdahl carbon fixation pathway, protein–protein interactions between methyltransferase (MeTr) and corrinoid iron–sulfur protein (CFeSP) are required for the transfer of a methyl group. While crystal structures have been determined for MeTr and CFeSP both free and in complex, solution structures have not been established. Here, we examine the transient interactions between MeTr and CFeSP in solution using anaerobic small-angle X-ray scattering (SAXS) and present a global analysis approach for the deconvolution of heterogeneous mixtures formed by weakly interacting proteins. We further support this SAXS analysis with complementary results obtained by anaerobic isothermal titration calorimetry. Our results indicate that solution conditions affect the cooperativity with which CFeSP binds to MeTr, resulting in two distinct CFeSP/MeTr complexes with differing oligomeric compositions, both of which are active. One assembly resembles the CFeSP/MeTr complex observed crystallographically with 2:1 protein stoichiometry, while the other best fits a 1:1 CFeSP/MeTr arrangement. These results demonstrate the value of SAXS in uncovering the rich solution behavior of transient protein interactions visualized by crystallography.

Structural Basis for Electron and Methyl-Group Transfer in a Methyltransferase System Operating in the Reductive Acetyl-CoA Pathway

Several anaerobic acetogenic, methanogenic, hydrogenogenic, and sulfate-reducing microorganisms are able to use the reductive acetyl-CoA (Wood–Ljungdahl) pathway to convert CO2 into biomass. The reductive acetyl-CoA pathway consists of two branches connected by the Co/Fe-containing corrinoid iron–sulfur protein (CoFeSP), which transfers a methyl group from a methyltransferase (MeTr)/methyltetrahydrofolate (CH3-H4 folate) complex to the reduced Ni–Ni–[4Fe–4S] cluster (cluster A) of acetyl-CoA synthase. We investigated the CoFeSP and MeTr couple of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans and show that the two proteins are able to catalyze the methyl-group transfer reaction from CH3-H4 folate to the Co(I) center of CoFeSP. We determined the crystal structures of both proteins. The structure of CoFeSP includes the previously unresolved N-terminal domain of the large subunit of CoFeSP, revealing a unique four-helix-bundle-like architecture in which a [4Fe–4S] cluster is shielded by hydrophobic amino acids. It further reveals that the corrinoid and the [4Fe–4S] cluster binding domains are mobile, which is mandatory for the postulated electron transfer between them. Furthermore, we solved the crystal structures of apo-MeTr, CH3-H4-folate-bound MeTr, and H4-folate-bound MeTr, revealing a substrate-induced closure of the CH3-H4 folate binding cavity of MeTr. We observed three different conformations of Asn200 depending on the substrate bound in the active site, demonstrating its conformational modulation by hydrogen-bonding interactions with the substrate. The observed flexibility could be essential to stabilize the transition state during methyl-group transfer. The conformational space and role of Asn200 are likely conserved in homologous cobalamin-dependent MeTrs such as methionine synthase.

Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium Difficile

The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron–sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50 mg MeTr was produced per liter Luria–Bertani broth media. The recombinant methyltransferase was well characterized by SDS–PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway.

Metal centers in the anaerobic microbial metabolism of CO and CO2.

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO(2), for harnessing 'green' energy and producing biofuels. One strategy is to convert CO(2) into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO(2) and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO(2), we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO(2) and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe(4)S(4) clusters, catalyzes the addition and elimination of CO(2) during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron-sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B(12) and a Fe(4)S(4) cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co(3+) intermediate. Studies of CO and CO(2) enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C-C and C-S bond formations.

Infrared and EPR Spectroscopic Characterization of a Ni(I) Species Formed by Photolysis of a Catalytically Competent Ni(I)-CO Intermediate in the Acetyl-CoA Synthase Reaction

Acetyl-CoA synthase (ACS) catalyzes the synthesis of acetyl-CoA from CO, coenzyme A (CoA), and a methyl group from the CH(3)-Co(3+) site in the corrinoid iron-sulfur protein (CFeSP). These are the key steps in the Wood-Ljungdahl pathway of anaerobic CO and CO(2) fixation. The active site of ACS is the A-cluster, which is an unusual nickel-iron-sulfur cluster. There is significant evidence for the catalytic intermediacy of a CO-bound paramagnetic Ni species, with an electronic configuration of [Fe(4)S(4)](2+)-(Ni(p)(+)-CO)-(Ni(d)(2+)), where Ni(p) and Ni(d) represent the Ni centers in the A-cluster that are proximal and distal to the [Fe(4)S(4)](2+) cluster, respectively. This well-characterized Ni(p)(+)-CO intermediate is often called the NiFeC species. Photolysis of the Ni(p)(+)-CO state generates a novel Ni(p)(+) species (A(red)*) with a rhombic electron paramagnetic resonance spectrum (g values of 2.56, 2.10, and 2.01) and an extremely low (1 kJ/mol) barrier for recombination with CO. We suggest that the photolytically generated A(red)* species is (or is similar to) the Ni(p)(+) species that binds CO (to form the Ni(p)(+)-CO species) and the methyl group (to form Ni(p)-CH(3)) in the ACS catalytic mechanism. The results provide support for a binding site (an "alcove") for CO near Ni(p), indicated by X-ray crystallographic studies of the Xe-incubated enzyme. We propose that, during catalysis, a resting Ni(p)(2+) state predominates over the active Ni(p)(+) species (A(red)*) that is trapped by the coupling of a one-electron transfer step to the binding of CO, which pulls the equilibrium toward Ni(p)(+)-CO formation.

Metalloproteins/metalloenzymes for the synthesis of acetyl-CoA in the Wood-Ljungdahl pathway

This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway, including formate dehydrogenase (FDH), corrinoid iron sulfur protein (CoFeSP), acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH, a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate, catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP, as a methyl group transformer, accepts the methyl group from CH3-H4 folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO2 to CO and ACS functions for acetyl-coenzyme A synthesis through condensation of the methyl group, CO and coenzyme A, to finish the whole pathway. This paper introduces the structure, function and reaction mechanisms of these enzymes.

Enzymology of the Wood–Ljungdahl Pathway of Acetogenesis

The biochemistry of acetogenesis is reviewed. The microbes that catalyze the reactions that are central to acetogenesis are described and the focus is on the enzymology of the process. These microbes play a key role in the global carbon cycle, producing over 10 trillion kilograms of acetic acid annually. Acetogens have the ability to anaerobically convert carbon dioxide and CO into acetyl-CoA by the Wood-Ljungdahl pathway, which is linked to energy conservation. They also can convert the six carbons of glucose stoichiometrically into 3 mol of acetate using this pathway. Acetogens and other anaerobic microbes (e.g., sulfate reducers and methanogens) use the Wood-Ljungdahl pathway for cell carbon synthesis. Important enzymes in this pathway that are covered in this review are pyruvate ferredoxin oxidoreductase, CO dehydrogenase/acetyl-CoA synthase, a corrinoid iron-sulfur protein, a methyltransferase, and the enzymes involved in the conversion of carbon dioxide to methyl-tetrahydrofolate.

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase: EVIDENCE FOR A RANDOM MECHANISM OF METHYL AND CARBONYL ADDITION

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.

Structural insights into methyltransfer reactions of a corrinoid iron–sulfur protein involved in acetyl-CoA synthesis

The cobalt- and iron-containing corrinoid iron-sulfur protein (CoFeSP) is functional in the acetyl-CoA (Ljungdahl-Wood) pathway of autotrophic carbon fixation in various bacteria and archaea, where it is essential for the biosynthesis of acetyl-CoA. CoFeSP acts in two methylation reactions: the transfer of a methyl group from methyltransferase (MeTr)-bound methyltetrahydrofolate to the cob(I)amide of CoFeSP and the transfer of the methyl group of methyl-cob(III)amide to the reduced Ni-Ni-[4Fe-4S] active site cluster A of acetyl-CoA synthase (ACS). We have solved the crystal structure of as-isolated CoFeSP(Ch) from the CO-oxidizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans at 1.9-A resolution. The heterodimeric protein consists of two tightly interacting subunits with pseudo-twofold symmetry. The large CfsA subunit comprises three domains, of which the N-terminal domain binds the [4Fe-4S] cluster, the middle domain is a (betaalpha)(8)-barrel, and the C-terminal domain shows an open fold and binds Cobeta-aqua-(5,6-dimethylbenzimidazolylcobamide) in a "base-off" state without a protein ligand at the cobalt ion. The small CfsB subunit also displays a (betaalpha)(8)-barrel fold and interacts with the upper side of the corrin macrocycle. Structure-based alignments show that both (betaalpha)(8)-barrel domains are related to the MeTr in the acetyl-CoA pathway and to the folate domain of methionine synthase. We suggest that the C-terminal domain of the large subunit is the mobile element that allows the necessary interaction of CoFeSP(Ch) with the active site of ACS(Ch) and the methyltetrahydrofolate carrying MeTr. The conformation in the crystal structure shields the two open coordinations of cobalt and likely represents a resting state.

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

Methyl transfer reactions are important in a number of biochemical pathways. An important class of methyltransferases uses the cobalt cofactor cobalamin, which receives a methyl group from an appropriate methyl donor protein to form an intermediate organometallic methyl-Co bond that subsequently is cleaved by a methyl acceptor. Control of the axial ligation state of cobalamin influences both the mode (i.e., homolytic vs heterolytic) and the rate of Co-C bond cleavage. Here we have studied the axial ligation of a corrinoid iron-sulfur protein (CFeSP) that plays a key role in energy generation and cell carbon synthesis by anaerobic microbes, such as methanogenic archaea and acetogenic bacteria. This protein accepts a methyl group from methyltetrahydrofolate forming Me-Co(3+)CFeSP that then donates a methyl cation (Me) from Me-Co(3+)CFeSP to a nickel site on acetyl-CoA synthase. To unambiguously establish the binding scheme of the corrinoid cofactor in the CFeSP, we have combined resonance Raman, magnetic circular dichroism, and EPR spectroscopic methods with computational chemistry. Our results clearly demonstrate that the Me-Co3+ and Co2+ states of the CFeSP have an axial water ligand like the free MeCbi+ and Co(2+)Cbi+ cofactors; however, the Co-OH2 bond length is lengthened by about 0.2 angstroms for the protein-bound cofactor. Elongation of the Co-OH2 bond of the CFeSP-bound cofactor is proposed to make the cobalt center more "Co1+-like", a requirement to facilitate heterolytic Co-C bond cleavage.

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

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.

Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum.

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate. Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe(4)S(4)](2+) clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway.

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.