Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The corrinoid iron-sulfur protein (CFeSP) from Clostridium thermoaceticum functions as a methyl carrier in the Wood-Ljungdahl pathway of acetyl-CoA synthesis. The small subunit (33 kDa) contains cobalt in a corrinoid cofactor, and the large subunit (55 kDa) contains a [4Fe-4S] cluster. The cobalt center is methylated by methyltetrahydrofolate (CH3-H4folate) to form a methylcobalt intermediate and, subsequently, is demethylated by carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). The work described here demonstrates that the [4Fe-4S] cluster is required to facilitate the reactivation of oxidatively inactivated Cob(II)amide to the active Co(I) state. Site-directed mutagenesis of the large subunit gene was used to change residue 20 from cysteine to alanine, which resulted in formation of a cluster with EPR and redox properties consistent with those of [3Fe-4S] clusters. The midpoint potential of the cluster in the C20A variant was approximately 500 mV more positive than that of the [4Fe-4S] cluster in the native enzyme. Accordingly, it was found that the Co center in the C20A mutant protein could be reduced artificially but was severely crippled in its ability to be reduced by physiological electron donors. This is probably because the reduced cluster of the C20A protein cannot provide the driving force needed to reduce Co(II) to Co(I), since the Co(II/I) midpoint potential is -504 mV. The C20A variant also was unable to catalyze the steady-state synthesis of acetyl-CoA when CH3-H4folate or methyl iodide were provided as methyl donors and CO and CODH/ACS as reductants. Addition of chemical reductants rescued the catalytically crippled variant form in both of these reactions. On the other hand, in single-turnover reactions, the methyl-Co state of the altered protein was fully active in methylating H4folate and in synthesizing acetyl-CoA in the presence of CO and CoA. The combined results strongly indicate that the FeS cluster of the CFeSP is necessary for reductive activation of Co(II) to Co(I) by physiological reductants but is not required for catalysis, e.g., demethylation of CH3-H4folate or methylation of CODH/ACS. We propose that, during reductive activation, electrons flow from the reduced electron-transfer protein (e.g., CODH/ACS or reduced ferredoxin (Fd)) to the FeS cluster which then directs electrons to the cobalt center for catalysis. These results also support earlier hypotheses that the methylation and demethylation reactions involving the CFeSP are SN2-type nucleophilic displacement reactions and do not involve radical chemistry.
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PMID:Role of the [4Fe-4S] cluster in reductive activation of the cobalt center of the corrinoid iron-sulfur protein from Clostridium thermoaceticum during acetate biosynthesis. 954 55

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.
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PMID:Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage. 1067

Acetyl-CoA synthase from Clostridium thermoaceticum (ACS(Ct)) is an alpha(2)beta(2) tetramer containing two novel Ni-X-Fe(4)S(4) active sites (the A and C clusters) and a standard Fe(4)S(4) cluster (the B cluster). The acsA and acsB genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37(o)C under anaerobic conditions with Ni supplementation. The isolated recombinant His-tagged protein (AcsAB) exhibited characteristics essentially indistinguishable from those of ACS(Ct), from which Ni had been removed from the A cluster. AcsAB migrated through nondenaturing electrophoretic gels as a single band and contained a 1:1 molar ratio of subunits and 1.0-1.6 Ni/alphabeta and 14-22 Fe/alphabeta. AcsAB exhibited 100-250 units/mg CO oxidation activity but no CO/acetyl-CoA exchange activity. Electronic absorption spectra of thionin-oxidized and CO-reduced AcsAB were similar to those of ACS(Ct), with features typical of redox-active Fe(4)S(4) clusters. Partially oxidized and CO-reduced AcsAB exhibited EPR signals with g values and low spin intensities indistinguishable from those of the B(red) state of the B cluster and the C(red1) and C(red2) states of the C cluster of ACS(Ct). Upon overnight exposure to NiCl(2), the resulting recombinant enzyme (ACS(Ec)) developed 0. 06-0.25 units/mg exchange activity. The highest of these values is typical of fully active ACS(Ct). When reduced with CO, ACS(Ec) exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACS(Ct). Variability of activities and signal intensities were observed among different preparations. Issues involving the assembly of these metal centers in E. coli are discussed.
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PMID:Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli. 1105 Jan 60

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the "NiFeC" EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)-S(3) site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu-S bond length of 2.25 A and a metal neighbor at 2.65 A, consistent with the Cu-Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu-S(3)Se environment with a 2.4-A Se-Cu bond, strongly implicating a Cu-SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.
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PMID:Functional copper at the acetyl-CoA synthase active site. 1258 21

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. One structure of the Moorella thermoacetica enzyme revealed that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bridged to a binuclear CuNi center with Cu at the proximal metal site (M(p)) and Ni at the distal metal site (M(d)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity and Cu content, we had proposed that the Cu-containing enzyme is active [Seravalli, J., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3689-3694]. Here we have reexamined this proposal. Enzyme preparations with a wider range of Ni (1.6-2.8) and Cu (0.2-1.1) stoichiometries per dimer were studied to reexamine the correlation, if any, between the Ni and Cu content and ACS activity. In addition, the effects of o-phenanthroline (which removes Ni but not Cu) and neocuproine (which removes Cu but not Ni) on ACS activity were determined. EXAFS results indicate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examined to investigate the electronic state of this proposed intermediate in the ACS reaction mechanism. The combined results strongly indicate that the CuNi enzyme is inactive, that the NiNi enzyme is active, and that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](2+) (net S = 0) cluster bridged to a Ni(1+) (S = (1)/(2)) at M(p) that is bridged to planar four-coordinate Ni(2+) (S = 0) at M(d), with the spin predominantly on the Ni(1+). Furthermore, these studies suggest that M(p) is inserted during cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieved with NiFeC signals of approximately 0.8 spin/mol.
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PMID:Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not. 1504 2

The Ni(II)-dicarboxamido-dithiolato complexes (Et4N)2[Ni(NpPepS)] (1) and (Et4N)2[Ni(PhPepS)] (2) were used as Nid metallosynthons in the construction of higher nuclearity dinuclear Ni-Cu and Ni-Ni species to model the bimetallic Mp-Nid site of the A-cluster of acetyl coenzyme A synthase/CO dehydrogenase (ACS/CODH). Reaction of 1 with [Cu(neo)Cl] and [Ni(terpy)Cl2] in MeCN affords the dinuclear complexes (Et4N)[Cu(neo)Ni(NpPepS)] (3) and [Ni(terpy)Ni(NpPepS)] (4), respectively. Reaction of 2 with [Ni(dppe)Cl2] in MeCN yields [Ni(dppe)Ni(PhPepS)] (6). The Ni-Cu complex 3 exhibits no redox chemistry at the Nid site and no reaction with CO. In contrast, the Nip sites in 4 and 6 are readily reduced (characterized by their Ni(I) EPR spectra) and bind CO, exhibiting nuco bands at 2044 and 1997 cm-1, respectively, indicating terminal CO binding. The present Ni-Ni systems replicate the structural and chemical properties of the A-cluster site in ACS/CODH and support the presence of Ni at Mp in the catalytically active enzyme.
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PMID:Structural models of the bimetallic subunit at the A-cluster of acetyl coenzyme a synthase/CO dehydrogenase: binuclear sulfur-bridged Ni-Cu and Ni-Ni complexes and their reactions with CO. 1553 84

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.
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PMID:EPR and infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO. 1619 Jul 5

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.
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PMID:Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase. 1788 77

Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria and by the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex in Archaea. In both systems, a nickel- and Fe/S-containing active site metal center, the A cluster, catalyzes acetyl C-C bond formation/breakdown. Carbonyl group exchange of [1-(14)C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO(2) was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidation to CO(2) at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by analysis of three recombinant A cluster proteins, ACDS beta subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACS(Ch)), and truncated ACS(Ch) lacking its 317-amino acid N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni(+)-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C-C bond fragmentation. Protein conformational changes, related to "open/closed" states previously identified crystallographically, were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni(2+)-acetyl intermediate, controlling Ni(2+)-acetyl fragmentation and Ni(2+)(CO)(CH(3)) condensation. EPR spectral changes likely reflect variations in the Ni(+)-CO equatorial coordination environment in closed buried hydrophobic and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, versus interdomain contacts in ACS, ensures that CO is not released from the ACDS beta subunit in the absence of appropriate interactions with the alpha(2)epsilon(2) CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO(2).
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PMID:Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila: acetyl C-C bond fragmentation at the a cluster promoted by protein conformational changes. 2020 35

A combined theoretical and experimental study was performed on diarylethenes and diarylethene-capped sexithiophenes aiming at an improved understanding of the electrochemical and photochemical ring-opening and ring-closing mechanisms. Theoretical calculations, based on DFT and TDDFT, suggested that the spatial distribution and the occupancy of the frontier orbitals determine and control the diarylethenes' ring-opening and ring-closing upon photoirradiation and electrochemical oxidation. Optimized geometries, potential energy surfaces, and activation energies between the open-ring and closed-ring forms were calculated for diarylethenes in the neutral ground state, excited states, and mono- and dicationic states. Analysis of the frontier orbitals was employed to understand the cyclization and cycloreversion of diarylethenes and to predict and explain the switching properties of diarylethene-capped sexithiophene molecular wires. The TDDFT data were verified with experimentally measured UV/vis spectra. The DFT calculations estimated open-shell ground states of diarylethene-capped sexithiophene dications, which were verified with EPR spectroscopy, and the broadening of the peaks in the EPR spectra were explained with the calculated singlet-triplet splitting. The good agreement of experiment and theory allows for the understanding of switching behavior of diarylethenes in solutions, in metal break junctions, in monolayers on metal surfaces, and as a part of complex organic molecular wires.
ACS Nano 2011 Feb 22
PMID:Electrochemical and photochemical cyclization and cycloreversion of diarylethenes and diarylethene-capped sexithiophene wires. 2120 63


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