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Acetyl-CoA synthase (ACS) is a central enzyme in the carbon and energy metabolism of certain anaerobic species of bacteria and archaea that catalyzes the direct synthesis and cleavage of the acetyl CC bond of acetyl-CoA by an unusual enzymatic mechanism of special interest for its use of organonickel intermediates. An Fe 4 S 4 cluster associated with a proximal, reactive Ni p and distal spectator Ni d comprise the active site metal complex, known as the A cluster. Experimental and theoretical methods have uncovered much about the ACS mechanism, but have also opened new unanswered questions about the structure and reactivity of the A cluster in various intermediate forms. Here we report a method for large scale isolation of ACS with its A cluster in the acetylated state. Isolated acetyl-ACS and the two-electron reduced ACS, produced by acetyl-ACS reaction with CoA, were characterized by UV-visible and EPR spectroscopy. Reactivity with electron acceptors provided an assessment of the apparent E m for two-electron reduction of the A cluster. The results help to distinguish between alternative electronic states of the reduced cluster, provide evidence for a role of the Fe/S cluster in catalysis, and offer an explanation of why one-electron reductive activation is observed for a reaction cycle involving 2-electron chemistry., Competing Interests: Declaration of Competing Interest The authors declare no competing financial conflict of interest with the contents of this article. Any opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the view of the Department of Defense or the Uniformed Services University of the Health Sciences., (Published by Elsevier Inc.)

Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO 2 molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a Δ acsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD + "acetobutyrogenesis," requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile , explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon. IMPORTANCE Clostridium difficile is an anaerobic, multidrug-resistant, toxin-producing pathogen with major health impacts worldwide. It is the only widespread pathogen harboring a complete set of Wood-Ljungdahl pathway (WLP) genes; however, the role of the WLP in C. difficile is poorly understood. In other anaerobic bacteria and archaea, the WLP can operate in one direction to convert CO 2 to acetic acid for biosynthesis or in either direction for energy conservation. Here, conditions are defined in which WLP levels in C. difficile increase markedly, functioning to support metabolism of carbohydrates. Amino acid nutritional requirements were better defined, with new insight into how the WLP and butyrate pathways act in concert, contributing significantly to energy metabolism by a mechanism that may have broad physiological significance within the group of nonclassical acetogens.

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO.

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).

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found in methanogens and certain other Archaea that carries out the overall synthesis and cleavage of the acetyl C-C and C-S bonds of acetyl-CoA. The reaction is involved both in the autotrophic fixation of carbon and in the process of methanogenesis from acetate, and takes place at a unique active site metal center known as the A cluster, located on the beta subunit of the ACDS complex and composed of a binuclear Ni-Ni site bridged by a cysteine thiolate to an Fe4S4 center. In this work, a high rate of acetyl-CoA synthesis was achieved with the recombinant ACDS beta subunit by use of methylcobinamide as an appropriate mimic of the physiological base-off corrinoid substrate. The redox dependence of acetyl-CoA synthesis exhibited one-electron Nernst behavior, and the effects of pH on the observed midpoint potential indicated that reductive activation of the A cluster also involves protonation. Initial burst kinetic studies indicated the formation of stoichiometric amounts of an A cluster-acetyl adduct, further supported by direct chromatographic isolation of an active enzyme-acetyl species. Titration experiments indicated that two electrons are required for activation of the enzyme in the process of forming the enzyme-acetyl intermediate. The results also established that the A cluster-acetyl species undergoes reductive elimination of the acetyl group with the simultaneous release of two, low potential electron equivalents. Thus, the one-electron Nernst behavior can be interpreted as the sum of two separate, low potential, one-electron steps. The results tend to exclude reaction mechanisms involving either one- or three-electron reduced forms of the A cluster as immediate precursors to the acetyl species. A scheme involving a [Fe4S4]1+-Ni1+ species is favored over a [Fe4S4]2+-Ni0 form. The role of proton uptake in the possible formation of a Ni2+-hydride intermediate is also discussed. Trapping of electrons during the formation of the A cluster-acetyl species from substrates CO and methylcobinamide was found to be highly favorable, thus presenting a means for extensive activation of the enzyme under otherwise nonpermissive physiological redox potentials.

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates.

The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of acetyl-CoA in methanogens that metabolize acetate to CO(2) and CH(4), and also carries out acetyl-CoA synthesis during growth on one-carbon substrates. The ACDS complex contains five subunits, among which beta possesses an Ni-Fe-S active-site metal cluster, the A-cluster, at which reaction with acetyl-CoA takes place, generating an acetyl-enzyme species poised for C-C bond cleavage. We have used Ni and Fe K fluorescence XANES and EXAFS analyses to characterize these metals in the ACDS beta subunit, expressed as a C-terminally shortened form. Fe XANES and EXAFS confirmed the presence of an [Fe(4)S(4)] cluster, with typical Fe-S and Fe-Fe distances of 2.3 and 2.7 A respectively. An Fe:Ni ratio of approximately 2:1 was found by Kalphabeta fluorescence analysis, indicating 2 Ni per [Fe(4)S(4)]. Ni XANES simulations were consistent with two distinct Ni sites in cluster A, and the observed spectrum could be modeled as the sum of separate square planar and tetrahedral Ni sites. Treatment of the beta subunit with Ti(3+) citrate resulted in shifts to lower energy, implying significant reduction of the [Fe(4)S(4)] center, along with conversion of a smaller fraction of Ni(II) to Ni(I). Reaction with CO in the presence of Ti(3+) citrate generated a unique Ni XANES spectrum, while effects on the Fe-edge were not very different from the reaction with Ti(3+) alone. Ni EXAFS revealed an average Ni coordination of 2.5 S at 2.19 A and 1.5 N/O at 1.89 A. A distinct feature at approximately 2.95 A most likely results from Ni-Ni interaction. The methanogen beta subunit A-cluster is proposed to consist of an [Fe(4)S(4)] cluster bridged to an Ni-Ni center with one Ni in square planar geometry coordinated by 2 S + 2 N and the other approximately tetrahedral with 3 S + 1 N/O ligands. The electronic consequences of two distinct Ni geometries are discussed.

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl(2). The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni(2)[Fe(4)S(4)] arrangement at the active site. A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel.

Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+). Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.

The amount of oxidative phosphorylation enzymes in mitochondrial encephalomyopathy patients has been studied by two-dimensional electrophoresis (blue native PAGE/Tricine-SDS-PAGE). Only 20 mg muscle was required to identify and analyse complexes I, III, IV, and V after Coomassie staining. In most cases reduced amounts of the involved complex(es) correlated well with decreased enzyme activities. The reliability of the method was reflected by the constant mutual ratio of the complexes found in all controls. Deviations from normal ratios were found to be more sensitive indicators for a defect than the absolute quantities, which varied considerably within the control group both in the enzymic and in the electrophoretic analysis. The effect of the mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes on the amount of oxidative phosphorylation complexes was demonstrated for the first time directly on the protein level. In patients without known DNA mutations, specific defects of single complexes were identified. The new technique is a sensitive method for the identification of oxidative phosphorylation defects, complementary to enzymic measurements.

Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain-reaction-amplified fragment. The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W. & Weiss, H. (1989) Nature 339, 147 - 149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase.

Serratia marcescens HY bacteria must be lysogenic with either prophage y or psi to make it possible for phage kappa to form plaques unless they carry a so-called ink mutation. Genes in y and psi termed any and anp were identified that after infection of ink+ cells are necessary for an effective propagation of these phages as well as of coinfecting kappa phage. When kappa infects y and/or psi-lysogenic cells it transactivates the respective prophage genes by means of two early genes termed tay and tap. It appears that on infection of nonlysogenic ink+ cells kappa damps its own development, provided the regulatory region of the responsible gene is undermethylated. After kappa infection duly to achieve the special methylation of this region seems to be the function of any and anp. There are some more genes in y and psi prophage under the control of tay and tap, concerning in both cases a Dam methylation (recognition sequence GATC) of kappa DNA, a recombination proneness under restricting conditions of kappa DNA not modified by the modification enzyme of HY, and the kappa plaque size. By hybridization studies a region of homology common to y and psi was demonstrated which from its size might comprise all the transactivated genes. The view is supported by genetic data indicating an affinity among the any and anp genes. Investigation of various any mutants were indicative of DNA inversions in this region of the y genome. Surprisingly some of the any mutants had become sensitive in their plaque forming ability to an inhibitory activity exerted by prophage psi. Mutants of psi unable to interfere but still able to lysogenize were isolated. A model is presented accounting for the formation of pleiotropic and nonpleiotropic mutations with Any phenotype and their reversion types. Possible functions of the y genes and their counterparts in psi are discussed.

The nosology of the inborn errors of myelin metabolism has been stymied by the lack of molecular genetic analysis. Historically, Pelizaeus-Merzbacher disease has encompassed a host of neurologic disorders that present with a deficit of myelin, the membrane elaborated by glial cells that encircles and successively enwraps axons. We describe here a Pelizaeus-Merzbacher pedigree of the classical type, with X-linked inheritance, a typical clinical progression, and a pathologic loss of myelinating cells and myelin in the central nervous system. To discriminate variants of Pelizaeus-Merzbacher disease, a set of oligonucleotide primers was constructed to polymerase-chain-reaction (PCR) amplify and sequence the gene encoding proteolipid protein (PLP), a structural protein that comprises half of the protein of the myelin sheath. The PLP gene in one of two affected males and the carrier mother of this family exhibited a single base difference in the more than 2 kb of the PLP gene sequenced, a C----T transition that would create a serine substitution for proline at the carboxy end of the protein. Our results delineate the clinical features of Pelizaeus-Merzbacher disease, define the possible molecular pathology of this dysmyelinating disorder, and address the molecular classification of inborn errors of myelin metabolism. Patients with the classical form (type I) and the more severely affected, connatal variant of Pelizaeus-Merzbacher disease (type II) would be predicted to display mutation at the PLP locus. The other variants (types III-VI), which have sometimes been categorized as Pelizaeus-Merzbacher disease, may represent mutations in genes encoding other structural myelin proteins or proteins critical to myelination.