Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not

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.     

Crystallographic evidence for a CO/CO<Subscript>2</Subscript> tunnel gating mechanism in the bifunctional carbon monoxide dehydrogenase/acetyl coenzyme A synthase from<Emphasis Type="Italic"> Moorella thermoacetica</Emphasis>

Acetyl coenzyme A synthase (ACS) acts in concert with carbon monoxide dehydrogenase (CODH) to catalyze the formation of acetyl-coenzyme A from CO₂-derived CO and CH₃⁺ molecules. Recent crystal structures have shown that the three globular domains constituting the ACS subunit may be arranged in either a closed or an open conformation. A long hydrophobic tunnel network allows diffusion of CO between the CODH and the ACS active sites in the closed form, but it is blocked in the open form. On the other hand, the active site of ACS is only accessible for coenzyme A and the methyl donating protein in the open domain conformation. Although several metal compositions have been observed for this active site, present consensus is that it consists of a Ni-Ni-[Fe₄S₄] cluster. The observed conformational changes of ACS and the resulting different substrate accessibilities of the catalytic central nickel are reviewed here in the context of a putative CO₂/CO tunnel gating mechanism.     

Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase

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.     

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO₂. A unique [NiFe₄S₄] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe₃S₄] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.     

Spectroscopic and computational insights into the geometric and electronic properties of the A-cluster of acetyl-coenzyme A synthase

For the last two decades, the bifunctional enzyme acetyl-coenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica has been the subject of considerable research aimed at elucidating the geometric and electronic properties of the A-cluster, which serves as the active site for ACS catalysis. While the recent success in obtaining high-resolution X-ray structures of this enzyme solved many of the mysteries regarding the number, identities, and coordination environments of the metal centers of the A-cluster, fundamental questions concerning the catalytic mechanism of this highly elaborate polynuclear active site have yet to be answered. This Commentary summarizes relevant information obtained from spectroscopic and computational studies on the oxidized, reduced, and CO-bound forms of the A-cluster and highlights some of the key issues regarding the electronic properties and reactivity of this cluster that need to be addressed in future studies.     

Acetyl-coenzyme A synthase: the case for a Ni<Subscript>p</Subscript><Superscript>0</Superscript>-based mechanism of catalysis

Acetyl-CoA synthase (also known as carbon monoxide dehydrogenase) is a bifunctional Ni-Fe-S-containing enzyme that catalyzes the reversible reduction of CO₂ to CO and the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl group donated by a corrinoid iron-sulfur protein. The active site for the latter reaction, called the A-cluster, consists of an Fe₄S₄ cubane bridged to the proximal Ni site (Ni_p), which is bridged in turn to the so-called distal Ni site. In this review, evidence is presented that Ni_p achieves a zero-valent state at low potentials and during catalysis. Ni_p appears to be the metal to which CO and methyl groups bind and then react to form an acetyl-Ni_p intermediate. Methyl group binding requires reductive activation, where two electrons reduce some site on the A-cluster. The coordination environment of the distal Ni suggests that it could not be stabilized in redox states lower than 2+. The rate at which the [Fe₄S₄]²⁺ cubane is reduced is far slower than that at which reductive activation occurs, suggesting that the cubane is not the site of reduction. An intriguing possibility is that Ni_p²⁺ might be reduced to the zero-valent state. Reinforcing this idea are Ni-organometallic complexes in which the Ni exhibits analogous reactivity properties when reduced to the zero-valent state. A zero-valent Ni stabilized exclusively with biological ligands would be remarkable and unprecedented in biology.Electronic Supplementary Material Supplementary Material is available in the online version of this article at     

Autocatalytic activation of acetyl-CoA synthase

Acetyl-CoA synthase (ACS ACS/CODH CODH/ACS) from Moorella thermoacetica catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group of a corrinoid-iron-sulfur protein (CoFeSP). A time lag prior to the onset of acetyl-CoA production, varying from 4 to 20 min, was observed in assay solutions lacking the low-potential electron-transfer agent methyl viologen (MV). No lag was observed when MV was included in the assay. The length of the lag depended on the concentrations of CO and ACS, with shorter lags found for higher [ACS] and sub-saturating [CO]. Lag length also depended on CoFeSP. Rate profiles of acetyl-CoA synthesis, including the lag phase, were numerically simulated assuming an autocatalytic mechanism. A similar reaction profile was monitored by UV-vis spectrophotometry, allowing the redox status of the CoFeSP to be evaluated during this process. At early stages in the lag phase, Co²⁺FeSP reduced to Co⁺FeSP, and this was rapidly methylated to afford CH₃-Co³⁺FeSP. During steady-state synthesis of acetyl-CoA, CoFeSP was predominately in the CH₃-Co³⁺FeSP state. As the synthesis rate declined and eventually ceased, the Co⁺FeSP state predominated. Three activation reductive reactions may be involved, including reduction of the A- and C-clusters within ACS and the reduction of the cobamide of CoFeSP. The B-, C-, and D-clusters in the subunit appear to be electronically isolated from the A-cluster in the connected subunit, consistent with the ~70 Å distance separating these clusters, suggesting the need for an in vivo reductant that activates ACS and/or CoFeSP.Abbreviations ACS acetyl-CoA synthase, also known as CODH (carbon monoxide dehydrogenase) or CODH/ACS or ACS/CODH - CH₃-Co³⁺FeSP, Co²⁺FeSP, and Co⁺FeSP corrinoid-iron-sulfur protein with the cobalamin in the methylated 3+, unmethylated 2+, and unmethylated 1+ states - CoA coenzyme A - DTT dithiothreitol - H-THF or THF tetrahydrofolic acid or tetrahydrofolate - MT methyl transferase - MV methyl viologen     

Tridentate N(2)S ligand from 2,2'-dithiodibenzaldehyde and N,N-dimethylethylenediamine: Synthesis, structure, and characterization of a Ni(II) complex with relevance to Ni Superoxide Dismutase

Nickel Superoxide Dismutase (NiSOD) and the A-cluster of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase (CODH/ACS) both feature active sites with Ni coordinated by thiolate and amide donors. It is likely that the particular set of donors is important in tuning the redox potential of the Ni center(s). We report herein an expansion of our efforts involving the use of 2,2′-dithiodibenzaldehyde (DTDB) as a synthon for metal-thiolate complexes to reactions with Ni complexes of N,N-dimethylethylenediamine (dmen). In the presence of coordinating counterions, these reactions result in monomeric square-planar complexes of the tridentate N₂S donor ligand derived from the Schiff-base condensation of dmen and DTDB. In the absence of a coordinating counterion, we have isolated a Ni(II) complex with an asymmetric N₂S₂ donor set involving one amine and one imine N donor in addition to two thiolate donors. This latter complex is discussed with respect to its relevance to the active site of NiSOD.     

Function of the tunnel in acetylcoenzyme A synthase/carbon monoxide dehydrogenase

Acetylcoenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) contains two Ni–Fe–S active-site clusters (called A and C) connected by a tunnel through which CO and CO₂ migrate. Site-directed mutants A578C, L215F, and A219F were designed to block the tunnel at different points along the region between the two C-clusters. Two other mutant proteins F70W and N101Q were designed to block the region that connects the tunnel at the ββ interface with a water channel also located at that interface. Purified mutant proteins were assayed for Ni/Fe content and examined by electron paramagnetic resonance spectroscopy. Analyses indicate that same metal clusters found in wild-type (WT) ACS/CODH (i.e., the A-, B-, C-, and probably D-clusters) are properly assembled in the mutant enzymes. Stopped-flow kinetics revealed that these centers in the mutants are rapidly reducible by dithionite but are only slowly reducible by CO, suggesting an impaired ability of CO to migrate through the tunnel to the C-cluster. Relative to the WT enzyme, mutant proteins exhibited little CODH or ACS activity (using CO₂ as a substrate). Some ACS activity was observed when CO was a substrate, but not the cooperative CO inhibition effect characteristic of WT ACS/CODH. These results suggest that CO and CO₂ enter and exit the enzyme at the water channel along the ββ subunit interface. They also suggest two pathways for CO during synthesis of acetylcoenzyme A, including one in which CO enters the enzyme and migrates through the tunnel before binding at the A-cluster, and another in which CO binds the A-cluster directly from the solvent.     

Tunnel mutagenesis and Ni-dependent reduction and methylation of the α subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase

Two isolated alpha subunit mutants (A110C and A222L) of the alpha(2)beta(2) acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the alpha subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated alpha subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the "majority" activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe(4)S(4) cubane in the apo-alpha subunit and the Ni-activated alpha subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-alpha subunit reduction was monophasic while Ni-activated alpha subunit reduction was biphasic, with the more rapid phase coincident with that of apo-alpha subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe(4)S(4)](2+) cubane. An upper limit of two electrons per alpha subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe(4)S(4)](2+) cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.     

Xenon in and at the end of the tunnel of bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase

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.     

Infrared studies of carbon monoxide binding to carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme that catalyzes the reversible reduction of carbon dioxide into carbon monoxide and the coupled synthesis of acetyl-CoA from the carbon monoxide produced. Exposure of CODH/ACS from Moorella thermoacetica to carbon monoxide gives rise to several infrared bands in the 2100-1900 cm(-1) spectral region that are attributed to the formation of metal-coordinated carbon monoxide species. Infrared bands attributable to M-CO are not detected in the as-isolated enzyme, suggesting that the enzyme does not contain intrinsic metal-coordinated CO ligands. A band detected at 1996 cm(-1) in the CO-flushed enzyme is assigned as arising from CO binding to a metal center in cluster A of the ACS subunit. The frequency of this band is most consistent with it arising from a terminally coordinated Ni(I) carbonyl. Multiple infrared bands at 2078, 2044, 1970, 1959, and 1901 cm(-1) are attributed to CO binding at cluster C of the CODH subunit. All infrared bands attributed to metal carbonyls decay in a time-dependent fashion as CO(2) appears in the solution. These observations are consistent with the enzyme-catalyzed oxidation of carbon monoxide until it is completely depleted from solution during the course of the experiments.     

CO-dependent H2 evolution by Rhodospirillum rubrum: Role of CODH:CooF complex

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H₂ evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H₂ is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe₄S₄ clusters. The observation of 2 Fe₄S₄ clusters for CooF contradicts the prediction of 4 Fe₄S₄ clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H₂ evolution indicates that ∼ 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H₂ evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H₂ evolution activity.     

Channeling of carbon monoxide during anaerobic carbon dioxide fixation

Carbon monoxide is an intermediate in carbon dioxide fixation by diverse microbes that inhabit anaerobic environments including the human colon. These organisms fix CO(2) by the Wood-Ljungdahl pathway of acetyl-CoA biosynthesis. The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) catalyzes several key steps in this pathway. CO(2) is reduced to CO at a nickel iron-sulfur cluster called cluster C located in the CODH subunit. Then, CO is condensed with a methyl group and coenzyme A at cluster A, another nickel iron-sulfur cluster in the ACS subunit. Spectroscopic studies indicate that clusters A and C are at least 10-15 A apart. To gain a better understanding of how CO production and utilization are coordinated, we have studied an isotopic exchange reaction between labeled CO(2) and the carbonyl group of acetyl-CoA with the CODH/ACS from Clostridium thermoaceticum. When solution CO is provided at saturating levels, only CO(2)-derived CO is incorporated into the carbonyl group of acetyl-CoA. Furthermore, when high levels of hemoglobin or myoglobin are added to remove CO from solution, there is only partial inhibition of the incorporation of CO(2)-derived CO into acetyl-CoA. These results provide strong evidence for the existence of a CO channel between cluster C in the CODH subunit and cluster A in the ACS subunit. The existence of such a channel would tightly couple CO production and utilization and help explain why high levels of this toxic gas do not escape into the environment. Instead, microbes sequester this energy-rich carbon source for metabolic reactions.     

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.     

The role of nickel in acetyl-CoA synthesis by the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase: enzymology and model chemistry

 CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO₂ at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. Received: 10 April 1996 / Accepted: 4 July 1996     

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

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.     

Redox, haem and CO in enzymatic catalysis and regulation

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 ? (1 ?=0.1?nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.     

QM/MM computations reveal details of the acetyl-CoA synthase catalytic center

The “open” (A_open) and “closed” (A_closed) A-clusters of the acteyl-CoA synthase (ACS) enzyme from Moorella thermoacetica have been studied using a combined quantum mechanical (QM)/molecular mechanical (MM) approach. Geometry optimizations of the oxidized, one- and two-electron reduced A_open state have been carried out for the fully solvated ACS enzyme, and the CO ligand has been modeled in the reduced models. Using a combination of both α_open and α_closed protein scaffolds and the positions of metal atoms in these structures, we have been able to piece together critical parts of the catalytic cycle of ACS. We have replaced the unidentified exogenous ligand in the crystal structure with CO using both a square planar and tetrahedral proximal Ni atom. A one-electron reduced A-cluster that is characterized by a proximal Ni atom in a tetrahedral coordination pattern observed in both the A_open (lower occupancy proximal Ni) and A_closed (proximal Zn atom) geometries with three cysteine thiolates and a modeled CO ligand demonstrates excellent agreement with the crystal structure atomic positions, particularly with the displacement of the side chain ring of Phe512 which appears to serve as a structural gate for ligand binding. The QM/MM optimized geometry of the A-cluster of ACS with an uncoordinated, oxidized proximal nickel atom in a square planar geometry demonstrates poor agreement with the atomic coordinates taken from the crystal structure. Based on these calculations, we conclude that the square planar proximal nickel coordination that has been captured in the A_open structure does not correspond to the ligand-free, oxidized [Fe₄S₄]²⁺ − Ni_p²⁺ − Ni_d²⁺ state. Overall, these computations shed further light on the mechanistic details of protein conformational changes and electronic transitions involved in the ACS catalytic cycle.