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Carbon monoxide-dependent transcriptional changes in a thermophilic, carbon monoxide-utilizing, hydrogen-evolving bacterium Calderihabitans maritimus KKC1 revealed by transcriptomic analysis
research-article
Author(s):
Masao Inoue ,
Hikaru Izumihara ,
Yuto Fukuyama ,
Kimiho Omae ,
Takashi Yoshida ,
Yoshihiko Sako
Publication date (Electronic):
9 May 2020
Journal:
Extremophiles
Publisher:
Springer Japan
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Abstract
Calderihabitans maritimus KKC1 is a thermophilic, carbon monoxide (CO)-utilizing, hydrogen-evolving bacterium that harbors seven cooS genes for anaerobic CO dehydrogenases and six hyd genes for [NiFe] hydrogenases and capable of using a variety of electron acceptors coupled to CO oxidation. To understand the relationships among these unique features and the transcriptional adaptation of the organism to CO, we performed a transcriptome analysis of C. maritimus KKC1 grown under 100% CO and N 2 conditions. Of its 3114 genes, 58 and 32 genes were significantly upregulated and downregulated in the presence of CO, respectively. A cooS–ech gene cluster, an “orphan” cooS gene, and bidirectional hyd genes were upregulated under CO, whereas hydrogen-uptake hyd genes were downregulated. Transcriptional changes in anaerobic respiratory genes supported the broad usage of electron acceptors in C. maritimus KKC1 under CO metabolism. Overall, the majority of the differentially expressed genes were oxidoreductase-like genes, suggesting metabolic adaptation to the cellular redox change upon CO oxidation. Moreover, our results suggest a transcriptional response mechanism to CO that involves multiple transcription factors, as well as a CO-responsive transcriptional activator (CooA). Our findings shed light on the diverse mechanisms for transcriptional and metabolic adaptations to CO in CO-utilizing and hydrogen-evolving bacteria.
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Most cited references54
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Database resources of the National Center for Biotechnology Information
Richa Agarwala, Tanya Barrett, Jeff Beck … (2017)
Abstract The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts for published life science journals. The Entrez system provides search and retrieval operations for most of these data from 39 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. New resources released in the past year include PubMed Data Management, RefSeq Functional Elements, genome data download, variation services API, Magic-BLAST, QuickBLASTp, and Identical Protein Groups. Resources that were updated in the past year include the genome data viewer, a human genome resources page, Gene, virus variation, OSIRIS, and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.
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Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle
A critical step in the biogeochemical cycle of sulfur on Earth is microbial sulfate reduction, yet organisms from relatively few lineages have been implicated in this process. Previous studies using functional marker genes have detected abundant, novel dissimilatory sulfite reductases (DsrAB) that could confer the capacity for microbial sulfite/sulfate reduction but were not affiliated with known organisms. Thus, the identity of a significant fraction of sulfate/sulfite-reducing microbes has remained elusive. Here we report the discovery of the capacity for sulfate/sulfite reduction in the genomes of organisms from 13 bacterial and archaeal phyla, thereby more than doubling the number of microbial phyla associated with this process. Eight of the 13 newly identified groups are candidate phyla that lack isolated representatives, a finding only possible given genomes from metagenomes. Organisms from Verrucomicrobia and two candidate phyla, Candidatus Rokubacteria and Candidatus Hydrothermarchaeota, contain some of the earliest evolved dsrAB genes. The capacity for sulfite reduction has been laterally transferred in multiple events within some phyla, and a key gene potentially capable of modulating sulfur metabolism in associated cells has been acquired by putatively symbiotic bacteria. We conclude that current functional predictions based on phylogeny significantly underestimate the extent of sulfate/sulfite reduction across Earth’s ecosystems. Understanding the prevalence of this capacity is integral to interpreting the carbon cycle because sulfate reduction is often coupled to turnover of buried organic carbon. Our findings expand the diversity of microbial groups associated with sulfur transformations in the environment and motivate revision of biogeochemical process models based on microbial community composition.
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Structure, Function, and Mechanism of the Nickel Metalloenzymes, CO Dehydrogenase, and Acetyl-CoA Synthase
1 Introduction 1.1 Research on Catalytic Reactions Involving CO and CO2: Relationship to Energy, the Environment, Biogeochemistry, Toxicology, Health, and Technology The major environmental- and energy-related problems facing our planet directly relate to carbon dioxide and the carbon biogeochemical cycle, which includes the biological fixation of CO2 into organic carbon and the oxidation of fixed carbon back to CO2. Thus, the ultimate source of all fossil fuels is CO2, which has been fixed into organic carbon and deposited in the earth’s crust over the last 4 billion years. Because modern life is so reliant on energy, particularly on fossil fuels, there is intense competition for these nonrenewable resources, thus creating a problem that has significant economic and political impacts. 1 A related problem of growing concern is the rising level of greenhouse gases, especially CO2 and methane. In nature, fixation of CO2 occurs on a huge scale, with photosynthetic CO2 fixation occurring at a rate of 200 gigatons per year. 2 There are six known pathways by which CO2 is fixed, 3 with the Calvin cycle and photosynthesis providing most of this fixed carbon. Under anaerobic conditions, the Wood–Ljungdahl pathway is a predominant CO2 sink, and CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS), the subjects of this review, are the key enzymes in this pathway. 4 The oxidation of organic carbon to CO2 slightly outpaces CO2 fixation, leaving a balance in the atmosphere. In May 2013 at Mauna Loa Observatory, the atmospheric CO2 levels reached 400 ppm—their highest value since records began—and the levels are increasing at a rate exceeding 2 ppm per year. 5 Further increases are predicted to produce large and uncontrollable impacts on the world climate, and evidence suggests that these changes are underway. 5,6 Thus, it is important to develop renewable nonfossil energy supplies that are CO2-neutral and easily stored, distributed, and used. CODH/ACS and the Wood–Ljungdahl pathway of CO and CO2 fixation could play a role in this development. Our ability to deal with these environmental- and energy-related problems will depend upon our understanding of the biology related to the global carbon cycle, especially those processes that lead to and limit CO2 fixation. One might imagine biotechnological solutions to both the greenhouse gas and energy-limitation problems. For example, supplying limiting nutrients, e.g., iron fertilization in the Ironex experiments, can stimulate CO2 fixation in the ocean. 7 Similarly, given the high efficiency and rates of enzymatic CO2 activation and fixation, principles borrowed from nature are being explored to design better CO2-reactive catalysts. 8 While CO2 is a relatively inert and nontoxic product of the complete oxidation of carbon, CO is a reactive, toxic gas that is produced naturally in some anaerobic bacteria by the two-electron reduction of CO2 and in aerobic organisms by heme oxygenase-catalyzed decomposition of porphyrins. 9 CO also is generated anthropogenically by the incomplete combustion of organic materials, predominantly by the oxidation of methane and other hydrocarbons. In the United States, poisoning by CO is responsible for ∼1000 accidental deaths, 10 while more than 50 000 people per year seek medical attention for CO poisoning. 11 Faulty furnaces, inadequately ventilated heating sources, and engine exhaust exposure are the main culprits of CO poisoning. The mode of toxicity appears to be inhibition by binding tightly to the metallocenters in heme proteins, such as hemoglobin, myoglobin, and cytochrome oxidase. 12 CO emissions lead to atmospheric levels of CO ranging from 0.05 ppm in rural areas to as high as 350 ppm in some urban settings. 13 Though this level is below the toxicity threshold, the OSHA limit for CO is 50 ppm continuous exposure for 8 h. Mild effects of CO poisoning are observed in humans when CO levels remain as high as 200 ppm for 2–3 h and exposure to 1000 ppm for 1 h is fatal. Though it may seem counterintuitive, given its reputation as “silent killer” and environmental pollutant, CO, at low levels, is cytoprotective and therapeutic applications for cardiovascular diseases, inflammatory disorders, and organ transplantation are being explored. 14 This strategy follows the recognition that heme oxygenase-1 is induced during tissue injury and oxidative stress. 15 Diverse microbes can grow on CO as their sole source of carbon and electron-equivalents. 16 This includes anaerobes such as Moorella thermoacetica, 17 some purple sulfur bacteria akin to Rhodospirillum rubrum, 18 and Carboxydothermus hydrogenoformans, 19 as well as some aerobic carboxydobacteria like Oligotropha carboxidovorans. 20 These are the organisms in which CO metabolism has been most thoroughly studied. As indicated by its low half-cell potential (−0.52 V, below), CO is a potent electron donor—approximately 1000-fold stronger than NADH—and life forms have probably utilized that property of CO as an energy source ever since life emerged 4 billion years ago in the archaean eon. Approximately 1 century ago, Haldane 21 and Leduc 22 suggested that the earliest organisms were likely to have been anaerobic autotrophs, and it has been proposed that life emerged within anaerobic hydrothermal vents by exploiting CO as a carbon and energy source. 23 The early atmosphere, which was formed by outgassing from the earth’s interior by volcanoes and hydrothermal vents, is expected to have a similar composition to that of modern volcanoes and vents, with little to no O2 and relatively high concentrations of CO2, CO and CH4. Hydrothermal vents, which contain dissolved CO at about 100 nM concentrations, 24 still support diverse populations of anaerobic CO oxidizers. 16,25 It has been suggested that a version of the Wood–Ljungdahl pathway may have been the first metabolic sequence to emerge, with early organisms metabolizing CO and CO2 using ancestral forms of CODH/ACS. 3,26 Contemporary bacteria that use this pathway, such as M. thermoacetica and C. hydrogenoformans, have been proposed as models for these early chemolithotrophs. 19 Anthropogenic CO production amounts to about 2 billion tons per year, 27 while microbial CO metabolism is partly responsible for maintaining the ambient CO below toxic levels by removing an approximately equal amount of CO from the Earth’s atmosphere. 28 As described in more detail below, the microbial enzymes responsible for CO oxidation can operate at rates as high as 40 000 (mol CO)(mol enzyme)−1 s–1 and catalytic efficiencies reaching 2 × 109 M–1 s–1. 29 Anaerobic microbes can grow on CO by use of CODH/ACS, the topic of this review, to initiate metabolism by the Wood–Ljungdahl pathway, while aerobes use a Cu Mo-pyranopterin CODH that is coupled to the Calvin–Benson pathway. 30 CO is also of great importance in the chemical industry and its reactivity is linked to formation of metal–CO bonds. For example, the M–CO complex plays a key role in industrial organometallic catalysis reactions, including the Monsanto process for acetate synthesis; the industrial Reppe process leading to the carbonylation of alkenes, alkynes, and conjugated dienes; Fischer–Tropsch reaction; hydroformylation; homologation; the water–gas shift (WGS); and hydrogenation using water as the hydrogen source. 8,31 Furthermore, we feel it is likely that interdisciplinary research on the enzymology of CO oxidation will lead to the development of novel catalysts that follow principles used by the natural catalysts for carbonylation (ACS) and reversible CO2 reduction (CODH). 1.2 CO and CO2 Chemistry Carbon dioxide is the final product of the compete oxidation of carbon. A comprehensive review on CO2 activation and reduction is available; 8 thus, we will summarize only those aspects of CO and CO2 reactivity that are most relevant for the present review on CODH/ACS. CO2 is very abundant in the atmosphere and stored as various forms of carbonate, yet it is relatively inert, which raises the stakes for researchers to describe strategies to convert CO2 to useful products. This review focuses on a two-enzyme complex that couples two extremely important reactions in biology and industry. CODH catalyzes CO2 reduction to CO and ACS catalyzes C–C bond formation using CODH-generated CO and a methyl group to generate the key metabolic intermediate acetyl-CoA. This coupled reaction is a highly efficient biochemical equivalent of coupling the WGS reaction to the Monsanto process in a single reaction mixture. Here we will briefly review the chemical principles related to the activation and reduction of CO2 and to the use of CO in carbonylation and C–C bond-forming reactions. 1.3 Introduction to CODH- and ACS-Dependent Microbial CO and CO2 Fixation The use of CO, a toxic gas to animals, as a metabolic building block is an interesting property of certain classes of diverse organisms that can fix CO2 and are capable of converting CO into CO2. CODH reversibly oxidizes CO to CO2. This activity allows organisms to grow on CO as a sole source of carbon and energy. The CO2 is then fixed into cellular carbon by one of the six known reductive CO2 fixation pathways. 3 A review is available that covers the history of microbial CO oxidation and our understanding of the catalytic mechanism of CODH and ACS up until ∼2003. 30 Aerobic CO metabolism is performed by carboxydotrophic bacteria, which are aerobic microbes that grow on CO as their sole source of carbon and energy, 16 fixing CO according to eq 1. 32 Aerobic CO oxidizing bacteria are taxonomically diverse, including α-, β-, and γ-proteobacteria; Firmicutes; and Actinobacteria, including pathogenic and nonpathogenic mycobacteria. 16,33 These microbes transfer the electrons derived from CODH-catalyzed CO oxidation to O2 through an electron transport chain involving quinones. 34 The CO2 is assimilated into cell carbon through the Calvin–Benson–Basham pathway. 16,35 The enzyme responsible for CO oxidation is called MoCu–CODH because it contains a binuclear Mo–Cu center in which the Cu is thiolate ligated to a molybdopterin center. 36 The CODH of Oligotropha carboxidovorans is the most thoroughly characterized MoCu–CODH enzyme. 36,37 This three-subunit enzyme also contains two [2Fe–2S] clusters and FAD, in common with other members of the xanthine oxidoreductase family. 36,38 1 The Ni–CODH plays a similar role in anaerobic microbes that the Mo–Cu enzyme plays in aerobic metabolism, allowing organisms to grow autotrophically on CO by coupling CO oxidation to CO2 fixation. Purple sulfur bacteria like Rhodospirillum rubrum and Rubrivivax gelatinosus(18) couple CO oxidation to the Calvin–Benson–Basham cycle, while methanogenic Archaea and sulfate reducing and acetogenic bacteria use the Wood–Ljungdahl pathway. 30 The CODH from the latter organisms contains a tightly associated ACS, which is purified either as an α2β2 complex containing a central core of two CODH subunits that are associated on either side by two ACS subunits 39 or as a larger complex containing other components of the Wood–Ljungdahl pathway (e.g., the corrinoid iron–sulfur protein and methyltransferase). 40 The association of CODH with ACS confers the ability to utilize the Wood–Ljungdahl pathway to perform diverse reactions in the carbon cycle (Figure 1). As shown in the top left panel, CODH/ACS allows organisms to grow autotrophically on CO and CO2. In this pathway, CODH catalyzes CO2 reduction into CO; then, ACS catalyzes the condensation of in situ generated CO with CoA and a methyl group bound to the cobalt center in a B12-containing protein, to generate the key metabolite, acetyl-CoA. This mode of autotrophic growth is used by a variety of anaerobic microbes, including acetogenic bacteria and methanogenic Archaea. The Wood–Ljungdahl pathway is found in a wide distribution of phylogenetic classes, including Clostridia, Deltaproteobacteria, Chloroflexi, and Spirochaetes, and is also found in two domains (Archaea and Bacteria); however, it is found in only a few species within these classes, suggesting that this pathway was distributed by horizontal gene transfer of the core genes (CODH, ACS, MeTr, CFeSP). 41 The marker genes for the Wood–Ljungdahl pathway are acsB (ACS) and the two subunits of the CFeSP (acsC and acsD); as the only genes that co-occur and are co-omitted among the sequenced bacterial genomes, 41 these enzymes are undoubtedly crucial for acetogenesis. Figure 1 The Wood–Ljungdahl pathway of CO/CO2 fixation and its involvement in acetogenesis and methyltrophy, as well as in the oxidation of acetate to methane. The methanogenic CODH/ACS is often called ACDS, acetyl-CoA synthase decarbonylase. In acetogenic bacteria, this pathway generates acetate (eq 2), conserving energy through electron transfer-linked phosphorylation. As shown in the bottom left panel (Figure 1), coupling methanogenesis to this pathway (operating in reverse) gives organisms the ability to convert acetate to methane. Sulfate-reducing bacteria can utilize the eight electrons generated during acetate oxidation (again using this pathway in reverse, bottom right scheme) to reduce sulfate to H2S. The pathway also allows organisms to grow on various methyl donors, such as methanol and aromatic methyl ethers (top right panel). Furthermore, any oxidative pathway that generates CO2 can potentially couple to the Wood–Ljungdahl pathway. For example, heterotrophic growth on sugars allows organisms to stoichiometrically convert glucose into 3 mol of acetate by capturing the reducing equivalents and the 2 mol of CO2 generated by glycolytic oxidation of pyruvate to acetyl-CoA and generating a third mole of acetyl-CoA. 2 42 2 CO Dehydrogenase 2.1 Redox Chemistry and Enzymology Involving CO and CO2 Because there is such a large reservoir of CO2 and its potential for conversion into useful products, there is much interest in the activation and reduction of CO2. The energetic requirements for CO2 reduction (eqs 3–8) at pH 7 vs NHE depend on the number of electrons in the redox half-reactions, as shown in eq 3–8. 3 4 5 6 7 8 9 10 The one-electron reduction of CO2 (eq 3) requires very negative potentials, due in part to the energy required for structural rearrangement of linear CO2 to form the bent CO2 anion radical. 43 The high overpotential (cell potentials in excess of 2.0 V) associated with formation of this radical anion intermediate remains the major obstacle to rapid and efficient heterogeneous electrochemical reduction of CO2. 8 On the other hand, the two-electron reduction to either CO or to formate occurs under much less demanding redox conditions (eqs 4,5). Both CO and formate formation are pH- and solvent-dependent, 44 being more favorable at low pH. The optimal catalyst for CO2 reduction to either CO or formate should avoid the highly energetically unfavorable formation of an anion radical as a catalytic intermediate. This principle has been demonstrated with an enzyme-based CO2 electroreduction catalyst, which rapidly generates CO via eq 4 at −0.52 V, i.e., without any overpotential. 45 In biology, the two CO2 reduction reactions to CO and formate are catalyzed by CO and formate dehydrogenase, respectively, which contain metal clusters to aid in CO2 activation and electron transfer. These reactions are important in the global carbon cycle and are keys to the activation of CO2 under anaerobic conditions. 8,46 Similarly, the synthetic catalysts that promote these reactions contain metals that bind CO2 and facilitate electron transfer. Homogeneous catalysts provide one mechanism for the reduction of CO2, by hydrogenation to formate, yet to increase its reductant potential, high H2 pressures and/or bases are used to drive the reaction. 8 The chemical interconversion between CO and CO2 (eq 9) is an important industrial reaction called the WGS reaction, which, in the reverse direction, provides fuel-cell-grade H2 from steam reforming. 47 In this direction, the reaction is marginally favorable with a ΔH o of −41.2 kJ mol–1 and a ΔG o 298 of −28.6 kJ mol–1 48 and is typically performed at temperatures greater than 200 °C using d-metal catalysts on oxide supports. 49 Because of the industrial importance, a number of laboratories in academia and industry are developing catalysts that rapidly and efficiently produce H2 from CO and water; for example, Ru3(CO)12 and a recent Au–CeO2 nanomaterial were described with a reactivity of 0.01 (mol H2) s–1 (mol metal carbonyl)−1 at 160 °C 50 and between 0.3 and 3.9 site–1 s–1 at 240 °C, 49b respectively. The WGS reaction is very similar to the reaction catalyzed by the enzyme CODH. In comparison, an enzyme (CODH)-based electrocatalyst yields a value for CO oxidation of >3.2 s–1 at 30 °C. 51 In solution, the enzymatic oxidation of CO by CODH I from C. hydrogenoformans (CODH Ch I) occurs with a turnover frequency of ∼40 000 s–1; 29,45a however, in the enzymatic reaction, electrons are transferred to redox proteins (e.g., ferredoxin) that couple to other redox enzymes like hydrogenase with proton reduction being a very slow side reaction. 52 By coadsorbing the C. hydrogenoformans CODH and Escherichia coli hydrogenase to conducting graphite particles, 51 highly efficient CO-dependent H2 production has been observed with a turnover frequency at 30 °C comparable to that of conventional high-temperature WGS catalysts (>2.5 s–1) (see section 1.2). 51 This biochemical reaction performed on purified enzymes is similar to the mode by which some anaerobic microbes grow. C. hydrogenoformans is an anaerobic organism that can live on CO as sole carbon source, evolving H2 as a byproduct. 29 A number of other microbes have been discovered that also adopt this seemingly extreme life style. 25,30,53 Multielectron reduction of CO2 is a very important reaction. Given that this process is more thermodynamically favorable than the two-electron reduction, it is somewhat surprising that such a reaction has not been discovered in nature, which instead uses discrete two-electron steps. For example, methanogenic archaea specialize in catalyzing CO2 reduction to methane (eq 8), which, when coupled to H2 oxidation, is thermodynamically favorable and provides energy for cellular growth. 54 Similarly, acetogenic bacteria catalyze the reduction of CO2 to acetic acid (eq 2), coupled to the oxidation of H2 or other electron donors. In nature, these eight-electron reduction reactions occur by discrete two-electron steps through the formate (CO), formaldehyde, methanol, and methane oxidation levels with the carbon from CO2 bound to and transferred among organic or metallic cofactors during the process. There are at least two reasons for the natural strategy of using enzymes that catalyze discrete two-electron-transfer steps. One is that the intermediates in the one-carbon metabolism branch off into various directions to make important cellular metabolites. Another is that the microbe is producing the final product (CH4 or CH3COOH) as a byproduct, with energy being conserved as ATP (through electron-transfer-linked phosphorylation) in the most thermodynamically favorable reaction(s) in the sequence. In synthetic systems, multielectron CO2 reduction has had limited success and the catalysts generally require large overpotentials, are unstable, and exhibit low product selectivity and yields, with the predominant industrial pathway for multielectron reduction being through CO. 8 CO is readily available as syngas (a mixture mainly of CO, CO2, and H2), which is produced by steam reforming (or other gasification processes) of reduced carbon-containing compounds like natural gas, coal, and biomass; however, these processes require high temperatures and are energy intensive. Thus, development of a highly efficient process for converting CO2 to CO would have high impact on hydrocarbon production from CO2. Interestingly, there are no known enzymatic catalysts for multielectron CO reduction; however, nitrogenase, which functions in nature to catalyze the eight-electron reduction of N2 and two protons to form H2 and ammonia, providing fixed nitrogen into the global nitrogen cycle, 55 has been modified by mutagenesis to catalytically reduce CO directly, albeit very slowly. 55a,56 The related vanadium-based nitrogenase slowly reduces CO to form a variety of short chain hydrocarbons, including ethylene, ethane, propane, and propylene. 57 In the formation of hydrocarbons from CO by nitrogenase, CO binds to Fe atom(s) on one face of FeMo-cofactor. 58 A number of chemical catalysts have been developed for multielectron reduction of CO, though most require high temperatures and pressures and produce mixtures of products. 8 For example, Fischer–Tropsch conversion of CO to methanol and other hydrocarbons using Cu/ZnO catalysts is a well-developed and efficient process. 59 2.2 Characteristics of Ni–CODHs 2.2.1 Enzymatic Activities Ni–CODHs can catalyze the reversible conversion of CO to CO2 with specific activities as high as 15 756 U/mg (k cat of ∼39 000 s–1) reported at pH 8 and 70 °C for CODH I from C. hydrogenoformans (CODH Ch I) using conventional kinetic assays. 29 Other two well-studied Ni–CODHs, CODH from R. rubrum (CODH Rr ) and CODH/ACS from M. thermoaceticum (CODH/ACS Mt ), are reported to oxidize CO at k cat values of ∼10 000 and ∼3000 s–1, respectively. 60,60b The high catalytic rates and their wide range among different CODHs attract significant interest; however, various properties of these enzymes have made it difficult to perform mechanistic investigations and structural studies. Perhaps the most challenging issue is that the Ni–CODH is extremely oxygen-sensitive; therefore, growth of the organism and purification and manipulation of the enzyme require the strict avoidance of contact with oxygen. This is most easily accomplished by performing studies within an anaerobic chamber whenever possible and by using Schlenck line techniques for any investigations outside the chamber. For example, the glovebox within the authors’ laboratory maintains the oxygen level below 2 ppm. The rapid catalytic turnover frequencies pose problems because most stopped-flow and freeze-quench instruments have dead times in the 1 ms range, while under optimal catalytic conditions, the half-time for all intermediate steps in the reaction cycle must be greater than 0.2 ms (0.7/3000) for even one of the least active enzymes (that from M. thermoacetica). Yet these issues have been mostly overcome by performing rapid kinetic experiments at low temperatures and/or at suboptimal pH values. 61 CO2 becomes a substrate for CODHs at redox potentials below ca. −300 mV, and the turnover frequency is in the range of 10 s–1, which is significantly lower than the k cat values for CO oxidation. 62,63 Electrochemical studies showed that CODH/ACS Mt catalyzes CO2 reduction very efficiently with almost no overpotential. 64 Reduction of CO2 to CO plays a key role in the Wood–Ljungdahl pathway 65 (Figure 1) and could allow fuel production if an efficient large-scale enzymatic electrocatalyst could be achieved. Experiments with electrode-immobilized CODH are described below in section 2.5. Catalytic reactions reported for Ni–CODHs are not limited to CO/CO2 conversion. CODH Rr produces formate as a slow side reaction during CO2 reduction in its nickel-containing and nickel-deficient forms. 66 CODH/ACS Mt can convert nitrous oxide to dinitrogen in the presence of a low-potential electron donor. 67 CODH/ACS Mt has been shown to catalyze the anaerobic reduction of 2,4,6-trinitrotoluene, a dangerous pollutant. 68 Furthermore, CODH/ACS Mt can catalyze the oxidation of n-butyl isocyanide (n-BIC) to n-butyl isocyanate (n-BICt). 69 In addition, the C531A and H265 V variants of recombinant CODH Rr catalyze H2 oxidation and hydroxylamine reduction, respectively. 70 2.2.2 Structural and Spectroscopic Properties, Metal Clusters, and Redox Chemistry The X-ray structures of five Ni–CODHs have been reported. These include structures of three bacterial (M. thermoacetica, C. hydrogenoformans, and R. rubrum) and one archaeal (Methanosarcina barkeri) enzyme. 39,71 The bacterial enzymes have sequence similarities between 46% (C. hydrogenoformans and R. rubrum) and 63% (M. thermoacetica and R. rubrum) and structures that are nearly identical (RSMD of ∼0.95 Å according to PDB 1MJG and 1JQK). Crystal structures clearly reveal the presence of five metal clusters per homodimeric enzyme, two nickel–iron–sulfur clusters, called the C-clusters, one Fe4S4 D-cluster; and two Fe4S4 B-clusters, as shown in Figure 2. 39b,71b,39a,71a The structures also reveal why all CODHs are dimeric—there is a single D-cluster that bridges the two subunits; furthermore, the C-cluster of one subunit and the B-cluster of the other are closer than those from the same subunit. Thus, a functional dimer is required for rapid electron transfer. The methanogenic CODH contains two more Fe4S4 clusters (E- and F-clusters) than the bacterial enzymes. Since one subunit is positioned over the D-cluster of this enzyme, E- and F-clusters are proposed to be part of the electron transfer chain. 71i This proposal is supported by the high sequence similarity between the FeS domain bearing E- and F-clusters and M. barkeri pyruvate ferredoxin oxidoreductase, electron donor for ferredoxin, and the location of these clusters between the surface and the B-cluster. Structures of the B-, C-, and D-clusters are shown in Figure 2. Magnetic circular dichroism (MCD), resonance Raman (rR), and electronic absorption spectroscopic studies on the nickel-deficient CODH Rr support the presence of two different types of [Fe4S4]2+/+ clusters, presumably consisting of the bridging D-cluster and the two B-clusters. 71b,71a,72 The midpoint potential of the B-clusters, between −300 and −530 mV, is consistent with an electron transfer role. 72 Interestingly, the D-cluster adopts a diamagnetic 2+ state at potentials higher than −530 mV. 72 Although the D-cluster shows an unusually low redox potential, its proximity to the surface and the B-cluster would be consistent with an electron transfer role in the CODH mechanism, though the role of this cluster has not been established. Reversible CO/CO2 conversion was shown to occur at the C-cluster; 61,62a,73 thus, there is much interest in characterizing this metal center, which is composed of an iron–sulfur cluster combined with a nickel atom. 74−76 Four different oxidation states for the C-cluster have been suggested: a catalytically inactive and EPR-silent Cox state; a one-electron reduced Cred1 state, which binds CO and has an electron paramagnetic resonance (EPR) spectroscopic signal with g-values at 2.01, 1.81, and 1.65 (g av = 1.82); a two-electron-reduced EPR-silent Cint state; 77 and a three-electron-reduced form, Cred2, which binds CO2 and has a distinct EPR signature with g-values of 1.97, 1.87, and 1.75 (g av = 1.86). 74,78 The electronic structure of these redox states is not clear yet; however, the majority of unpaired electron spin density is localized on Fe in both Cred1 and Cred2, which exhibit large 57Fe and small 61Ni hyperfine values. 79 The g-values and midpoint redox potentials for the metal clusters of CODHs from various organisms are shown in Table 1. Figure 2 (A) Structure of CODH Rr in cartoon representation, (B) distances between the metal clusters, (C) structure of the D-cluster, (D) structure of the B-cluster, and (E) structure of the C-cluster. Atom colors: dark gray (iron), orange (sulfide), red (oxygen), blue (nitrogen), white (carbon), dark green (nickel). Generated using Pymol from PDB 1JQK. The nickel, iron, and sulfide content; molecular structure; and redox properties of the C-cluster have been the subject of many spectroscopic and structural studies (Figure 3). 74,75,79b,80−85 The X-ray diffraction structures and anomalous dispersion experiments revealed that Ni in the C-cluster is a part of a slightly distorted iron–sulfur cubane. Another iron atom in the C-cluster, but outside the cubane, was assigned as ferrous component II (FCII) (also called unique iron and the pendant Fe), according to a Mossbauer study. 85b For the Cred1 state, a ferrous component III (FCIII) was also described while other two irons were assigned to be mixed valence Fe2+Fe3+. 74 Thus, according to this scenario, Cred1 would consist of three ferrous and one ferric iron. The initial crystal structure of C. hydrogenoformans CODH II (CODH Ch II) included a bridging sulfido ligand connecting nickel and the pendant iron, indicating the cluster composition as [NiFe4S5], 71b with the bridging sulfide proposed to serve an undetermined catalytic role. 71c However, crystal structures for CODH Rr , 71a CODH/ACS Mt , 39a,39b and another CODH Ch II crystal structure 86 do not include the bridging sulfide. Furthermore, sulfide appears to reversibly inhibit CODH Rr and CODH/ACS Mt . 87,88 Inhibition by sulfide and other ligands, which bind to different oxidation states of the C-cluster, will be discussed in more detail below in section 2.2.3. It is now accepted by the community that there is no bridging sulfide between Ni and the pendant Fe in the active form of the C-cluster. This Fe-bound hydroxide is viewed as the nucleophile that attacks a Ni–CO to generate a metal-bound carboxylate during the catalytic cycle. 71d,85c,94 It has been suggested that sulfide acts as a reversible inhibitor by replacing the catalytically important hydroxide. 87,88 Crystallographic studies of the carboxylate-bound state, 71a observation of COS as a substrate, 95 and weak CO-dependent hydrogen evolution activity of CODHs 96 support this proposal. The CODH structure in its Cred1 state reported by Jeoung and Dobbek also is interpreted to have a bridging hydroxide between Ni and pendant Fe. ENDOR spectroscopy of Cred1 reveals the proton from the metal-bound hydroxyl group while Cred2 appears to lack this spectral feature. 85c On the other hand, in the Cred2 state, a bridging hydride was proposed upon computational calculations. 97,98 Structural changes upon catalytic activity will be discussed later. Table 1 Spectroscopic and Electrochemical Data for the Ni–CODHs from Different Sources A-cluster B-cluster C-cluster g-values (−CO) E 0′ g-values E 0′ g-values E 0′ R. rubrum(74,89) 2.04, 1.94, 1.89 –418 2.03, 1.88, 1.71 –110 1.97, 1.87, 1.75 C. hydrogenoformans(88) 2.04, 1.93, 1.89 2.01, 1.89, 1.73 1.96, ?, 1.77 M. thermoaceticum(79b) 2.08, 2.07, 2.03 2.04, 1.94, 1.90 –440 2.01, 1.81, 1.65 –220 2.06, 2.05, 2.03 –530 1.97, 1.87, 1.75 –530 M. thermoaceticum with azide 90 2.34, 2.07, 2.03 2.34, 2.11, 2.04 M. barkeri(91) 2.05, 1.94, 1.90 –390 2.01, 1.91, 1.76 –35 ?, ?, 1.73 M. soehngenii(92) 2.05, 1.93, 1.86 –410 2.01, 1.89, 1.73 –230 M. thermophila(93) 2.06, 2.05, 2.03 2.04, 1.93, 1.89 –444 2.02, 1.87, 1.72 –154 ?, ?, 1.79 Figure 3 Structure of C-cluster including only one coordinating residue, cysteine, and the ligands from (A) CODH Rr (PDB 1JQK), (B) CODH Ch II (PDB 1SU8), (C) CODH Ch II at 320 mV (PDB 3B53), (D) CODH Ch II at 600 mV (PDB 3B51), (E) cyanide-bound CODH Ch II at 320 mV (PDB 3I39), (F) CO2-bound CODH Ch II at 600 mV (PDB 3B52), (G) cyanide-bound CODH/ACS Mt (PDB 3I04), (H) CODH/ACS Mt (PDB 3I01), (J) n-BICt-bound CODH/ACS Mt (PDB 2YIV), (K) CO-bound CODH Mb (PDB 3CF4). Atom colors: Dark gray (iron), orange (sulfide), red (oxygen), blue (nitrogen), white (carbon), dark green (nickel). Accessory proteins (CooC, CooT, and CooJ), whose genes are part of a CODH-containing gene cluster in R. rubrum, appear to be required for assembly of the C-cluster. 99 Deletion of CooC, which has ATPase and GTPase activity and a nucleotide-binding P-loop region, leads to a C-cluster that contains the Fe–S but lacks Ni components of the cluster. 99,100 This Ni-deficient form of CODH Rr can be activated in vitro by incubation of the reduced protein with NiCl2. 101 However, a similar role for AcsF, the M. thermoacetica homologue of CooC, could not be established. 102 On the basis of homology with HypC, CooT may be involved in metal ion discrimination. 99 CooJ has a histidine-rich C-terminus and binds up to four nickel ions per monomer. 103 As shown in Figure 2, the C-cluster is deeply buried inside the enzyme with the C-, B-, and D-clusters aligned as an efficient redox wire with 10–11 Å intercluster distances to allow rapid electron transfer. 71b The structures of CODH/ACS Mt , CODH Rr , and CODH Ch II are very similar, with strict conservation of all amino acid residues that ligate the metal clusters (Figure 2, Table 2). Other residues that are thought to be important in acid–base chemistry are also identified in Table 2. Table 2 Key Residues in the Primary and the Secondary Coordinating Spheres of the Metal Centers in Different Ni–CODHs organism (PDB ID) A-cluster B-cluster C-cluster D-cluster His-tunnel acid–base Rr (1JQK) C50 C300, C338 C41, C49 H95 K568 C53 C451, C481 C41′, C49′ H98 H95 C58 C531, H265 H101 D223 C72 W575 Ch (3B51) C48 C295, C333 C39, C47 H93 K563 C51 C446, C476 C39′, C47′ H96 H93 C56 C526, H261 H99 D219 C70 H102 W570 Mt (1OAO) C506, C509 C68 C317, C355 C59, C67 H113 K587 C518, C528 C71 C470, C500 C59′, C67′ H116 H113 C595, C597 C76 C550, H283 H119 D241 G596 C90 H122 W594 2.2.3 Inhibition of CODH Enzymatic Activity Several molecules including nitrous oxide, sulfide, azide, thiocyanate, cyanate, cyanide, and n-BIC are known to inhibit the catalytic activity of CODHs. 60b,67,69,71c,88,90,104 Here we will describe research on these inhibitors that has helped to enlighten the CODH catalytic mechanism. Electrochemical studies combined with EPR spectroscopy showed that cyanate, an analogue of CO2, binds the Cred2 state and inhibits CO2 reduction. 88 Most likely it binds to the active site in a similar fashion as CO2 and could be used in structural studies. Inhibition of CO oxidation is limited to a very narrow potential range, with almost no inhibition occurring at potentials more positive than −0.4 V. 88 Binding of cyanate is slow, requiring several seconds with millimolar concentrations. On the other hand, isocyanides (e.g., n-BIC), which have been previously used as CO analogues, 36,105 can act both as a substrate and an inhibitor of CODH/ACS Mt . 69,71h Since CODH catalyzes the oxidation of n-BIC to n-BICt much more slowly (105-fold) than CO oxidation, n-BIC behaves as a rapidly binding competitive inhibitor of CO oxidation with a K i value of 1.66 mM. 69 The crystal structure of CODH Ch II treated with n-BIC reveals the C-cluster in an n-BICt-bound state containing a Ni–C bond and a hydroxyl group attached to the pendant iron (Figure 3J). 71h A hydrogen-bonding network that likely plays a role in stabilizing the C-cluster-bound CO2 includes the iron-bound hydroxyl, a free water molecule, the oxygen of the n-BICt, and two residues, His93 and Lys563. Cyanide, an analogue of CO, is a reversible inhibitor of CODH. 71f,71g,82,94,104,106,107 Depending on the conditions, cyanide can act as a rapid reversible inhibitor or a slow binding inhibitor. 106a When cyanide binds to the C-cluster in the Cred1 state, it forms a complex with an EPR spectrum that exhibits a g av of 1.72 (g = 1.55, 1.78, 1.87). 104,106a,108 CN– does not interact with the Cred2 state nor does it inhibit reduction of CO2. 88 Several studies suggested the nickel as the binding site for the cyanide, 104,106a,109 while, based on the results of ENDOR 85c and Mossbauer 74 studies, the iron was proposed as the binding site. Furthermore, it was proposed that cyanide may bind to multiple sites. 94 Furthermore, different binding modes, bent 71f or linear, 71g are suggested according to different crystal structures (Figure 3E,G). In the bent binding mode, there is still a water molecule bound to the pendant Fe, while there is no pendant Fe-bound water in the linear cyanide binding mode. A rearrangement is suggested to occur upon the rapid reversible binding of cyanide to yield a more stable cyanide adduct represented by the linear binding mode. 71g,94 ENDOR and Mossbauer results, previously interpreted as an evidence for cyanide binding to the pendant Fe, most likely represent a change on the water binding/leaving due to the linear binding mode of cyanide. Sulfide (S2-, HS–, or H2S) has been proposed to act both as inhibitor 87,88 and as activator, 104,110 and its existence and role as a bridging ligand between Ni and the pendant Fe in the C-cluster have been controversial (as mentioned in the previous section). Sulfide inhibits CO oxidation, but not CO2 reduction, as expected given that there were no significant changes in the EPR spectrum upon its addition to CODH in its Cred2 state. 87,88,104 Furthermore, Wang et al. showed that sulfide binds the inactive Cox state of the C-cluster inhibiting catalytic activity in the −50 and −250 mV potential range. 88 2.3 Catalytic Mechanism of CO Oxidation and CO2 Reduction 2.3.1 Metal-Based Catalysis of the Water–Gas Shift Reaction The proposed CO/CO2 conversion mechanism discussed here is analogous to the water–gas shift reaction described in Scheme 1. In both reaction mechanisms, CO and hydroxide ion are bound to two different metal centers that should be positioned in a proper geometry during the catalysis to allow the hydroxide to attack the M–CO intermediate, resulting in the formation of M–COOH. Release of the CO2 from the metal complex is coupled to a hydride shift, leaving a metal hydride that undergoes protonation to generate H2. Scheme 1 Mechanism of the Water–Gas Shift Reaction Scheme 2 Proposed Catalytic Mechanism of Reversible Carbon Monoxide Dehydrogenase The most well-characterized ferredoxin (Fd) from M. thermoacetica and many other organisms contains two [Fe4S4] clusters and thus can accept two electrons. For a Fd containing a single cluster, two Fd would be required. 2.3.2 Enzymatic Mechanism of CODH Besides the metal binding and positioning effects of the WGS catalysts, CODH is able to increase the reaction rate by optimizing the ligand binding geometry, controlling the acid–base reactions in and around the active site, enhancing substrate and product transport, and using the metal clusters as a wire to achieve a very fast electron transfer to the corresponding electron acceptors. 71b,111 In the description below, all residue numbers refer to the CODH Ch II. Oxidation of CO in the C-cluster occurs by a ping-pong reaction as shown in Scheme 2. In the first half reaction, the Cred1 state of the C-cluster binds and undergoes reduction by CO and then transfers electrons from the reduced C-cluster (Cred2) through the B- and D-clusters in the enzyme. However, we should point out that this electron transfer role for D-cluster has not been established. Furthermore, the D-cluster is not reducible at potentials as low as −530 mV, indicating that it may serve a structural, instead of an electron-transfer role. 72 In the second half-reaction, electrons are transferred to the external redox partners, e.g., ferredoxin. The midpoint reduction potential of the Cox/Cred1 redox couple is −200 mV, while it was reported as −530 mV for the Cred1/Cred2 redox couple. Cred1/Cred2 redox couple reduction potential matches well for the CO/CO2 redox potential. Similar to the water–gas shift reaction, the first catalytic step is the binding of CO and water to the metal centers (Scheme 2). On the basis of the results of ENDOR spectroscopic 85c and X-ray crystallographic 71a,71b studies, the catalytic water (hydroxide) molecule binds to the pendant Fe site of the C-cluster and also associates through H-bonding interactions with Lys563, His93, and His263 (Figure 4). These residues are proposed to participate in acid–base reactions, including formation of active Fe(II)–hydroxide. 71a,71b Site-directed substitutions of Lys563 and His113 abolish enzymatic activity, confirming the importance of these residues in catalysis. 112 A histidine tunnel composed of histidine residues located on sequential turns of a helix starting near the C-cluster and ending at the protein surface is proposed to facilitate transfer of protons during the reaction (Figure 4). 71a,112 Steady-state kinetic studies conducted using NMR spectroscopy support the presence of a rich proton reservoir inside the enzyme. 94 Figure 4 Structure of the C-cluster from CODH Ch II at 600 mV including only one coordinating residue: histidine and the ligands proposed to be important in catalytic activities. Atom colors: Dark gray (iron), orange (sulfide), red (oxygen), blue (nitrogen), white (carbon), dark green (nickel). Unbound red spheres represent the water molecules. Generated using Pymol from PDB 3B51. CO binds to the Cred1 state of the C-cluster with a diffusion-controlled rate constant greater than 2 × 108 M–1 s–1 (a value that is 10-fold faster than k cat/K m) according to rapid freeze quench EPR, 61 NMR, and steady-state kinetic studies. 94 However, the rate of reduction of the B-cluster (3000 s–1) 61 is only slightly higher than the steady-state k cat, indicating that this step is partially rate-limiting in the CODH mechanism. On the basis of NMR and steady-state kinetic studies, release of CO2 has also been proposed to be partly rate-limiting. 94 Binding of CO to CODH/ACS is associated with Fourier transform infrared (FTIR) bands at 1901, 1959, 1970, 2044, and 2078 cm–1, assigned to the Ni–CO stretching mode. 85a The absence of any IR bands in this region for the as-isolated CODH/ACS Mt suggests that the intrinsic Ni–CO ligand seen in hydrogenases 113 is not present in CODH. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals the presence of Ni2+ in the as-isolated Cred1 state of CODH Ch . Treatment of the enzyme with CO or Ti3+ changed the Ni K-edge shape slightly but does not shift the edge significantly. In both cases, the average Ni–S distance increases to 2.25 Å, making the Ni site more tetrahedral. Similarly, significant changes in the EXAFS analysis upon CO treatment suggest a structural rearrangement in the C-cluster, but without any changes in the Ni oxidation state. The only crystal structure that depicts a Ni–CO complex in a CODH is that of the CODH (CODH Mb ) portion of M. barkeri ACDS (Figure 3K), which, like the other CODHs, shows a water ligand bound to the pendant Fe. 71i CO is bound to the Ni in a bent fashion, with an angle of 103°, which could contribute to the high turnover numbers by destabilizing the ground state of the Ni–CO intermediate. The crystal structure of the complex between cyanide and CODH/ACS Mt reveals a similarly bent Ni–CN bond (Figure 3G), 71f supporting a bent Ni–CO bond with the substrate. It was proposed that a conserved isoleucine residue very close to the bound-CO could sterically block the linear binding of the CO. 71f It should be pointed out that an independent scrutiny of the crystallographic data, including a recalculation of the electron density, did not find evidence for the CO-ligand in the CODH Mb structure and for the CN ligand in CODH Mt structure. 114 In another structure of the CN complex, in this case with CODH Ch , the Ni–CN is linear (Figure 3E). A computational study indicated that Ile567 (Figure 4) plays a steric role and that Lys563 and the histidine residues are involved in acid–base chemistry during CO oxidation. 111 In the second step of the catalytic cycle, the Fe-bound hydroxide attacks the Ni–CO. FTIR studies support the formation of a metal carboxylate. 85a On the basis of the crystal structure of a bicarbonate-soaked CODH Ch II crystal, the Ni and Fe subcomponents of the C-cluster are bridged by a carboxylate, indicating that this could be a catalytic intermediate formed by attack of the hydroxide to the Ni–CO (Figure 3F). 86 Superimposition of the C-clusters of CO-bound CODH Mb with CO2-bound CODH Ch II suggests a significant shift in the carbon atom’s position, which is proposed to change the nickel coordination from tetrahedral to square planar in the CO2-bound form. The third step includes the generation and release of CO2 and a proton, and the reduction of the C-cluster from Cred1 to the Cred2 state, which thus should be two electrons more reduced than Cred1. While reduction of Cred1 to Cred2 upon reaction with CO is very fast (>2 × 108 M–1 s–1), 61,84 release of CO2 is proposed to be slow on the basis of NMR and steady-state kinetic studies. 94 Note that in the WGS reaction (above), this step involves a hydride migration, leaving the metal center in the same redox state. For several reasons, including the similarity of the EPR signals of Cred1 and Cred2, it was proposed that a metal hydride is also formed during this part of the CODH reaction cycle. 114 A related proposal is that two-electron reduction of the C-cluster generates a Ni0 state. 115 Because Ni(0) would be a diamagnetic species in a spin system with most of the electron density in the Fe–S cluster, formation of this low-valent Ni state would also be consistent with the minimal EPR spectral differences between the Cred1 and Cred2 states. In the fourth step, the C-cluster returns to its resting Cred1 state upon transfer of two electrons to the B- and D-clusters. The distance between the metal clusters is approximately 11 Å (Figure 2B), making it a good electron transfer route. 71b,116 Rapid kinetic studies show that, at high (>K m ) CO concentrations, internal electron transfer (from the C-cluster to B- and D-clusters) can be rate-limiting during the first half-reaction; 84 however, the final step (the pong stage) of the mechanism appears to be rate-limiting during steady-state turnover. 61,84 Step 5 involves electron transfer to the final electron acceptor. CODH interfaces with many electron carriers that support different specific activities, 29,117 including small redox proteins (ferredoxin, flavodoxin, rubredoxin); cofactors [FAD and FMN, but not NAD(P)]; redox enzymes (couple directly to CODH), like pyruvate:ferredoxin oxidoreductase (PFOR); hydrogenase; and artificial electron acceptors, like bipyridyl (viologen) dyes and methylene blue. 52,82 2.3.3 CO and Water Channels Given that the CODH active site is buried deeply inside the protein and the catalysis rates are very high, there must be highly efficient routes to achieve optimal substrate and product flow. A very long hydrophobic channel starting from the surface of the protein directing above the apical coordination site of nickel in the C-cluster was proposed to be the substrate channel, while another channel starting approximately at the end of the proposed substrate channel and ending at the enzyme surface near the B- and D-clusters was also proposed to be the water channel. 71b Although the recently published crystal structures support the presence of the channels, experimental support for these channels in monofunctional Ni–CODHs has been lacking. A recent X-ray crystallographic study of the interaction of CODH Ch II with the inhibitor and slow substrate n-BIC revealed the presence of two different channels: one similar to the substrate channel found in the CODH component of CODH/ACS and another substrate channel unique for monofunctional Ni–CODHs. 71h This unique channel is blocked by several residues in bifunctional Ni–CODHs, most likely to avoid the escape of the substrates. Molecular dynamics and density functional theory computations have provided evidence for a dynamically formed gas channel in CODH/ACS for diffusion of CO2 from solvent to the C-cluster. 118 Two cavities that are not apparent in the X-ray structures and are transiently created by protein fluctuations are proposed to form this channel. 2.4 Inorganic Modeling for CODH 2.4.1 Structural Models for the C-Cluster Spectroscopic studies had initially been interpreted to exclude the possibility of Ni being within a cube. 119 Thus, the first publication of the crystal structure of CODH was surprising to the bioinorganic chemistry community, because it revealed the C-cluster to contain a NiFe3S4 cubane cluster bridged to another iron. 71a,71b This heterometallic cluster has proven to be one of the most difficult metal centers to model. Holm and co-workers successfully prepared the first [NiFe3S4] cubane model complex 2 (Scheme 3) by reacting 1 with Ni(PPh3)4. 120 Changing the Ni ligand resulted in the synthesis of many different complexes; for example, with Ni(SEt)4, 3 is obtained. Manipulation of the iron ligands by tailoring the starting linear ferric cluster 1 led to novel NiFe3S4 clusters, e.g., 4. 121 Another modeling approach began by preparing cuboidal Fe3S4 clusters, 122 5, and incorporating different metal ions into this center, to generate a series of [MXFe3LS3] [where LS3 is 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolythio)benzene(3−)] complexes. 123 In these model complexes, the iron atoms are bound to LS3 ligands, making them structurally analogous to the C-cluster. Several [NiFe3S4] complexes, including a square planar species, have been reported, e.g., 6. 124,124b However, among these synthetic structural models, none has yet been reported to be active in catalyzing the interconversion of CO and CO2. Furthermore, no NiFe3S4 complex bridged to a pendant Fe like that of the C-cluster has yet been reported. Scheme 3 Schematic Views of Model Complexes Mimicking the C-Cluster 2.4.2 Functional Models for CODH As described in a recent US Department of Energy (DOE) report, 125 “The major obstacle preventing efficient conversion of carbon dioxide into energy-bearing products is the lack of catalysts...”; thus, the development of effective catalysts for the activation, reduction, and conversion of CO2, an abundant greenhouse gas, to fuels and chemicals would have enormous economic and environmental impact. As described in the introduction, CO2 reduction is difficult because of both thermodynamic (the low redox potential required) and kinetic (the chemical inertness of CO2) issues. The largest barrier that the model complexes have to overcome is the very high activation energy of the one-electron reduction of CO2 to the radical anion (see the Introduction, the electrochemistry section below, and a recent review 8 for details). Two detailed reviews covering catalytic CODH models are available. 8,126 Thus, here we will briefly describe important conclusions from the catalytic modeling efforts and how they relate to the enzymology of CODH, as well as suggest how principles uncovered from studies of the enzyme might inform the next generation of CO2 reduction (or CO oxidation) catalysts. Initial efforts to accomplish CO2 reduction included the synthesis of Co+ and Ni+ compounds of cyclam and its variants. 127,128 These studies showed the importance of the metal reduction potential, solvent effects, and intermolecular and intramolecular hydrogen bonding on CO2 binding affinity and kinetics. 129,130 In the enzyme, these factors are optimized to promote proper H-bonding, salt bridge and hydrophobic interactions among residues in the overall protein structure, and appropriate geometries and distances for metals and ligands at the active site, as well as in the secondary coordination sphere. Palladium phosphine complexes have been designed to be highly active molecular catalysts of CO2 reduction to CO. 131−133 In these complexes, Pd2+ is coordinated by three phosphorus atoms, RP(CH2CH2PR′2)2, where R and R′ can be alkyl or aryl groups, and a solvent molecule. According to the proposed catalytic cycle, 126 the initial step includes reduction of the metal center from (2+) to (1+) oxidation state, as shown in Scheme 4. Then, in the rate determining step (at low pH values), CO2 binds to the Pd+ to form a metal carboxylate at a rate that depends on the reduction potential of the metal center, with rates increasing as the potential decreases. 134 Similar initial steps are observed in Fe, Co, and Ni catalysts that require very negative potentials for one-electron reduction; however, they exhibit different rate-determining steps. 135−137 The next step is the protonation of the metal carboxylate, which promotes C–O bond cleavage and presumably is the origin of the increase in rate of CO2 reduction as the acidity of the reaction mixture increases. 138 Then, solvent (a coordinating organic molecule, e.g., dimethylformamide) dissociates from the metal center upon another 1e– reduction of the CO2H-bound complex, leaving a vacant site on the metal. 131 Protonation of this complex forms LPd–COOH2 followed by C–O bond cleavage and separation of CO and H2O on the metal center. At low acid concentrations, the C–O bond-cleavage step becomes rate-determining. 126 In the last step, water and carbon monoxide are released from the complex and solvent coordinates again to the metal. Dissociation of the M–CO bond is very fast, since the CO affinity of Pd2+ is very low. 131,133 In order to increase the CO2 affinity of the Pd catalysts (and unwittingly generate an intermediate(s) like that observed in CODH), the bimetallic compound 7 (Scheme 4) was prepared. 139 While one Pd binds the carbon atom of CO2, the other acts as a general base to bind the oxygen. This complex exhibits CO2 reduction activity as high as 104 M–1 s–1; however, it becomes inactivated after several turnovers, most likely due to Pd–Pd bond formation. We surmise that formation of a Ni–Fe bond would also be inhibitory to the enzymatic reaction and that this is prevented in Ni–CODHs due to the different reduction potentials of the metal centers. The general bimetallic theme is not necessarily conducive to catalysis in that compound 8, which has a Ni–Fe bimetallic model like CODH, has no CO2 reduction activity. 140 As a result, there is still a need to prepare and explore metallic catalysts to efficiently and economically reduce CO2. Scheme 4 Schematic View of the Proposed Intermediates in CO2 Reduction on Palladium Catalyst 2.5 Electrochemical and Environmental Application Efforts Given an abundant source of CO2, an important aim for technology would be to achieve rapid and efficient CO2 reduction to any of its reduction products using energy provided by electricity or solar sources. Electrochemical considerations are important in each case; a reversible electrocatalyst operates close to the reversible potential and is therefore by definition the most efficient, and efficiency is important given the cost of electricity and the need to exploit the visible region of the solar spectrum. The first 2 equiv stage of CO2 reduction, namely, its conversion to CO or formate, formally takes us into organic chemistry, but this stage is the most demanding in terms of electrochemical potential. There are numerous efforts to find suitable catalysts for CO2 reduction that are based on first-row transition metals; so far, the most successful electrocatalyst is Cu, although a sizable overpotential is required to drive conversions to several products. Other catalysts include polymeric Ru carbonyl complexes, compounds based on other transition elements, and even pyridinium ions, but they all fall far short of the performance observed in electrochemical studies of CODH. Protein film electrochemistry (PFE) refers to a suite of electrochemical techniques used to study an enzyme that is attached tightly to a suitable electrode surface, usually by simple physical adsorption. 141 The electrode is rotated at various speeds in an enclosed cell containing a small volume of buffered electrolyte and connected to a gas supply that goes through the headspace and equilibrates with solution. Reagents can also be injected into the solution through a septum. Many redox enzymes have now been investigated by PFE, revealing detailed information on their catalytic activity in both oxidizing and reducing directions, as a direct function of electrode potential (E). The primary observable is the catalytic current (i), negative or positive for net reduction or net oxidation, respectively, which is directly proportional to the turnover rate at the particular electrode potential that is applied by the instrument. Specifically, the current i (E) observed at a particular potential is related to net turnover frequency k cat(E) at that particular potential by i (E) = k cat(E). nFAΓ, where n is the number of electrons involved (2 for CODH), F is the Faraday constant, A is the electrode area, and Γ is the electroactive coverage of enzyme. The latter is usually 420 nm). In each case, 5 mg of nanoparticles (20 mg in the case of ZnO) was modified with CODH Ch I (total 2.56 nmol) and RuP (total 56 nmol). The buffer in each experiment was 0.20 M MES, pH 6, 20 °C. (C) Production of CO by visible light using direct band gap excitation of various types of cadmium sulfide attached to CODH Ch I. QD = quantum dot, NR = nanorod; calcined CdS was heated at 450 °C for 45 min. The buffer in each experiment was 0.35 M MES, pH 6, at 20 °C. Adapted from refs (146a) (copyright 2011 The Royal Society of Chemistry) and (147) (copyright 2012 The Royal Society of Chemistry) with permission. The first of experiments used CODH Ch I attached to various nanoparticles for which the natural band gaps E G exceed the energy available from visible light; consequently, the nanoparticles were modified by coattachment of the photosensitizing complex “RuP” = [Ru2+(bpy)2(4,4′-(PO3H2)2-bpy)]2+ (λmax 455 nm), analogous to technology introduced by Michael Grätzel for dye-sensitized photovoltaic cells. ref146 The relevant conduction band potentials E CB (measured for bulk materials) are as follows: TiO2 (anatase), −0.52 V (note E CB = 3.1 eV, hence the need to use UV irradiation when RuP is not coattached); TiO2 (rutile), −0.32 V; ZnO, ca. −0.5 V; SrTiO3, −0.72 V. For comparison the standard reduction potential for the CO2/CO couple at pH 6.0 is −0.46 V. The results depicted in panel B show that CO production by dye-sensitized visible light excitation depends greatly on the nature of the metal oxide semiconducting nanoparticles. Anatase is clearly supreme: the nanoparticles known as P25 are a composite of anatase with some rutile phase, although rutile itself is inactive (as expected, since E CB is too positive to drive CO2 reduction) and SrTiO3 is possibly inactive because E CB is so negative that electrons easily transfer back to RuP. The best rate, obtained with P25 and calculated on the basis of total CODH Ch I used, equates to a CO production rate of approximately 0.15 s–1 per molecule of CODH. 146 This rate is much slower than that achieved for a hydrogenase at the same material (50 s–1), a fact that is still not resolved. One important difference between the conventional electrochemical and photoexcitation experiments is that, in the latter, electrons may recombine before being used by the catalyst. The tentative conclusion is that a good photoelectrocatalyst should be one that traps all the electrons required to carry out the reaction and restricts their return to the semiconductor and inevitable recombination. Using semiconducting materials with a smaller band gap, it is possible to use visible light with the need for dye sensitization. Experiments similar to those with RuP-modified anatase, but using band gap excitation, were carried out using different types of nanoparticle formed from cadmium sulfide, CdS. As a rough guide, for bulk CdS, E g = 2.3 eV (corresponding to λ = 540 nm) and E CB = −0.87 V. Using CdS nanoparticles (nanorods, NR) or CdS quantum dots (QD), slightly higher rates were achieved, 0.25 s–1 compared to the results obtained with anatase (panel C). 147 The CdS quantum dots have a typical radius that is half that of CODH; thus, in principle, up to 10 QDs may bind to one CODH molecule, reversing the size ratio indicated in panel A. Thermal calcination of CdS nanoparticles, which results in irregular clusters of larger particle size, resulted in no activity when CODH was attached. The success of these artificial photosynthesis experiments gives strong encouragement for pursuing research in this area and for the role that enzymes play in providing a reversible catalyst in which many different properties can be modified by genetic engineering and tested quantitatively by electrochemical methods. 3 Acetyl-CoA Synthase 3.1 Chemistry and Biochemistry of C–C Bond-Forming Reactions Involving CO2 and CO Developing an industrial process that efficiently couples CO2 reduction to CO with a carbonylation reaction would be an important advance in the chemical industry because carbon–carbon formation by reactions with CO is instrumental in many industrial processes. 148 CODH/ACS catalyze such a coupled process as an important component of the biological carbon cycle. 46 If fuels could be made from CO2, these C–C bond-forming reactions will be of even more importance in energy generation. Industrial processes involving carbonylation chemistry include the Monsanto process, hydroformylation, and the Reppe process. As has been pointed out elsewhere, 149 the intermediate steps in the Monsanto process for acetic acid formation from methanol and CO are nearly identical to those in the catalytic mechanism of ACS, as described in the Introduction. Both the biological and homogeneous catalysts use organometallic mechanisms that feature low-valent metal centers [e.g., Rh(I) vs Ni(I)] to react with CO and form a metal–carbonyl bond (M–CO) or with a methyl donor and generate a methyl–metal bond (M–CH3). The key carbon–carbon bond-forming reactions involve a migratory insertion of the metal-bound CO and methyl groups to generate an acyl–metal intermediate that undergoes reductive elimination by a coordinated iodide in the chemical reaction or by the thiolate of CoA in ACS to generate acetyl-CoA. 150 Acetyl-CoA then serves as a source of energy and cell carbon. 30 M–alkyl and M–CO are also key intermediates in the hydroformylation reaction, to convert alkenes to aldehydes. Similar organometallic intermediates are formed in the Pd-based Reppe process. 31b 3.2 Characteristics of CODH/ACS 3.2.1 Enzymatic Activity The gene encoding ACS (acsB) is a marker for the Wood–Ljungdahl pathway, and whenever it occurs in a microbial genome, it is within a gene cluster containing other pathway genes. 41 ACS associates tightly in a complex with CODH and utilizes the product of the CODH reaction (CO) as its substrate in a kinetically coupled reaction linked to generation of acetyl-CoA via eq 11. 61,71b,151 The second substrate of ACS is a methyl group donated by a methylated B12 protein, the corrinoid iron–sulfur protein (CFeSP). The third substrate is CoA, which reacts with CO and the Co-bound methyl group to make acetyl-CoA, a cellular carbon and energy source. As shown in Figure 1, ACS can catalyze this reaction reversibly. Thus, in aceticlastic methanogens, it catalyzes the disassembly of acetyl-CoA, breaking both the C–C and C–S bonds to form CoA, the methylated CFeSP, and CO. 152 A convenient assay for ACS is to measure that rate of exchange of 14C from [1-14C] acetyl-CoA with 12CO. For ACS (ACS Ch ) and CODH/ACS (CODH/ACS Ch ) from C. hydrogenoformans, exchange rates were reported to be 2.4 or 5.9 μmol of CO per min per mg, respectively, at 70 °C and pH 6 in the presence of 3 mM Ti(III) citrate. 110 The exchange rate reported for CODH/ACS Mt is 0.16 μmol of CO per min per mg at 55 °C and pH 6, without addition of any external reducing agent. 153 ACS Ch and CODH/ACS Ch also catalyze acetyl-CoA synthesis from CFeSP, methylcobalamin, CoA, and CO with activities of 0.14 and 0.91 U/mg μmol of acetyl-CoA production per min per mg. 110 11 3.2.2 Active Site Metal Cluster and the Importance of Nickel in ACS The active site of ACS, so-called the A-cluster, was the first NiFeS cluster reported, 154 although the specific role of nickel in ACS activity was established later. 155,156 In the A-cluster, a Fe4S4 cluster is bridged to a nickel, called the proximal nickel (Nip) because of its proximity to the cluster, and also thiolate-bridged to the distal nickel (Nid), which is coordinated by two cysteine and two backbone amides as shown in Figures 10 and 11. The Nid, stabilized due to its square planar geometry and oxidation state (2+), is adjacent to a cavity that can accommodate the substrate and products. Nip is coordinated by three S atoms in an apparent T-shaped environment. Another ligand, which completes a distorted square planar coordination, has been assigned as an oxygen ligand donated by water 110 or an acetyl 39a group, though, in the latter case, the structure was of an enzyme containing Cu at the Nip site. The Nip is labile (i.e., easily replaced by other metals) and is thought to be the sole metal that is directly involved in binding the substrates. Two different crystal structures showed copper or zinc located at the Nip site (Figure 11), 39a,39b and early studies indicated a positive correlation between the copper content and ACS activity; thus, copper was suggested to be a component of the active cluster. 157 However, studies over a much wider range of Ni contents demonstrated that activity was positively correlated with Ni and negatively related to the Cu content; 110,158 furthermore, copper was not responsible for, and even inhibited, the activity of the enzyme. 159 The active methanogenic enzyme was shown to contain two Ni per active center. Thus, it is clear that the active A-cluster contains two Ni and four Fe atoms. In almost all of the studies utilizing recombinant ACS, the enzyme is activated by nickel reconstitution. CO binding to the A-cluster upon the reduction by dithionite results in an EPR-active species called NiFeC species, due to its hypefine broadening by 61Ni, 57Fe, and 13CO, and is used to determine the nickel incorporation into the A-cluster. Figure 10 Structure of CODH/ACS Mt . (A) Overall structure of CODH/ACS. Green units in the center are the two CODH homodimers; the left unit is the ACS in open conformation, and the right unit is the ACS in closed conformation. Closer views of the A-cluster pocket in (B) open conformation and (C) closed conformation. Atom colors: Brown (iron), orange (sulfide), red (oxygen), blue (nitrogen), light green (carbon), dark green (nickel), white (unassigned). Generated using Pymol from PDB 1OAO. Figure 11 Structure of A-cluster from PDB (A and B) 1OAO, (C) 1MJG, and (D) 2Z8Y. Generated using Pymol. 3.3 Structure of the CODH/ACS 3.3.1 Inner Channel in CODH/ACS The gene encoding ACS is generally contiguous with that encoding CODH. This genetic linkage parallels tight enzymatic coupling of CODH and ACS. Kinetic coupling has been established by several experiments, including one in which CO2 was used as a substrate and the incorporation of in situ-formed CO into the carbonyl group of acetyl-CoA was monitored. Unlabeled CO in solution does not decrease the rate or extent of incorporation of labeled 14CO2 into acetyl-CoA. 160 Although CO is a substrate for the CODH/ACS, absence of CO in the solution did not affect acetyl-CoA synthase activity, while CO2 had a major impact on the reactivity. 63 Similarly, addition of hemoglobin or myoglobin to the assay mixture as a CO scavenger only marginally inhibited acetyl-CoA synthesis. 63,160 These and other results 63,160,161 suggest that CO produced in the CODH subunit from CO2 remains sequestered within the enzyme without equilibrating with solution as it is transferred to the ACS active site, and it was proposed that CO migrates through an inner channel within the CODH/ACS complex from the CODH to that ACS active site. 63,160 The crystal structure of CODH/ACS Mt showed that the A- and C-clusters are separated by 67 Å, which would seem to be too long to allow kinetic coupling of the CODH- and ACS-catalyzed reactions (Figure 10A). 39 However, interior surface calculations and diffraction experiments on Xe-treated crystals disclosed the presence of a continuous 140 Å long hydrophobic tunnel that connects the active sites of CODH and ACS, the C- and A-clusters, respectively (Figure 12). 39,71e Since the van de Waals radius of Xe (2.16 Å) and CO (∼2 Å) are similar, Xe can be considered as a good mimic for CO. A total of 19 Xe atoms were located in this hydrophobic tunnel. Examination of the residues within 5 Å of Xe atoms shows an insignificant degree of sequence homology but supports a highly conserved pattern of hydrophobic residues (except for the positions and orientations of C468 and T593, which are located near the C-cluster). The tunnel is composed of a series of interconnected hydrophobic pockets that can be conceptualized as a pinball plunger where launching of each ball (gas molecule) from the trough into the playfield releases another ball into the launching lane. Of course, with CODH/ACS, multiple balls are at play in the channel and each has only one target, the A-cluster. In each of the ACS subunits, one Xe atom was found 3.5 Å from Nip (Figure 11D). Further experimental support for a CO-binding pocket near the A-cluster is the finding that, when CO-bound ACS is subjected to photolysis, the energy barrier for recombination of Nip with CO is only 1 kJ/mol. 162 Figure 12 Structure of CODH/ACS Mt crystallized in the presence of high pressures of Xe (PDB 2Z8Y) (shown as the blue spheres) to reveal the hydrophobic CO tunnel. Adapted with permission from ref (71e). Copyright 2008 American Chemical Society. When residues (A578, L215, A219, A110, A222, A265) that are located within the hydrophobic channel in CODH/ACS Mt were substituted, ACS activity with CO2 as substrate was severely diminished. 163 These results support the importance of the tunnel for CO migration to the A-cluster. Furthermore, the variants exhibit little inhibition of acetyl-CoA synthesis by CO, in contrast to the wild-type proteins, indicating that the channel plays an important role in cooperative inhibition of A-cluster activity by CO. It was suggested that there may be at least two ways for CO to reach the A-cluster: through the channel and from the solvent. A water channel close to the ββ interface is proposed to be the second way for the CO, 163a but this idea is not well established yet. The role of the CO channel is most likely to prevent the loss of energetically expensive CO in the solution and to efficiently direct this gaseous substrate to its site of reactivity at the A-cluster. 3.3.2 Conformational Changes As shown in Figure 10, ACS consists of three main domains. The first domain, which interacts with CODH, starts with helices and continues with a Rossman fold. This domain contains a ferredoxin interaction domain. 164 The second domain includes six Arg residues near Trp418 (Figure 10A). These residues are involved in CoA binding according to fluorescence-quenching studies of Trp418 and inhibition studies of CoA binding upon modification of Arg residues. 165 The final domain bears the A-cluster. This domain undergoes structural rearrangements during turnover (Figure 10B,C). ACS binds three substrates of vastly different sizes: CO (30 Da), CoA (770 Da coenzyme), and methylated CFeSP (88 kDa dimeric protein). CODH/ACS Mt is crystallized in two different forms that are thought to be related to the catalysis: closed 39a and open 110 conformations. Another structure depicts both conformations (one in each CODH/ACS dimer) (Figure 10). 39b In its closed conformation, the channel is open, allowing CO to pass through the tunnel to the A-cluster; however, there is no apparent access to the methylated CFeSP. In the open configuration, one of the domains (domain 3) of ACS rotates, which partially exposes the A-cluster, enabling interaction with the CFeSP and closure of the CO tunnel. Although the catalytic importance and the main trigger of this conformational change are not yet well established experimentally, there appear to be at least four discrete conformations. Throughout all of these conformational changes, both CO and the A-cluster must be protected from exposure to solvent, because CO does not equilibrate with solvent during catalysis. 160,63 In one closed conformation, poised for binding CO, the CO channel is open to allow the CO to reach the A-cluster, which is buried and unable to access the CFeSP (Figure 10C). In an open conformation, ready to bind the methyl group, the A-cluster is rotated to interface with the CFeSP and the CO channel is blocked to avoid CO release (Figure 10B). Another closed (solvent-excluded) conformation is required during formation of the acetyl–metal complex to avoid hydrolysis of the acetyl–metal center. Then, the A-cluster must be rotated into a more open conformation to allow CoA binding, thiolytic cleavage of the acetyl group, and acetyl-CoA release. A crystal structure of the truncated ACS Mt is proposed to represent the CoA binding conformation of the enzyme. 166 While there is concrete crystallographic proof for the first two conformations, more work is needed to reconcile the other two conformations. Experiments performed on the methanogenic acetyl-CoA decarbonylase/synthase (ACDS) suggested that the N-terminal region of ACS is involved in C–C bond cleavage. 167 On the basis of kinetic and spectroscopic data for different ACS enzymes, it appears that conformational changes directly impact stability of the Ni–acetyl intermediate. Steric hindrance around the Nip due to conformational changes of a proximal phenylalanine (F512) is proposed to facilitate C–C bond cleavage and to affect interaction of CO with the enzyme. 168 Thus, conformational changes clearly affect ACS enzymatic activity, and studies are needed to better understand these impacts on the catalytic mechanism. 3.4 Catalytic Mechanism of Acetyl-CoA Synthesis The chemistry of the ACS reaction is catalyzed by the A-cluster; surprisingly, even though this center contains six redox-active metals, substrate binding seems to be confined to a single metal center, Nip. Pulse-chase studies indicate that the steady-state mechanism involves random order binding of the methyl group and CO, followed by ordered binding of CoA. 169 The two competing mechanisms that have been proposed differ in the oxidation state of the Nip. The “paramagnetic mechanism” proposes a Nip(I) catalyst and Ni(I)–CO [or methyl–Ni(III) and methyl–Ni(II) for the other branch of the random mechanism] and acetyl–Ni(II) intermediates (Scheme 6), 170 while the “diamagnetic mechanism” proposes a Ni(0) active catalyst with Ni(0)–CO and methyl–Ni(II) [without the paramagnetic methyl–Ni(III)] intermediates. 115 However, both mechanisms include organometallic methyl–Ni, acetyl–Ni, and thiolytic cleavage of the acetyl–Ni species by CoA. Because of their similarity, we will focus here on the paramagnetic mechanism and include relevant aspects of the Ni(0)-based mechanism. As described elsewhere, the mechanism of acetyl-CoA formation resembles the Monsanto process, where acetic acid is produced by the reaction of methanol and CO on a rhodium complex through organometallic complexes. 171,172 Scheme 6 Proposed Paramagnetic Mechanism of Acetyl-CoA Synthesis Catalyzed by the A-Cluster The essential role of the tetrameric α2β2 CODH (as it was known until 1985) in acetyl-CoA synthesis was predicated on studies of the isotope exchange reaction between CO and the carbonyl group of acetyl CoA. 151 In this reaction, the C–C and C–S bonds of acetyl-CoA are cleaved to generate enzyme-bound methyl, carbonyl, and CoA groups, allowing the central carbonyl group to exchange with free CO; finally, the C–S and C–C bonds must be resynthesized. That CODH/ACS alone (and it was subsequently shown that ACS alone is required for this reaction 61 ) is required clearly demonstrated that this enzyme is responsible for the key step in the Wood–Ljungdahl pathway: condensation of methyl, CO, and CoA to form acetyl-CoA. Because CODH/ACS catalyzes an exchange reaction between CoA and acetyl-CoA much faster than the CO/acetyl-CoA exchange reaction, CoA was proposed to be the final substrate that reacts with the bound acetyl group to form acetyl-CoA. 173 Since methylation of CODH/ACS by the methylated CFeSP can occur without any CO or CoA and also faster than the overall acetyl-CoA synthesis reaction, the methyl group was suggested to be the first substrate to bind Nip. 174−177 Since the back-π-donation upon CO binding to the metal is expected to decrease the electron density on the metal center, its reactivity with methyl could be decreased if methyl is bound as the second intermediate. However, CO can also bind the enzyme in the absence of a methyl donor or CoA; furthermore, a pulse-chase study of acetyl-CoA synthesis with CODH/ACS Mt and with ACS-only clearly indicated that either CO or methyl can bind first during catalysis. 169 In this pulse-chase (or isotope dilution) study, CODH/ACS Mt is incubated with equimolar amounts of a labeled substrate (14CH3–CFeSP, 14CO, or 3′-dephospho-CoA) and then mixed with a solution containing either (1) the other two substrates at high concentrations or (2) all three substrates at high concentrations. Incorporation of the label into product is measured. If the mechanism is strictly ordered with labeled substrate being first to bind, addition of that unlabeled substrate in excess will not lead to dilution of the isotope. On the other hand, if it is actually the second substrate in the sequence, it must dissociate to allow the true first substrate to bind before it can form a productive complex. Dissociation leads to isotope dilution, as detected in the product. This method is valuable because one can determine how ordered (or how random) the reaction is. Nearly complete dilution of dephospho-CoA in the pool of excess CoA is observed. However, there is no measurable isotope dilution when ACS is treated with 14CH3–CFeSP or 14CO. Thus, the first substrate can be either the methyl group or CO group, and the third substrate is CoA. However, it is important to note that CO but not CO2 was used as the source of the carbonyl group of acetyl-CoA; thus, possible regulatory effects of the tunnel and the possible effects of the coupled reaction on the mechanism were not addressed in this study. For illustration, we show the mechanistic scheme as an ordered reaction with CO as the first binding substrate (Scheme 6). Before substrate binding, a reductive activation by Ti(III) citrate or another low-potential electron donor is required. The oxidized state of the A-cluster, which has a configuration of [Fe4S4]2+Nip 2+Nid 2+, cannot accept a methyl from the CFeSP 176,178 or bind CO. 177 This Nip(I) intermediate was trapped by photolysis of the Ni(I)–CO species and its EPR spectrum was recorded, exhibiting g-values of 2.56, 2.10, and 2.01. 162 Then, in a kinetically coupled reaction, Nip(I) binds CO as the first step in the mechanism. For this to occur, the tunnel must be open to allow migration of the CO that is produced from CO2 in the C-cluster. Two reduced states have been observed by Mossbauer spectroscopy: [Fe4S4]+ [Nip]+ and [Fe4S4]2+ [Nip]+. 179 The Nip–CO species is proposed to form the well-characterized NiFeC species. 30 DFT calculations combined with EPR, 154 ENDOR, 180 Mossbauer, 85b,181 IR, 182 and X-ray experiments 183,184 indicate that NiFeC species consists of a [Fe4S4]2+ cluster bridged to a dinuclear Ni center, Nip +–CO, and Nid 2+. 185 According to the EPR spectral properties, the unpaired electron density is delocalized over the Nip, the [Fe4S4] cluster, and the terminal carbonyl group. 154,186 Various experiments indicate the catalytic competence of the NiFeC species. It forms at the same rate and decays 6-fold faster than the steady-state rate of acetyl-CoA synthesis. 177 The rate of the formation of NiFeC species monitored by EPR equals the rate of the Ni–CO bond formation probed by IR, indicating that Ni–CO is the only metal–carbonyl species formed upon the reaction of ACS and CO. 182b Controlled potential enzymology studies revealed the need for only a single electron transfer with a midpoint potential of −511 mV 187 to activate the A-cluster, a value that is very similar to that reported for the formation of NiFeC species from acetyl-CoA (−541 mV). 188 Ferredoxin-II (Fd-II), which enhances the isotopic exchange rate, 154 is shown to activate the A-cluster most likely by forming this Ni+ species. 187 In the diamagnetic mechanism, formation of a Ni(0) intermediate is proposed 115 and is supported by the ability of a model Ni(0)–phosphine complex to accept a methyl group from a Co3+–CH3 complex; 189 however, a Ni(0) state on ACS has never been observed or reported. Furthermore, two-electron reduction of Ni(0) to Ni2+ would be extremely difficult, since even the reduction potential for Ni2+–CO/Ni+–CO is already very negative, below −550 mV. The presence of a Ni(0) in a highly electropositive environment formed by Nid 2+ and [Fe4S4]2+ seems unlikely because electron transfer to the cluster or the Nid would be favored. The second step of the mechanism is binding of the methyl group to the A-cluster. In this step, the protein is most likely in its open conformation, where the A-cluster is accessible to the large CFeSP. Rapid kinetic studies utilizing a chiral methyl donor suggested the transfer of a methyl cation through an SN2 mechanism where Nip attacks to the Co3+–CH3 on the CFeSP to leave behind a Co1+ and a methylated Nip. The methyl, like CO, appears to bind to the Nip. 155,158,190 Although a radical methyl transfer is suggested according to the model studies, 191,192 this is not feasible in biology, since the reduction potential of Co3+–CH3/Co2+–CH3 is below −1 V, which would be too low for physiological electron transfer. 193 Rapid kinetic studies indicate that both methylated 177,190 and acetylated ACS 194 species are EPR-silent. This represents a challenge for the paramagnetic mechanism, since the SN2 addition of methyl cation to the Nip + should result in a Nip 3+. However, since the Nip 3+ state is predicted to be highly oxidizing and unstable, it should readily be reduced to the Ni2+ state. Since acetyl-CoA synthesis does not require net electron transfer from the environment, 151 this reduction could be achieved by an internal electron transfer, as shown in Scheme 6. Fd-II is shown to donate an electron to the proposed Nip 3+ intermediate and to accept an electron during the cleavage of the Nip–acetyl intermediate, most likely by interfacing with an internal electron shuttle. 187 However, this internal electron shuttle has not yet been identified. Such an internal electron transfer is not necessary for the diamagnetic mechanism, since Ni(0) is converted to Ni2+–CH3. However, as mentioned above, the diamagnetic mechanism has its own challenges. The next step involves a methyl migration (carbonyl insertion) to form an acetyl–metal complex. A crystal structure of ACS Mt is proposed to represent the CoA binding conformation of the enzyme. 166 Addition of CoA is followed by the thiolytic cleavage of the acetyl-CoA product and also the internal transfer of electrons. 3.5 Structural and Functional Models of ACS Modeling efforts for the A-cluster of ACS up until ∼2005 have been reviewed. 195 Thus, we will only briefly cover the Ni complexes reported (Scheme 7). Scheme 7 Schematic Views of Model Complexes of A-Cluster Since the [Fe4S4] complex and the distal Ni are thought to modulate the electronic and redox properties of the active site but not to bind any ligands, most model complexes have focused on imitating the Nip or the bimetallic Nip–Nid environment, omitting the [Fe4S4] complex. Initially, compound 10 was prepared by the reaction of 9 with Ni(cyclooctadiene)2 and CO as a very stable complex in anaerobic solution that undergoes immediate degradation upon air exposure. 196 The IR spectrum of 10 exhibits νCO bands at 1948 and 1866 cm–1. Crystallographic and NMR spectroscopic characterization of the compound indicates the presence of a Ni(0)Ni(2+) couple. The bimetallic Ni complexes, 12 and 14, have also been reported. 197 Compound 12 was synthesized from the reaction of 11 and (R2PCH2CH2PR2)NiCl2 (R = Et, Ph). Reaction of 11 with nickel chloride also yielded a trinuclear nickel complex upon the dimerization of two units of 11 around a nickel atom. The Ac–CycGlyCys–CONH2 is used as precursor for the synthesis of compound 11. Synthesis of compound 12 is a significant improvement, since it includes two sulfides and two phosphines to mimic the environment of Nip. That Ni can be reduced to form a Ni(1+)Ni(2+) complex. While the oxidized Ni(II) state cannot bind CO, the reduced state can and be reduced further to the Ni(0)Ni(2+) state. Compound 14, synthesized from 13, contains a coordinating ring pattern and donor set for Nid that is almost identical to that of the A-cluster. However, no ACS activity or ligand-binding properties were observed for this interesting compound. Furthermore, Harrop reported the synthesis and characterization of new complexes 15–18. 198−200 Treatment of compound 15 with Cu(2,9-dimethyl-1,10-phenanthroline)Cl resulted in a dinuclear Cu(I)–Ni(II) complex, which does not bind CO and does not include a reducible nickel center. 198 Neither compound 15 nor compound 16 can be reduced easily or can bind CO. Compounds 15 and 16 were utilized as precursors to prepare 17 and 18, respectively. Reduction of 17 with dithionite yields a five-coordinate Ni(I) complex in trigonal bipyramidal geometry with an axial EPR signature of g = 2.226, 2.125. The Ni(I) state of 17 binds CO to form a complex with a rhombic EPR spectra (g = 2.223, 2.218, 2.019), which is typical for six-coordinate Ni(I)–CO complexes 201 and with a Ni(I)–CO band at 2044 cm–1. Compound 18 can be reduced with dithionite or sodium borohydride to form a Ni(I) complex, based on its EPR spectrum. As expected, 18 binds CO in the Ni(I) state, exhibiting a strong Ni(I)–CO band at 1997 cm–1, a value that is very close to what is observed in A-cluster (1996 cm–1). 149 These studies show the stability and inertness of Nid 2+ and reducibility and ligand affinity of the Nip atom. A Ni(II)–Ni(I) compound, 19, was recently shown to accept methyl from methylcobaloxime and form thioester upon CO exposure. 202 This result indicates that a Ni(II)Ni(I) can afford the chemisty of the acetyl-CoA synthesis reaction in a proper coordination and electronic environment. Reactivity of a Nip(0) analogue, Ni(triphos)(PPh3) (compound 20), with a methyl–CFeSP analogue, 21, yields compound 22. 203 While compound 20 was methylated by 21 in approximately 1 h, no methylation or acylation was observed for compound 23, even 24 h after of reaction. Furthermore, reaction on compounds 24 and 25 with 21 and CO leads to acetylation of the S-ligand of the methylated nickel and dissociation of the thioester. 204,205 The viability of the Ni(II)/Ni(0) couple in Ni–acyl formation is further supported by another binuclear nickel compound, 26 (Dmp is 2,6-dimesitylphenyl), which forms the acetyl thioester upon reaction with CO. 206 The methyl group in compound 26 was donated either by compound 21 or MeI. These studies support the plausibility of the methyl ligand binding to the metal before CO binds. Similarly, a Ni(0)–CO complex, compound 27 was prepared and shown to accept methyl and to exhibit Ni–acyl bond formation. 207 As summarized above, inorganic model studies suggest that Ni(II) centers mimicking Nid are not reducible or catalytically active. Ni(0) and Ni(I) complexes can bind CO as well as mimic ACS activity. There are also examples of both Ni(I) and Ni(0) complexes that bind methyl followed by CO and vice versa. Further studies are necessary to clarify these mechanistic issues. Inclusion of the Fe4S4 cluster in the inorganic models would provide important information about the role of this redox-active center in the ACS reaction and perhaps would afford new catalysts to afford acetyl-CoA synthesis without enzymes. 4 Conclusions and Future Directions We have described studies on two remarkable metalloenzymes that have defined novel biochemical mechanisms involving organometallic chemistry to catalyze their reactions. CODH catalyzes CO2 reduction, a reaction that has important potential impact on the generation of energy-rich compounds and on the environment due to its involvement in the global carbon cycle. This is a catalyst that has optimized its kinetics and thermodynamics, operating at high rates and without an overpotential. These characteristics warrant further studies of CODH aimed at understanding the principles that guide these two enviable properties. Past studies outlined here have uncovered novel metal clusters to bind, activate, and transform substrates (CO and CO2) and macromolecular channels that enhance flux of precious substrates between catalytic sites. CODH also is a wonderful system to explore how chemical bond forming and breaking interfaces with redox chemistry. Future research will define the kinetic and structural properties and electronic states of the yet-to-capture intermediates in CO oxidation/CO2 reduction and reveal where the electrons reside during the two-electron redox interconversion. Future studies on this enzyme will be greatly enriched with the development of a well-defined and reproducible way to generate variants of CODH. This enzyme, especially coupled to ACS and other enzymes of the Wood–Ljungdahl pathway, offers great potential for biotechnology through the conversion of simple abundant compounds into needed chemicals and fuels. To realize this promise, host organisms must be developed or reconfigured to foster an anaerobic environment that includes all of the metallochaperones and accessory factors required to support the high activity observed in the native organisms. These factors and their roles need to be characterized. In order to tap the potential of CODH, ACS must be tamed. Above we have described the highly unusual metal center at the heart of this enzyme and provided information, gleaned by a mixture of biochemical, biological, and biophysical methods, on the modular way that this center forms organometallic (M–CO, M–CH3, M–acetyl, and M–S) bonds en route to generation of the compound at the center of our metabolic charts, acetyl-CoA. Though we have defined the novel modular approach to synthesis of this key metabolic building block, we do not yet understand the internal redox chemistry that drives C–C and C–S bond formation to generate acetyl-CoA. It is important to capture and characterize the yet undefined intermediates in the ACS catalytic cycle. We have learned to express ACS and reconstitute it in vitro to near full activity; however, the same challenges remain in developing a genetic system that produces a highly active enzyme. It will be extremely important to understand how the activities of CODH and ACS are coordinated in the complex and to increase our understanding of the dynamics and mechanics of the tunnel that carries CO from the C-cluster to the A-cluster. With both CODH and ACS, it is important to understand the movement of domains and how these proteins interact with other components of the Wood–Ljungdahl pathway, especially the CFeSP.Future high-impact papers will emerge that provide an understanding of the structures of complexes between CODH/ACS and the CFeSP, achievable by X-ray diffraction methods as well as other methods that can define conformational, ligation, and electronic states and measure distances among the redox centers in these various states. Finally, ACS has been found in the multidrug-resistant human pathogen Clostridium difficile, and better understanding of these enzymes could foster the discovery of new therapeutic solutions against C. difficile infections. 208
Author and article information
Contributors
Yoshihiko Sako: sako@kais.kyoto-u.ac.jp
Journal
Journal ID (nlm-ta): Extremophiles
Journal ID (iso-abbrev): Extremophiles
Title:
Extremophiles
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Springer Japan
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1431-0651
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(Electronic):
1433-4909
Publication date
(Electronic):
9
May
2020
Publication date PMC-release: 9
May
2020
Publication date
(Print):
2020
Volume: 24
Issue: 4
Pages: 551-564
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Article
Publisher ID: 1175DOI: 10.1007/s00792-020-01175-z
PMC ID: 7306483
PubMed ID: 32388815
SO-VID: 914979ec-f1e3-4085-bb1a-d9c260ca6b50
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Date
received
: 6
March
2020
Date
accepted
: 27
April
2020
Funding
Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
Award ID: 16H06381
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ScienceOpen disciplines: Microbiology & Virology