Enzymology of the Wood–Ljungdahl Pathway of Acetogenesis

STEPHEN W. R AGSDALE

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA

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

Key words: acetogenic ; carbon dioxide fixation; carbon monoxide; cobalamin

Introduction ern branch is unique to acetogens, methanogens, and sulfate reducers, and exhibits novel mechanistic fea- Some anaerobic microbes, including acetogenic tures. Acetogenic bacteria (e.g., acetogens or homoace- bacteria and methanogenic archaea, convert CO2 togens) synthesize acetic acid as their sole or primary to cellular carbon by the Wood–Ljungdahl pathway metabolic end-product. Globally, acetogens produce , 13 (FIG.1).1 2 The global importance of acetogens is cov- over 10 kg (100 billion U.S. tons) of acetic acid an- ered fully in Harold Drake’s chapter.3 Acetogenic bac- nually,10 which dwarfs the total output of the world’s teria use this pathway as their major means of gener- chemical industry. ating energy for growth. Moorella thermoacetica,isolated FIGURE 1 depicts growth of acetogens on glucose. in 1942,4 is the model and is the organism However, these organisms can use a variety of sub- on which most studies of the Wood–Ljungdahl have strates, including the biodegradation products of most been performed; its genome was recently sequenced. natural polymers, such as cellulose, lignin (sugars, alco- Methanogens growing on H2/CO2 use the pathway hols, aromatic compounds), and inorganic gases (CO, for generating cell carbon; however, those that can H2,CO2). When acetogens grow on H2/CO2,car- grow on acetate, essentially run the pathway in reverse bon enters the Wood–Ljungdahl pathway at the CO2 and generate energy by oxidizing acetate to 2 mol of reduction step, with H2 servingastheelectrondonor. 5 CO2. Acetoclastic methanogens also convert acetate Acetogens are important in the biology of the soil, of into acetyl-CoA for cell carbon synthesis through the extreme environments, and of organisms that house combined actions of acetate kinase6,7 and phospho- them in their digestive tract, such as humans, termites, transacetylase.8 and ruminants.11–13 The Wood–Ljungdahl pathway contains an Eastern (inred)andaWestern (in blue) branch (FIG. 1), as orig- Pyruvate Ferredoxin Oxidoreductase inally described.9 The Eastern branch is essentially the folate-dependent one-carbon metabolic pathway Pyruvate ferredoxin oxidoreductase (PFOR) was re- that is present from bacteria to humans and recapitu- viewed relatively recently.14 Catalyzing the oxidative lated with methanopterin in methanogens. The West- decarboxylation of pyruvate to form acetyl-CoA and CO2, PFOR links heterotrophic to the Wood–Ljungdahl pathway (FIG. 1). PFOR is also key Address for correspondence: Stephen W.Ragsdale, University of Michi- to cell carbon synthesis since, besides its catabolic func- gan Medical School, 1150 W. Medical Center Dr., 5301 MSRB III, Ann Arbor, MI 48109. tion, PFOR catalyzes pyruvate formation by reductive , [email protected] carboxylation of acetyl-CoA.15 16 Pyruvate can then

Ann. N.Y. Acad. Sci. 1125: 129–136 (2008). C 2008 New York Academy of Sciences. doi: 10.1196/annals.1419.015 129 130 Annals of the New York Academy of Sciences

ies show that it is a pi radical with spin density delo- calized over the aromatic thiazolium ring, as shown in the figure.28,29 In the next step of the PFOR mechanism, the HE-TPP radical transfers an electron to the Fe-S electron-transfer chain, presumably through the ox- idized A-cluster (FIG. 2). The rate of this electron- transfer reaction is CoA dependent.30 In the absence of CoA, the half-life of the HE-TPP radical intermediate is ∼2 min; however, in the presence of CoA, the rate of radical decay increases 100,000-fold.22 By study- ing various CoA analogues, it was shown that the thiol group of CoA alone lowers the transition state barrier for electron transfer by 40.5 kJ/mol. The final step in the PFOR mechanism is electron transfer through FIGURE 1. The Wood–Ljungdahl pathway with the East- Clusters A, B, and C (FIG.2)toferredoxin,20 the termi- ern and Western branches depicted in red and blue, nal electron acceptor for PFOR. This electron-transfer respectively. (In color in Annals on line.) reaction occurs extremely rapidly, with a second-order rate constant of 2–7 × 107 M−1 s−1.22,31 enter the incomplete tricarboxylic acid cycle17,18 to generate intermediates for cell carbon synthesis. PFOR is found in archaea, bacteria, and even anaerobic pro- CODH/ACS tozoa like Giardia.19 CODH/ACS was recently reviewed,32 so this part of the Wood–Ljungdahl pathway will be treated rather briefly. As shown in FIGURE 1, when aceto- gens are grown heterotrophically, the CO and elec- (1) 2 trons generated by the PFOR reaction are utilized by The structure of the Desulfovibrio africanus PFOR re- CODH/ACS and formate dehydrogenase to generate veals seven domains.20 Thiamine pyrophosphate (TPP) CO and formate, respectively.When they are grown on and a proximal [4Fe-4S] cluster (Cluster A) are deeply CO, CODH generates CO2, which is then converted buried within the protein, while the other two clus- to formate in the Eastern branch of the pathway and ters (B and C) lead to the surface (FIG. 2). The TPP CO is incorporated directly as the carbonyl group of binding site consists of two domains that bear strong acetyl-CoA. Both CO and CO2 are unreactive with- structural similarity to those in other TPP enzymes21 out a catalyst, but the enzyme-catalyzed reactions are and contains conserved residues that interact with fast, with turnover numbers as high as 40,000 s−1 re- Mg++, pyrophosphate, and the thiazolium ring. Rapid ported for CO oxidation by the Ni-CODH from Car- kinetic studies indicate that the initial steps in ox- boxydothermus hydrogenoformans at its physiological growth idative decarboxylation of pyruvate by PFOR are temperature.33 Even the least active CODHs catalyze similar to those of other TPP enzymes.22 Deproto- CO oxidation at rates of ∼50 s−1.34 There are two ma- nation of TPP yields the active ylide, which forms jor classes of CODHs: the aerobic Mo-Cu-Se CODH an adduct with pyruvate. Then, CO2 is released, from carboxydobacteria and the Ni-CODHs. Found in 35,36 forming the 2α-hydroxyethylidene-TPP adduct (HE- aerobic bacteria that oxidize CO with O2, the Mo- 23 37 TPP). In acetogens, the CO2 feeds into the Wood– CODH contains FAD, Fe/S centers, Cu, and 2 Mo , Ljungdahl pathway24 25 (FIG. 1) and, based on isotope- atoms bound by molybdopterin cytosine dinucleotide, exchange studies,26 may be channeled directly to CO and its structure has been solved.38 This enzyme will dehydrogenase/acetyl-CoA synthase (CODH/ACS). not be discussed further in this chapter, since only the After forming the HE-TPP intermediate, the nega- Ni-CODH/ACS is involved in the Wood–Ljungdahl tive charge on C-2 of HE-TPP promotes one-electron pathway. Ni-CODHs are divided into two classes: reduction of a proximal FeS cluster, forming an HE- the monofunctional nickel CODH, which catalyzes TPP radical intermediate (FIG. 2). Based on the crys- the reaction shown in Equation 2, and the bifunc- tal structure, this intermediate was proposed to be a tional CODH/ACS, which couples Equation 2 (CO novel sigma-type acetyl radical27; however,recent stud- formation) with Equation 3 (acetyl-CoA synthesis). Ragsdale: Enzymology of Acetogenesis 131

FIGURE 2. PFOR state including the HE-TPP radical, with the structure based on spectroscopic results, showing highly delocalized spin distribution (From Astashkin et al.29 Used with permission.), and the distance from HE-TPP radical to coupled cluster. Location of the clusters is based on the structure (PDB 1KEK).

The monofunctional CODH functions physiologically to a pyrolytic graphite electrode show complete re- in the direction of CO oxidation, allowing microbes versibility of CO oxidation and CO2 reduction; in fact, to take up and oxidize CO at the low levels found in at low pH values, the rate of CO2 reduction exceeds the environment, while the CODH in the bifunctional that of CO oxidation.47 protein converts CO2 into acetyl-CoA. The association of ACS with CODH forms a bi- functional CODH/ACS machine that is encoded by CO + 2H+ + 2e−  CO + H O 2 2 the acsA/acsB genes, respectively,and plays the key role ◦ E =−520 mV (2) in the Wood–Ljungdahl pathway.32,48,49 The CODH component catalyzes the conversion of CO2 to CO 25 CO + CH3−CFeSP + CoA (Eq. 2) to generate CO as a metabolic intermediate. The CO2 comes from the growth medium or from de- → acetyl − CoA + CFeSP (3) , carboxylation of pyruvate.25 30 Then, ACS catalyzes The crystal structures of the monofunctional NI- the condensation of CO,50 CoA, and the methyl group CODH and the CODH component of the bifunc- of a methylated corrinoid iron–sulfur protein (CFeSP) tional enzymes are very similar.39–42 These mushroom- to generate acetyl-CoA (Eq. 3),25 a precursor of cellular shaped, homodimeric enzymes contain five metal material and a source of energy. clusters per dimer: two C-clusters, two B-clusters, and CODH/ACS contains a 140-† channel that delivers a bridging D-cluster. The C-cluster is the catalytic site CO generated at the C-cluster to the A-cluster.40,51,52 for CO oxidation and is buried 18 † below the surface. The only metallocenter in ACS is the A-cluster, which This cluster can be described as a [3Fe-4S] cluster consists of a [4Fe-4S] cluster bridged to a Ni site (Nip) bridged to a binuclear NiFe cluster. The Rhodospirillum that is thiolate bridged to another Ni ion in a thiolato- rubrum CODH structure with a bridging Cys between and carboxamido-type N2S2 coordination environ- Ni and a special iron atom called ferrous component II ment.40,42,53 Thus, one can describe the A-cluster as (FCII), while the C. hydrogenoformans protein appears to a binuclear NiNi center bridged by a cysteine residue have a sulfide bridge between Ni and FCII.43,44 Fur- (Cys509) to a [4Fe-4S] cluster, an arrangement sim- thermore, there is evidence for a catalytically impor- ilar to the Fe-Fe hydrogenases in which a [4Fe-4S] tant persulfide at the C-cluster.45 Issues related to the cluster and a binuclear Fe site are bridged by a Cys different structures are discussed in a recent review.46 residue.87,88 Cluster B is a typical [4Fe-4S]2+/1+ cluster, while Clus- Two mechanisms for acetyl-CoA synthesis have ter D is a [4Fe-4S]2+/1+ cluster that bridges the two been proposed that differ mainly in the electronic struc- identical subunits, similar to the [4Fe-4S]2+/1+ cluster ture of the intermediates: one proposes a paramag- in the iron protein of nitrogenase. netic Ni(I)-CO species as a central intermediate,2 and The CODH mechanism involves a Ping-Pong reac- the other proposes a Ni(0) intermediate.54,55 A generic tion: CODH is reduced by CO in the “ping” step and mechanism that emphasizes the organometallic nature the reduced enzyme transfers electrons to an exter- of this reaction sequence is described in FIGURE 3. nal redox mediator like ferredoxin in the “pong” step. Step 1, as shown in the figure, involves the migration The reduced electron acceptors then couple to other of CO, derived from CO2 reduction, through the inter- reduced nicolinamide adenine dinucleotide phosphate subunit channel to bind to the Nip site in the A-cluster. (NAD(P)H) or ferredoxin-dependent cellular processes The binding of CO to ACS forms an organometallic that require energy. Details of the CODH reaction complex, called the NiFeC species that has been char- have been reviewed.32 Recent studies of CODH linked acterized by a number of spectroscopic approaches.2 132 Annals of the New York Academy of Sciences

FIGURE 3. ACS mechanism emphasizing the organometallic nature of the reaction sequence and the channel to deliver CO from the CODH active site to the A-cluster.

The electronic structure of the NiFeC species is performed in the Ljungdahl laboratory. The methyl described as a [4Fe-4S]2+ cluster linked to a Ni1+ cen- group of acetyl-CoA is formed by the six-electron ter at the Nip site, while the other Ni apparently re- reduction of CO2 in the reactions of the Eastern + , mains redox-inert in the Ni2 state.56 Lindahl’s group branch of the acetyl-CoA pathway (FIG.1).2 62 First, 63 has argued that the NiFeC species is not a true catalytic formate dehydrogenase converts CO2 to formate, intermediate in acetyl-CoA synthesis, that it may rep- which is condensed with H4folate to form 10-formyl- 64,65 resent an inhibited state, and that the Ni(0) state is the H4folate. The latter is then converted by a cyclohy- 54,55 catalytically relevant one. See the recent review for drolase to 5,10-methenyl-H4folate. Next, a dehydroge- 32 66 a discussion of these issues. Step 2 in the ACS mech- nase reduces methenyl- to 5,10-methylene-H4folate, 57 anism involves methylation of the A-cluster, which which is reduced to (6S)-5-CH3-H4folate by a reduc- involves the conversion of one organometallic species tase.67,68 (methyl-Co) to another (methyl-Ni). There is evidence that the methyl group binds to the Nip site of the A- cluster.58–60 Carbon-carbon bond formation, Step 3 Methyltransferase (MeTr, AcsE) in the catalytic cycle, occurs by condensation of the and Corrinoid Iron Sulfur Protein methyl and carbonyl groups to form an acetyl-metal (CFeSP, AcsCD) species. In the last step, CoA binds to ACS, triggering thiolysis of the acetyl-metal bond to form the C-S bond The methyl group of CH -H folate is transferred of acetyl-CoA, completing the reactions of the Western 3 4 to the cobalt site in the cobalamin cofactor bound branch of the Wood–Ljungdahl pathway. FIGURE 3in- to the CFeSP69,70 to form an organometallic methyl- dicates, for simplicity, that the ACS reaction sequence Co(III) intermediate in the Wood–Ljungdahl pathway is ordered with CO binding before the methyl group. (FIG. 1). This reaction is catalyzed by MeTr,encoded by Conversely, Lindahl has argued for a strictly ordered the acsE gene.24 MeTr belongs to the B -dependent binding mechanism, with methyl binding first, then 12 methyltransferase family that includes methionine CO, and finally CoA, as described in a recent review.55 synthase and related enzymes from methanogens.71 In the author’s opinion, there is insufficient evidence We have cloned, sequenced, and actively overex- to exclude the 1991 proposal that the carbonylation pressed MeTr72,73 and the CFeSP74 in Ecoli,making and methylation steps occur randomly61 (FIG.4). them amenable for site-directed mutagenesis studies. Tetrahydrofolate-Dependent Enzymes In collaboration with Cathy Drennan (MIT, Cam- bridge, Massachusetts), we also have determined the Most of the work on the folate enzymes involved structure of MeTr and a site-directed variant in its 75 76 in the Eastern branch of the pathway has been uncomplexed and CH3-H4folate–bound states. Ragsdale: Enzymology of Acetogenesis 133

FIGURE 4. Random mechanism of acetyl-CoA synthesis.

FIGURE 5. Proposed conformers and interactions of the CFeSP. (Modified from Svetlitchnaia et al.79)

It was concluded that CH3-H4folate binds tightly This 88-kDa heterodimeric protein contains a [4Fe- 77 2+/1+ 70,74 (Kd < 10 µM ) to MeTr within a negatively charged 4S] cluster and a cobalt cobamide. The crevice of the triose phosphate isomerase (TIM) bar- Fe-S cluster plays a role in reductive activation of rel.78 The structure of the CFeSP was also recently the cobalt to the active Co(I) state.83,84 Svetlitchnaia solved.79 et al.79 proposed that the C-terminal domain (FIG.5) A key step in the MeTr mechanism is activation of the large subunit is a mobile element that interacts of the methyl group of CH3-H4folate, since the re- alternatively with the A-cluster domain of ACS and action involves displacement at a tertiary amine and with MeTr. Three major conformers or complexes because the CH3-N bond is much stronger than the are described: (1) a methylation complex, in which product CH3-Co bond. Of the activation mechanisms the Co(I)-CFeSP binds MeTr and accepts the methyl that have been considered, protonation at N5 of the group of CH3-H4folate; (2) the methylated CFeSP; (3) pterin seems to be most plausible.71 Generation of a a complex between ACS and the methylated CFeSP.A positive charge on N5 would lower the activation bar- fourth conformation, which is not shown here, would rier for nucleophilic displacement of the methyl group be a reductive activation conformer, in which the cor- by the Co(I) nucleophile. There is significant experi- rinoid is in the inactive Co(II) state. This molecular mental support for protonation at N5 of the pterin, juggling proposed for the CFeSP shown in FIGURE 5 including proton uptake studies,77,80 pH dependen- has precedent in the related mechanism involving the cies of the steady state, and transient reaction kinetics various domains of methionine synthase, as shown by of MeTr81 and methionine synthase,82 and studies of the elegant structure–function studies of Matthews and variants that are compromised in acid–base catalysis.76 Ludwig.82,85,86 A question that has not been resolved is whether the proton transfer takes place upon formation of the bi- Summary nary complex, as indicated by studies with MeTr from , M. thermoacetica,49 77 or the ternary complex (with the Studies of the enzymes involved in the Wood– methyl acceptor), as concluded from studies on E. coli Ljungdahl pathway have elucidated new roles of methionine synthase.80 Recent studies indicate that this metal ions in biology (including the formation of protonation step relies on an H-bonding network, in- bioorganometallic intermediates, discovery of new stead of a single acid–base catalyst and that an Asn heterometallic clusters, and nucleophilic metal ions), residue is a key component of that network.76 uncovered novel substrate-derived radical intermedi- AsshowninFIGURE 1, the CFeSP interfaces ates, and revealed channeling of gaseous substrates. between CH3-H4folate/MeTr and CODH/ACS. These new outcomes and mechanisms will likely be 134 Annals of the New York Academy of Sciences

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