Kinetics of CO Insertion and Acetyl Group Transfer Steps, and a Model of the Acetyl-CoA Synthase Catalytic Mechanism

Acetyl-CoA synthases/carbon monoxide dehydrogenases are found in homoacetogenic bacteria, methanogenic archaea, and CO-utilizing hydrogenogenic bacteria.1–4 These O2-sensitive bifunctional enzymes allow such organisms to grow chemo-autotrophically on simple inorganic compounds. The enzyme from the homoacetogen Moorella thermoacetica (ACS/CODH) has been studied most extensively.5,6 The β subunits of this 310 kDa α2β2 tetramer catalyze the reversible reduction of CO2 to CO, while the α subunits catalyze the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from a corrinoid-iron sulfur protein (CoFeSP). At a buffered pH, this is represented by reaction 1. CO+CoA+CH3−Co3+FeSP⇄CH3−C(O)−CoA+Co1+FeSPKACS [1] The active-site for this reaction, called the A-cluster, consists of an [Fe4S4] cubane bridged via a cysteine residue to a Ni ion called proximal Nip. This Ni is also bridged (via 2 other cysteines) to a second Ni ion (distal Nid); thus, Nip is coordinated to 3 bridging thiolates. Nid has an N2S2 square-planar environment including coordination to two amide nitrogens derived from the protein backbone.7–9 Evidence suggests that CO and methyl groups bind to Nip during catalysis.10, 11 Although aspects of the ACS catalytic mechanism remain uncertain, our understanding of it is gradually improving. The components of the A-cluster in its most oxidized redox state (called Aox) appear to be in the {[Fe4S4]2+ Nip2+ Nid2+} electronic configuration.12, 13 This state is inactive for both catalysis and methyl group transfer but it can be activated by a 2-electron reduction corresponding to an apparent midpoint potential of ca. −540 mV vs. NHE at neutral pH.14 The resulting reductively-activated state apparently has the cubane and Nid in the 2+ states, suggesting the unprecedented {[Fe4S4]2+ Nip0 Nid2+} configuration.12,13,14 Although the occurrence of a zero-valent Ni atom in the reductively activated state is not established, we will use this nomenclature throughout in this paper, for convenience if for no other reason. For a full discussion of this issue, readers are referred to the literature.4,13–15 If preferred, “Ni0” can be viewed simply as an electron-counting formalism indicating the Aox state to which 2e− have been added. This formal view does not complicate or bias any interpretation, analysis, or conclusion presented here. Whether the methyl group or CO bind first to the enzyme remains contentious, and reasonable arguments have been made for both cases.5,6,11,16 The one-electron-reduced and CO-bound state of the A-cluster (the S = ½ Ared-CO state) has been proposed to be an intermediate of catalysis as well as an inhibitory state. Recent evidence that ACS/CODH need not pass through the Ared-CO state during catalysis and evidence that reductive activation requires 2 electrons14 compel us to favor the case where the methyl group binds first. It is also known that the reductively activated state accepts a methyl group in the absence of CO, as shown in reaction 2. Ni0+CH3−Co3+FeSP⇄Ni2+−CH3+Co1+FeSPKmet=k+metk−met [2] The resulting methylated state is stable and has been characterized.11,14,17,18 When exposed to CO, e.g. during catalysis, CO is thought to insert into the Ni-methyl bond in accordance with reaction 3. Ni2+−CH3+CO⇄Ni2+−C(O)CH3Kins=k+insk−ins [3] Reaction of the acetyl intermediate with CoA affords acetyl-CoA and regenerates the reductively-activated state, reaction 4. Ni2+−C(O)CH3+CoA⇄CH3C(O)−CoA+Ni0KCoA=k+CoAk−CoA. [4] Reactions 2 – 4 complete the catalytic cycle for the synthesis of acetyl-CoA. Of these steps, only methyl group transfer, reaction 2, has been studied specifically.19–21 In a stopped-flow study, ACS/CODH and Ti3+citrate were preincubated to generate the Ni0 state, and then reacted against CH3-Co3+FeSP (also preincubated in Ti3+citrate) and monitored at 390 nm where the product Co1+FeSP absorbs. Under these conditions, the reverse of reaction 2 (i.e. starting from Ni2+-CH3 and Co1+) could barely be detected, indicating that the equilibrium position lies on the products side.21 In contrast, the reverse reaction proceeded rapidly and to near completion when the Ni2+-CH3 was not preincubated with Ti3+citrate. Under these conditions, the equilibrium position appears to be on the reactants side of reaction 2. This reductant-dependent shift in the kinetic and thermodynamic properties of the methyl group transfer reaction is not understood mechanistically. Bhaskar et al. have examined the steady-state kinetics of the exchange reaction between acetyl-CoA and dephospho-CoA as catalyzed by the ACS/CODH homolog from Methanosarcina barkeri.22 Their results indicate a ping-pong mechanism in which the binding of acetyl-CoA to the enzyme is followed by the release of CoA and formation of the acetyl-intermediate. They proposed that acetyl-CoA binds to the oxidized form of the enzyme, followed by reduction. This was suggested because partially-reduced enzyme exhibited cooperative binding with acetyl-CoA whereas fully reduced enzyme showed simple hyperbolic binding. However, the same behavior would be observed if enzyme were first reduced and then bound with acetyl-CoA. This latter scenario would be congruent with a nucleophilic attack (e.g. by a Ni0 species) on the carbonyl of acetyl-CoA, as in the reverse of reaction 4. The alternative proposal of binding followed by reduction would seem to require attack by Ni2+, a non-nucleophilic metal ion. Using two methods, Bhaskar et al. measured the equilibrium constant for the reverse of reaction 4 to be ~ 0.2 (averaged value),22 suggesting KCoA ~ 5 for a homologous ACS/CODH from a methanogenic archaeon. To date, no direct studies of the CO insertion reaction 3 have been reported, nor have the kinetics of the reductive elimination of the acetyl group and CoA, reaction 4, been reported. The problem in studying these reactions has been to identify strategies for monitoring them. Using stopped-flow kinetics, we report here that reactions 3 and 4 can be monitored by starting with the methylated state of ACS/CODH. Resulting traces were used to construct a simple kinetic model describing the catalytic mechanism of acetyl-CoA synthase. In this paper we report these results and describe the model.