Evidence of a Molecular Tunnel Connecting the Active Sites for CO2Reduction and Acetyl-CoA Synthesis in Acetyl-CoA Synthase fromClostridiumthermoaceticum

Abstract

Autotrophic bacteria and archaea can grow on CO2 and H2 as their only source of carbon and energy.1 The central enzyme responsible for this process is acetyl-CoA synthase (ACS). ACS from Clostridium thermoaceticum catalyzes the reversible reduction of CO2 to CO, and the synthesis of acetyl-CoA from CO, coenzyme A (CoA), and the methyl group of a methylated corrinoid-iron-sulfur protein (CoFeSP).2 It also catalyzes the exchange of free CO with the carbonyl group of acetyl-CoA, presumably by a similar mechanism. ACS is an R2â2 tetramer containing two active-site Ni-X-Fe4S4 clusters.3-5 The active site for CO2/CO redox catalysis (the C-cluster) resides in â,3,6 while that for acetyl-CoA synthesis (the A-cluster) is located in R.4,5 The two sites appear to function independently, in that they can be separately inactivated.7 CO2 has a significant effect on the enzyme, possibly involving a protein conformational change.8 Exposure to CO2 alters the redox and CO-binding properties of the metal centers and abolishes the CO/acetyl-CoA exchange activity. Since C. thermoaceticum grows under a CO2 atmosphere, the CO2-altered form of ACS may be physiologically relevant. To address whether CO2 affects acetyl-CoA synthase activity, we examined initial rates of acetyl-CoA synthesis, using a CO concentration of 3 μM under a balance of Ar or CO2 (Figure 1, b and 2, respectively). Ti(III) citrate (Figure 1A) and methyl viologen (MV) (Figure 1B) were used as reductants.9 Initial rates were ACS dependent. Under Ar and using MV (Figure 1B, b), acetyl-CoA was initially synthesized at a rate of 0.04 ( 0.01 μmol min-1 mg-1 (1 μmol min-1 ) 1 unit) while under CO2 (Figure 1B, 2) the rate was 40 times faster (1.5 ( 0.3 units/mg). Similar initial rates were obtained using Ti(III) citrate (0.07 ( 0.02 units/ mg in Ar, Figure 1A b, and 1.2 ( 0.2 units/mg in CO2, Figure 1A 2). More importantly, similar initial rates were obtained in the absence of CO (Figure 1, 9). This indicates that CO generated from the reduction of CO2 at the C-cluster combined with the methyl group and CoA at the A-cluster to form acetyl-CoA. This raised the issue of whether CO dissociated from the C-cluster into solution and eventually bound the A-cluster once its concentration was sufficiently high. Assuming this and a kcat value of 1.3 s-1 for CO2 reduction,10 ∼120 h would be required to afford 3 μM CO. The absence of a lag period prior to the onset of acetyl-CoA synthesis (Figure 1, 9) is incompatible with this scenario.