Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the coenzyme A (CoA)-dependent oxidative decarboxylation of pyruvate. In many autotrophic anaerobes, PFOR links the Wood-Ljungdahl pathway to glycolysis and to cell carbon synthesis. Herein, we cloned and sequenced the M. thermoacetica PFOR, demonstrating strong structural homology with the structurally characterized D. africanus PFOR, including the presence of three [4Fe-4S] clusters per monomeric unit. The PFOR reaction includes a hydroxyethyl-thiamin pyrophosphate (HE-TPP) radical intermediate, which forms rapidly after PFOR reacts with pyruvate. This step precedes electron transfer from the HE-TPP radical intermediate to an intramolecular [4Fe-4S] cluster. We show that CoA increases the rate of this redox reaction by 10(5)-fold. Analysis by Marcus theory indicates that, in the absence of CoA, this is a true electron-transfer reaction; however, in its presence, electron transfer is gated by an adiabatic event. Analysis by the Eyring equation indicates that entropic effects dominate this rate enhancement. Our results indicate that the energy of binding CoA contributes minimally to the rate increase since the thiol group of CoA lends over 40 kJ/mol to the reaction, whereas components of CoA that afford most of the cofactor's binding energy contribute minimally. Major conformational changes also do not appear to explain the rate enhancement. We propose several ways that CoA can accomplish this rate increase, including formation of a highly reducing adduct with the HE-TPP radical to increase the driving force for electron transfer. We also consider the possibility that CoA itself forms part of the electron-transfer pathway.