Kinetic and thermodynamic characterization of the pyruvate ferredoxin oxidoreductase reaction mechanism

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the Coenzyme A (CoA)-dependent oxidative decarboxylation of pyruvate. The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO2 fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Moorella thermoacetica PFOR is a highly efficient pyruvate synthase. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of k cat/Km values for ferredoxin demonstrate that this Fe-S protein is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis. Herein, by cloning and sequencing the M. thermoacetica PFOR, we show the high degree of structural homology with the D. africanus PFOR, whose structure has been determined. Similarities between the two enzymes include the presence of 3 [4Fe-4S] clusters per monomer. The PFOR reaction includes a substrate-derived radical intermediate, called the hydroxyethyl-thiamine pyrophosphate (HE-TPP) radical, which forms rapidly after PFOR reacts with pyruvate. A key step in the PFOR reaction is electron transfer from the HE-TPP radical intermediate to an intramolecular [4Fe-4S] cluster. We show that CoA enhances the rate of this redox reaction 10 5-fold. Based on Marcus and Eyring analyses of this reaction we propose that the thiol group of CoA is the predominant contributor to this rate increase; whereas, the components of CoA, which afford most of the cofactor\u27s binding energy, contribute minimally to the rate enhancement. These results suggest a novel biochemical role for CoA in the PFOR reaction and we describe several ways that CoA could achieve its function