The redox landscape of pyruvate:ferredoxin oxidoreductases reveals often conserved Fe-S cluster potentials.

Here, we investigate the thermodynamic driving force of internal electron transfer of pyruvate:ferredoxin oxidoreductases (PFORs), by comparing the redox properties of a series of PFORs from Chlorobaculum tepidum, Magnetococcus marinus, Methanosarcina acetivorans, as well as revisiting the single historical precedent, the enzyme from Desulfovibrio africanus. These enzymes require a thiamine pyrophosphate cofactor, three [4Fe-4S] clusters, and CoA for activity and are found within anaerobic organisms that utilize the reverse tricarboxylic acid cycle, or other reductive pathways, performing carbon dioxide reduction and pyruvate synthesis. Yet, PFOR is often invoked as an oxidative enzyme responsible for generating reducing equivalents in the form of the redox carrier ferredoxin. Previous efforts to understand the mechanism of PFOR have relied upon a prior report of the iron-sulfur redox potentials derived from an incomplete redox titration. Here, we use direct protein film electrochemistry to provide a side-by-comparison of four PFOR enzymes, providing a new assessment of the iron-sulfur cluster redox potentials. As the Methanosarcina acetivorans PFOR is comprised of multiple polypeptides, our investigation of the recombinant PorD subunit allows us to construct a model, where the revised redox potentials are mapped to specific iron-sulfur clusters.