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 (ET) of pyruvate:ferredoxin oxidoreductases (PFORs), by comparing the redox properties of a series of PFORs from Chlorobaculum tepidum (Ct), Magnetococcus marinus (Mm), Methanosarccina acetivorans (Ma), as well as revisiting the single historical precedent, the enzyme from Desulfovibrio africanus. These enzymes require a thiamine pyrophosphate (TPP) cofactor, three [4Fe-4S] clusters, and Coenzyme A (CoA) for activity and are found within anaerobic organisms that utilize the reverse tricarboxylic acid (TCA cycle), or other reductive pathways, performing CO2 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 (PFE) to provide a side-by-comparison of four PFOR enzymes, providing a new assessment of the iron-sulfur cluster redox potentials. As the Ma PFOR is comprised of multiple polypeptides, our investigation of the recombinant PorD sub-unit allows us to construct a model where the revised redox-potentials are mapped to specific iron-sulfur clusters.