2-oxoacid:ferredoxin oxidoreductases (OFORs) utilize [4Fe-4S] cluster(s) and a thiamine pyrophosphate (TPP) for reversible CO2 fixation. The best-characterized OFOR, pyruvate:ferredoxin oxidoreductase (PFOR), cleaves pyruvate and releases electrons, which provide reducing power for central metabolism, including the Wood–Ljungdahl pathway, and other cellular processes, such as nitrogen fixation and sulfur reduction. Surprisingly, electron transfer between cofactors in PFOR is coenzyme A (CoA) -gated such that CoA binding accelerates electron transfer by 105-fold. Prior to this work, molecular details of how PFOR gates electron transfer in the absence of CoA are unknown. Here, we present the first crystallographic snapshot of pyruvate:ferredoxin oxidoreductase from Moorella thermoacetica (MtPFOR) with CoA bound, as well as structures of MtPFOR bound with two other reaction intermediates. These structural data reveal domain III, a domain whose function was previously unknown, as the binding site of CoA and establish consensus motifs for CoA-binding in the OFOR superfamily. MtPFOR structures further show that domain III undergoes a conformational change upon CoA binding that positions the thiolate of CoA directly adjacent to the TPP cofactor and seals off the active site. Strikingly similarities between MtPFOR and the only CoA-independent OFOR to date, oxalate oxidoreductase (OOR), further suggest a general electron transfer mechanism is retained; though, their structures are tailored for different reactions.

Binding site for coenzyme A revealed in the structure of pyruvate:ferredoxin oxidoreductase from Moorella thermoacetica

256th National Meeting (2018)

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