Abstract:

<p>2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a thiamine pyrophosphate (TPP) and [4Fe-4S] cluster-dependent enzyme from the reductive tricarboxylic acid (rTCA) cycle that fixes CO to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate OGOR operates bidirectionally (both CO-fixing and 2-oxoglutarate oxidizing), and that only one Fd (Fd1) supports efficient catalysis. Our 1.94-Å and 2.80-Å resolution crystal structures of native and substrate-bound forms of OGOR reveal the determinants of substrate specificity and CoA-binding in an OGOR, and illuminate the [4Fe-4S] cluster environment, portraying the electronic conduit allowing Fd1 to be wired to the bound-TPP. Structural and biochemical data further identify Glu45α as a mobile residue that impacts catalytic bias toward CO-fixation although it makes no direct contact with TPP-bound intermediates, indicating that reaction directionality can be tuned by second layer interactions. (149 of 150 words limit).</p>