A Reverse TCA Cycle 2-Oxoacid:Ferredoxin Oxidoreductase that Makes C-C Bonds from CO2

Abstract

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 2 to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of Magnetococcus marinus MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate that MmOGOR operates bidirectionally (both CO 2 -fixing and 2-oxoglutarate-oxidizing) and that only one Fd (MmFd1) supports efficient catalysis. Our 1.94-Å- and 2.80-Å-resolution crystal structures of native and substrate-bound forms of MmOGOR reveal the determinants of substrate specificity and CoA-binding in an OGOR and illuminate the [4Fe-4S] cluster environment, portraying the electronic conduit through which MmFd1 is wired to the bound TPP. Structural and biochemical data further identify Glu45α as a mobile residue that impacts catalytic bias toward CO 2 fixation, although it makes no direct contact with TPP-bound intermediates, indicating that reaction directionality can be tuned by second-layer interactions. The transformation of CO 2 into chemicals or fuels is a tantalizing chemistry at the heart of efforts to achieve sustainable carbon usage. The search continues for scalable catalysts that can be used to capture CO 2 in waste gas streams. Remarkably, microbial organisms contain enzymes that engage in CO 2 transformations routinely at ambient temperature and pressure, and often, such enzymes are also incredibly efficient in terms of minimal thermodynamic waste spent while engaging in catalytic chemistry. Further industrial promise is held in the implementation of engineered micro-organisms that contain CO 2 -reducing enzymes. Here, we investigate one of the reversible catalysts found widely in nature that can engage in the transformation of CO 2 into 2-oxoglutarate. The lessons from our study may impact how industrial microbiologists re-wire carbon pathways in scalable microbial bioreactors where CO 2 can be transformed into fuels or high-value molecules. We describe an oxoglutarate:ferredoxin oxidoreductase (OGOR) enzyme that is found natively in the reverse TCA cycle and demonstrate that it participates in the two-electron inter-conversion of CO 2 to 2-oxoglutarate while possessing only a single iron-sulfur cluster. X-ray crystal structures reveal how substrate specificity is generated, and determining the identity of the catalytically relevant redox partner yields insight into how electron-transfer steps may be key to guiding CO 2 reduction.