The Wood-Ljungdahl (WL) pathway represents Earth's most energy-efficient biological system for CO2 reduction, converting two equivalents of CO2 into acetyl-CoA via distinct methyl and carbonyl branches. Central to this process are three metalloenzymes: acetyl-CoA synthase (ACS), carbon monoxide dehydrogenase (CODH), and the corrinoid iron-sulfur protein (CFeSP). We will present our progress towards characterizing the organometallic intermediates formed in the active sites of ACS, CODH, and CFeSP during CO2 activation and fixation. Using synchrotron-based standard and high-resolution X-ray absorption spectroscopy (XAS) and theoretical methods combined with complementary characterization tools, we have successfully advanced our understanding of key catalytic intermediates, specifically the CO-bound (ANiFeC), methylated (AMe), and acetylated (AAc) species at the unique Ni-Fe-S A-cluster of ACS. Our findings suggest an electronic coupling mechanism within ACS that accommodates both paramagnetic and diamagnetic intermediates during catalysis. We have also identified through mutagenesis studies a hydrophobic alcove that is necessary for catalysis. Results from the characterization of resting and reduced states of CODH, which plays a critical role in reducing CO2 to CO at its specialized NiFeS C-cluster will be presented. We will show high-resolution spectral analysis from Ni/Fe K-edge XAS studies that reveal significant redox-dependent geometric rearrangements essential for substrate binding and controlled electron transfer. We have extended this methodology to CFeSP and our recent investigations have specifically addressed its extraordinary rate in transferring the methyl group from the methyl branch to the carbonyl branch. The corrinoid Co center in CFeSP utilizes a trans-axial ligand that significantly influences the methyl transfer reaction, demonstrating its impact on pathway efficiency and intermediate stabilization. Collectively, these studies combining advanced spectroscopy, structural biology, and computational modeling significantly deepen our understanding of the mechanistic details underlying CO2 fixation and carbon-carbon bond formation in the WL pathway.
The SSRL Structural Molecular Biology Program was supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences P30 program and by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Division, under contract FWP100593.