Project Summary The reactions of carbon-containing gas molecules (e.g., CO2 and CO) with metalloenzymes are vital biochemical processes. They are responsible for energy generation/storage and synthesis of biomass in many organisms. Additionally, the use and regulation of CO2 in our atmosphere has significant societal implications. In anaerobic microbes, the most prevalent enzymatic pathways for CO2 reduction and CO oxidation utilize nickel/iron-containing Carbon Monoxide Dehydrogenases (CODHes). Through spectroscopic and crystallographic studies of CODH, the active site of the enzyme was found to be an inorganic NiFe4S4 cluster, termed the C-cluster. Based on these experimental data, proposals for its mechanism have been formulated. However, discrepancies in (and different interpretations of) the experimental data have led to numerous structural/mechanistic hypotheses. Many important questions remain regarding the structure, function, and mechanism of the C-cluster. Synthetic bioinorganic modeling provides a valuable opportunity to learn about the electronic structure and reactivity of metalloenzymes. The well-defined environments in these models are far simpler to characterize spectroscopically and can be systematically varied to build structure–function relationships for fundamental insight into bioinorganic chemistry. Several synthetic models of the C-cluster have been developed and studied, including some with NiFe3S4 cores. However, none of these synthetic C-clusters had Ni in a biomimetic environment with three donor ligands, and none contained the important “unique” iron site outside the cubane core. Further, the influence of the second coordination sphere has not been investigated in C-cluster models. In this proposal, we seek to synthesize and study models of the C-cluster that contain biomimetic structural motifs hypothesized to be imperative for reactivity. The coordination environment around Ni will be systematically varied to elucidate the structure–function relationships of the metal centers and donors that comprise the C- cluster. These structural characteristics will be correlated with the reactivity of the cluster models toward CO and CO2, enabling us to study key proton and electron transfer events in a well-defined system. We will also study the validity of the various mechanistic hypotheses by synthesizing reactive intermediates of interest, including Ni hydrides. Most importantly, we will perform in-depth spectroscopic experiments on these model systems to shed light on the electronic structure and magnetic coupling of the reactive metal centers within the C-cluster. Bioinorganic spectroscopy will also be used to understand the redox events that occur during reaction with the gas molecules. In all cases, the reactivity and spectroscopic signatures will be compared with that of the C- cluster itself. These studies will help to elucidate the electronic structure and mechanism of the CODH C-cluster and will contribute to the ...