Enzyme and protein active sites containing two or more interacting metal centers now figure prominently in metallobiochemistry. Systems containing metal clusters composed of Fe atoms (e.g. ferredoxins, sulfite reductase, hemerythrin, ribonucleotide reductase, methane monooxygenase), Cu atoms (e.g. hemocyanin, tyrosinase, ceruloplasmin), Mn atoms (photosystem II, catalase) and more than one element, or heteropolynuclear clusters (e.g. nitrogenase-Mo,Fe; cytochrome oxidase-Fe,Cu; hydrogenase and carbon monoxide dehydrogenase-Ni,Fe), have been identified. The ultimate goals of this project are to elucidate the structures of metal clusters in metalloproteins, to determine the roles these clusters serve in the function of the protein, and to understand how nature designs a cluster for a specific purpose. This knowledge will provide a detailed understanding of biological processes and will aid in the design of pharmaceutical enzyme inhibitors and catalysts for various reactions. During the current report period, we have completed studies of the structural changes that occur in the Ni site in Thiocapsa roseopersicina hydrogenase during the binding of CO, a competitive inhibitor of the enzyme that has been shown to bind in a terminal fashion to the enzyme. The XAS studies do not show any evidence that the CO molecule binds directly to the Ni site. The CO complex is light sensitive and exhibits reversible photodissociation of the CO ligand. During photochemical cycling, the edge energy shifts by 0.5 eV in a cyclic fashion, suggesting that the CO binds near the Ni site and influences its electronic structure. A likely binding site is the Fe atom 2.7 A distant from the Ni in the recently published crystal structure of the D. gigas enzyme. These results support earlier studies indicating that Ni is not the substrate/inhibitor binding site in the enzyme.