This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal is part of our study on the critical metalloenzyme FeFe hydrogenase, and its mechanism of catalytic dihydrogen activation: 2H+ + 2e- = H2. We propose to use our high-resolution emission spectrometer on BioCAT to measure both site-selective EXAFS on purified enzyme as well as XES on simple inorganic "model" compounds. FeFe] H2ases are among the most efficient H2 catalysts known, with Kcat values up to 6000 H2 s-1 Despite the availability of excellent crystal structures and extensive spectroscopic study many questions about the molecular and electronic structure are still poorly understood. (1) What are the structural and conformational changes in the cluster during activation and catalysis?. (2) What are the oxidation states and spin-states of the dimeric Fe sub-sites. this more favorable towards hydrogen binding? If so how, and why? XES should allow us to resolve the oxidation state problem. Our previous on H-cluster models showed their low-spin Fe(I) nature, and that the valance region is sensitive to cluster oxidation state and/or the presence of a bridging CO. We propose to study further model compounds and extend the studies to purified protein, thus resolving the oxidation state issue. Site-selective EXAFS on purified protein should also help resolve the structural and conformational issues. As the cubane 4Fe are almost certainly high-spin, and the 2Fe subsite is low-spin, site-selective EXAFS should structurally characterize the expected dynamic changes of the low-spin 2Fe subsite separately from the 4Fe cubane.