EXCEED THE SPACE PROVIDED. We will continue to study structure-function relationships in a variety of metalloenzymes, using the technique of X-ray absorption spectroscopy. This synchrotron-radiation based technique provides direct local structural information about metal coordination environments at the active sites of metalloenzymes and also probes electronic structure (e.g., oxidation states) of these metal sites. XAS is applicable also to unique non-metal sites (e.g., Se, As, Sb) and we have used this capability to probe Se-substituted substrates and cofactors to .address mechanistic questions in a number of enzymes that utilize sulfur-containing compounds. XAS provides structural information complementary to that of other structural biology techniques, providing very accurate metal-ligand distances on frozen solution samples in a few hours. In this renewal, we will focus on the application of XAS to metal homeostasis systems, investigating both metalloregulation and metal transport and handling proteins. A number of proteins from both the ArsR and MerR families of metalloregulators will be the focus of our work. A second area of thematic effort is selenium in biology. First, we will continue our characterization of naturally occurring selenium-containing enzymes, in particular focusing on purine hydroxylase, a molybdoenzyme that contains a required dissociable selenium cofactor. Second, we will use selenium substitution for sulfur in S-adenosylmethionine (SAM) as a probe for the mechanisms involved in a newly recognized superfamily of SAM-dependent enzymes that function through radical generation. Multiple examples of this superfamily are targeted to allow comparative studies of the mechanisms involved. Third, we will use selenium substitution to investigate the interaction of coenzyme M and coenzyme B with the active-site FeS cluster of the heterodisulfide reductase from methanogenic archaea. In addition to these applications, we will conduct feasibility studies in preparation for a large-scale effort to develop the technology of high-throughput XAS (HT XAS). This technology, designed to characterize the 'metalloproteome', will allow us to interrogate the metal content and metal-site structures within each gene product of a given proteome and ask questions about how the expression of metalloproteins or the post-translational metal-loading of proteins depends on the growth conditions of an organism. This will be a parallel technology to the current developments in structural genomics. PERFORMANCE SITE ========================================Section End===========================================
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