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. The goal or this project is to understand the enzymatic reaction mechanism of diphthamide biosynthesis. Diphthamide is a posttranslationally modified His residue in eukaryotic translation elongation factor 2 (EF-2) a GTPase required for the translocation step of ribosomal protein synthesis. One of the most intriguing facts about diphthamide is that one of the genes involved in its biosynthesis is found to be a tumor suppressor gene in human. Recently, Lin group provided structural and biochemical evidence showing that the first step of diphthamide biosynthesis in archaea uses a novel iron-sulfur cluster enzyme, DPH2 (Zhu and others 2009). ESR spectroscopy at 4- 15K in ACERT played a crucial role in identifying the [4Fe-4S] iron-sulfur cluster. The results obtained by different methods in Lin's group suggest that unlike known radical SAM enzymes, DPH2 does not form 5[unreadable]-deoxyadenosyl radicals. Instead, it breaks the other C-S bond of SAM and transfers the 3-amino-3-carboxylpropyl group to EF-2, possibly via a radical mechanism. To prove or disprove the proposed radical mechanism, we plan to spin-trap and identify the intermediates by the shape of ESR spectra of their adducts with DMPO and possibly other spin-traps. We recently detected some spin trapping adducts and work on their identification. Also, our next step will be a study of other proteins involved in the biosynthesis of diphthamide: yeast DPH1-DPH2 dimer, and yeast DPH3, DPH4. We expect that all these proteins have a Fe-S cluster bound. To identify the cluster, its spin and oxidation state we will record low temperature EPR at a wide field range. It will allow us to detect higher spin states (S= 3/2, 5/2, 7/2) or mixtures thereof.