Radical catalysis in Fe/S cluster dependent dehydratases

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Typical dehydratases catalyze the cleavage of a carbon-oxygen bond yielding an unsaturated product via a,b-elimination of water. The a-hydrogen (pKa ~ 8) to be removed as proton is activated by an adjacent electron-withdrawing group, and the hydroxyl group leaves from the b-position. In contrast, several anaerobic bacteria employ oxygensensitive 2- or 4-hydroxyacyl-CoA dehydratases that use radicals to lower the pK of the unactivated b-protons by up to 26 units. These atypical dehydratases are the class of 2- hydroxyacyl-CoA dehydratases and the 4-hydroxybutyryl-CoA dehydratase. The former consist of two components: (a) a Fe/S-dependent ATPase component (activator) to generate the radical and (b) a Fe/S-dependent dehydratase catalyzing the actual dehydration. Recent spectroscopic investigations showed that a catalytically competent ketyl-radical anion is generated on the substrate to facilitate the dehydration. The research proposal focuses on elucidating the structural bases of (I.) nucleotide- and redox-dependent changes proposed for the activator, (II.) complex formation between the two components leading to radical generation on the dehydratase component and (III.) the unusual active site metal cluster of the dehydratase component. (IV.) Additionally, we want to extent our structural studies on the mechanism of 4-hydroxybutyryl-CoA dehydratases by studying substrate- and product-complexes.