Diiron carboxylate enzymes are a class of metalloproteins responsible for numerous reactions in cells such as fatty-acid desaturation and radical generation.  The goal of this research is to understand the importance of the active site structure in controlling the function of the diiron proteins rubrerythrin and symerythrin.  In contrast to the more well-studied diiron carboxylate proteins that contain two histidine and four carboxylate residues in their active sites, rubrerythrin and symerythrin have one and two additional carboxylate residues, respectively.  Starting from a de novo designed four-helix bundle protein created to mimic the 2-His/4-carboxylate diiron enzymes, we have successfully produced and purified rubrerythrin models that include an extra active site carboxylate residue, either aspartate (G14D) or glutamate (G14E).  The oxygen and hydrogen peroxide reactivity of these model systems have been investigated.  In addition, symerythrin model proteins that include a second additional carboxylate residue in the active site, either aspartate (G47D) or glutamate (G47E), have been produced and purified.  Metal binding, protein folding, and reactivity studies of these proteins suggest that they are excellent structural and spectroscopic models of the natural enzymes. Ultimately, these studies will give us a better understanding of how systematic changes to structural features of a protein affect its functionality.

Creation of rubrerythrin and symerythrin model proteins

247th National Meeting (2014)

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