The Rieske Protein is characterized by a [2Fe-2S] cluster that is ligated by two histidines (His154 and His134) and two cysteines (Cys151 and Cys132). The Rieske Protein is a subunit in complex III of the Electron Transport Chain (ETC) that helps shuttle electrons. Therefore, understanding its reduction potential is an important part of understanding the overall mechanism of the ETC. In the literature, reduction potential differences in Rieske have been proposed to be due to hydrogen bonding to the cluster and ligands as well as accessibility of the solvent and proximity of charged residues to the [2Fe-2S] cluster. A new hypothesis poses that changes to charges on distal residues to the cluster will affect the reduction potential. To probe this hypothesis, a series of mutants have been produced in the Rieske protein from Thermus thermophilus. These mutants are grouped into two categories. First, mutants that remove positively charged residues (Arg or Lys) and replace them with neutral or negatively charged residues, have been produced. These changes result in a protein that is overall more negative. The outcome of these mutations are proteins with reduction potentials that are lower, and also point to an effect of distance from the cluster. The second group of mutants remove negatively charged amino acids (Glu and Asp) and replace them with either neutral or positive amino acids. These mutations cause the protein to be overall more positively charged, which should cause an increase in the reduction potential. All of these mutants are characterized by measuring the reduction potential, determining the pKa and pI values for the protein, and reacting the protein with diethyl pyrocarbonate.

Exploring the effect of distal charges on the reduction potential of the Rieske protein from Thermus thermophilus

253rd National Meeting (2017)

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