The Rieske protein is found in the Cytochrome bc1 complex of the electron transport chain. It has a unique [2Fe-2S] cluster with two ligating histidines and two ligating cysteines. It is involved in the oxidation of ubiquinol during the Q cycle. The protein has a hydrophobic transmembrane domain that keeps it anchored in the complex, a soluble head domain that holds the cluster, and a flexible linker region that allows the head to move between a bound quinol and cytochrome c1. Rieske proteins have reduction potentials that are sensitive to the cluster’s environment and are dependent on pH. Mutations that alter the charges of residues in the protein can tune the reduction potential of the protein even when those mutations are distant from the cluster. To understand the effect of charges that are far from the [2Fe-2S] cluster, a series of mutations to the protein that alter the charge state of the protein have been produced. To accurately evaluate the charge state of these mutants by capillary gel electrophoresis, a charge ladder will be utilized. Production of the ladder is achieved by acetylating of the protein with acetic anhydride. Completed acetylation reactions are analyzed using isoelectric focusing gels, which separate proteins by isoelectric point (pI) the pH at which the protein is neutral, to evaluate the extent of acetylation. The optimized charge ladder will be used to accurately analyze the effect of charge distance from the cluster and the overall charge state on the reduction potential.

Determining the effect of distal charges on the reduction potential of the Rieske protein in thermus thermophilus

ACS Spring 2022

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