Aerobic organisms require respiration in order to survive, and the processes necessary for respiration are carried out through the electron transport chain (ETC). Reactive Oxygen Species (ROS), a byproduct of respiration, are biomolecules that can lead to cellular damage when present at elevated levels. The movement of electrons within Complex III of the ETC is regulated by electron transfer to and from the Rieske protein. Factors that tune the reduction potential of the Rieske protein may impact electron exchange through Complex III and decrease the efficiency of cellular respiration, resulting in cellular damage and death. Using cyclic voltammetry, reduction potentials for mutants of the Rieske protein were determined over a range of pH values in order to determine the effect of each mutation. For example, the voltammetry of a Rieske mutant, T102K, yielded a potential 8 mV less than that for the wild type at pH 5. This decrease is surprising as the positive charge of lysine was expected to stabilize a greater negative charge at the active site and increase the potential. We are investigating whether this distal mutation induces a structural change at the active site that has a greater effect than that of the positive charge. Alterations were also made to the Rieske protein through exposure to diethyl pyrocarbonate (DEPC), which reacts with a ligating histidine to label it with a polar group. The resulting DEPC-histidine adduct exhibits a new signal at a higher potential in the voltammetry. At pH 5 the ligating histidines are protonated, yet this signal still appears with nearly saturating amounts of DEPC. For comparison, only six equivalents of DEPC are needed at pH 7 to observe comparable signals. This work reveals that pH has an effect on reactivity, but active-site histidines are still susceptible to chemical modification under unexpected conditions.<br/>

Effect of active-site alterations on the reduction potential of the Rieske protein

2019 Spring National Meeting

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