Rieske proteins are electron transport proteins involved in key biological processes, such as respiration and photosynthesis. They have a [2Fe-2S] cluster ligated by 2 cysteine and 2 histidine ligands. In the respiratory chain, the Rieske protein picks up an electron and a proton from quinol and transfers it to cytochrom c1. Three aims will culminate in detailed information about the pH-dependent reduction potential of the Rieske protein. 1) The Rieske family of proteins is unique in that there is a highly conserved 3-dimensional structure, and yet there is a wide range of reduction potentials. Work will test how iron-sulfur proteins tune reduction potential through concerted spectroscopic and structural studies of mutants designed to alter reduction potential. 2) The reactivity of the histidine ligands to chemical modifiers will provide insight into how the protein carries both a proton and an electron. To probe the reactivity, diethyl pyrocarbonate (DEPC) or other modifiers will be reacted with the protein to establish where the modification occurs. How the reactivity changes with mutations that alter potential will tested the effect of the potential. 3) Rieske clusters are very stable to pH, yet the MitoNEET cluster, which is a [2Fe-2S] cluster ligated by 1 histidine and 3 cysteines, is not stable at low pH. Altering a Rieske protein to have a MitoNEET configuration will determine the role the protein fold plays in cluster stability. Broader Impacts: For the PI and the chemistry department at Trinity University, undergraduate research is an essential experience for aspiring chemists and biochemists. The students work on fundamental problems in science, but since they are inexperienced, the training needs to be extensive and thorough. Trinity has also been awarded a FAST grant and a McNair grant to increase participation the sciences, especially in those groups that are typically underrepresented and/or are first-generation college students. Trinity University is seeking to actively engage the surrounding communities in research and bring underrepresented groups into the sciences.