Abstract 2054 Investigating the Reduction Potential of the Rieske Protein in Spinacia oleracea and Thermus Thermophilus

The Rieske protein is of interest due to its unique properties. Located in the mitochondria, the metalloprotein is indispensable for many metabolic functions, helping drive the electrochemical gradient necessary for ATP synthesis. The characteristic property of the Rieske protein is the [2Fe-2S] cluster bound by two histidine and two cysteine residues. This cluster has an associated reduction potential, the ability to accept an electron, which allows the protein to perform an oxidation of quinol in its function in the Electron Transport Chain. The Hunsicker-Wang lab is interested probing and characterizing the structure which modulates reduction potential in two related projects. The goal of the first research project is to understand the effect different amino acids have on the [2Fe-2S] cluster in the Thermus thermophilus Rieske protein. Thus, this project utilizes a series of mutations to the protein. One protein that has been characterized adds a hydrogen bond to the cluster and a positive charge next to the cluster, and others alter the charge state of the protein. These proteins have been characterized using several techniques including pH-dependent circular dichroism, UV-Visible spectroscopy, and capillary electrophoresis. The goal of the second project is to purify and characterize wild-type spinach Rieske. The lab has performed research on the Rieske proteins in a bacterial system, Thermus thermophilus, and a eukaryotic system, Saccharomyces cerevisiae, but has only recently introduced research on Rieske in photosynthetic systems. The current project investigates the structure and function relationships in Spinacia oleracea, spinach, which will allow for characterization of a photosynthetic Rieske. In tandem, these studies will allow a better understanding of eukaryotic and bacterial systems through comparison. The purification of the spinach Rieske protein involves the adaptation of numerous protocols to achieve purification, which utilizes several new techniques to ensure the extraction of pure protein. The characterization of wild-type spinach will use two techniques: pH-dependent CD spectroscopy and DEPC modification. Each technique probes the reactivity of the histidines which ligate the [2Fe-2S] cluster in wild-type spinach. The Arnold and Mabel Beckman Foundation, The Trinity University Chemistry Department, The Welch Foundation.