Abstract 2055 "Mutating D111 in the Cu A protein to Determine the Reactivity of Ligating Histidines to Chemical Modifiers"

The electron transport chain (ETC) is a series of four protein complexes that couple redox reactions and the formation of an electrochemical gradient that leads to the formation of ATP. The electrons go through a chain of proteins (Complexes I- IV) with increasing reduction potentials and causes a release in energy. Complex IV, oxidizes cytochrome c and transfers the electrons to oxygen, the final electron acceptor. Complex IV in Thermus Thermophilus contains two heme groups and two copper sites in Subunit I and Subunit II. The primary focus for this project is Subunit II, where the soluble domain of the protein contains the binuclear Cu A site. The Cu A center contains two copper ions bridged by two cysteine thiolates; each copper is additionally coordinated by two histidine nitrogen atoms. Experiments have focused on the copper site and reactivity of the ligating, solvent exposed His 157. D111 is hypothesized to have a role in the reactivity of the ligating, histidine 157 because D111 is hydrogen bonded to the other ligating histidine. By mutating Asp 111 to asparagine, alanine, and serine, the change in proton exchange and reactivity of the histidine 157 can be determined by chemically modifying the mutants with diethyl pyrocarbonate (DEPC), 4-hydroxyl-trans-2-nonenal (HNE), and 4-oxo- 2-nonenal (ONE). DEPC is a commonly used chemical modifier but is not present in biological systems. HNE and ONE are endogenous lipid peroxidation products. High levels of reactiv oxygen species (ROS), oxidize nearby membrane lipids. HNE and ONE are such products that act as toxic second messengers of oxidative stress and covalently modify lipids, proteins, and DNA. The modifications on TtCu A mutants by DEPC, HNE and ONE will provide more conclusions about the reactivity of the ligating histidines and thus possible sites of modification in vivo during oxidative stress. Trinity Chemistry Department, The Welch Foundation, the McGavock Fund.