The Cu A center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the Cu A site (the TtCu A protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0-9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCu A increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the Cu A site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCu A through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.
The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain. In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center. Support or Funding Information Murchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation
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