| An understanding of the mechanism of electron and proton transfer in cytochrome c oxidase (CcO) is essential for a full understanding of the process of energy transduction in this key respiratory enzyme. Combining site-directed mutagenesis and EPR methods, some mutants at residue S44 associated with heme a were created and studied to determine its influence on the spectral characteristics of CcO. EPR data on S44G supports the prediction that this hydrogen bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial C cO. Of additional interest, the pH-dependence of the EPR spectrum of S44D and fast electron transfer studies showing dramatically inhibited electron transfer from CUA to heme a at high pH, indicate a proton-coupled electron transfer mechanism. This mutant could provide a new tool for studying proton and electron transfer reactions in cytochrome c oxidase.;Using the non-redox active Mg site of CcO, substituted with Mn, as a probe, proton/water movement was studied in its vicinity. Electron nuclear double resonance (ENDOR) studies provided us with detailed information on hyperfine coupling interactions between the Mn site and its liganded water molecules. Further studies of oxidized and reduced CcO, plus and minus cyanide or azide, with Electron spin echo envelope modulation (ESEEM) technique showed cyanide binding to the Mn in the enzyme. This finding supports the hypothesis that reduction of CuA causes one of the water ligands of the Mn to become a hydroxide ion, playing an important role in proton pumping in CcO. ESEEM simulations also provide support for this hypothesis.;As a more sensitive technique, high-field EPR was used to study C cO with the hope of gaining more information with higher resolution. The difference in Mn spectra of the reduced and the oxidized CcO was observed in Rs. CcO, which agrees well with findings in Pd. CcO. Further studies of super hyperfine coupling interactions between Mn and its water ligands in the oxidized and reduced enzymes showed some interesting data. The narrowing effect due to deuterium exchange was observed in the oxidized CcO, which can be potentially used to quantify the number of coupled protons. However, experiments with the reduced enzyme in H217O and 2H2O failed to show supposedly broadening and narrowing effects respectively. The possible reasons were explored and explained. |
