The primary objective of this research is to elucidate the mechanism of electron and proton transfer during the reduction of dioxygen to water by heme-copper oxidases.
Our specific aims will focus on four problems: 1. The mechanism of the reduction of dioxygen to water by bacterial heme-copper oxidases will be studied by the CO flow-flash method. Time-resolved multichannel optical absorption spectroscopy, in conjunction with singular value decomposition (SVD) and global exponential fitting analysis, will be used to follow the kinetics of electron and proton transfer and to deduce the UV-Vis spectra of the transient intermediates. These studies should provide new insight into the mechanism of the dioxygen reduction reaction by heme-copper oxidases. 2. We will investigate the reaction of dioxygen with bovine heart and bacterial oxidases in different oxidation states using dioxygen which is produced in situ by photodissociating synthetic dioxygen carriers. We will also extend this approach to rapid dioxygen binding and activation in ribonucleotide reductase (RNR), in which the reactions occur too rapidly to be monitored by conventional stopped-flow methods. 3. The intramolecular electron transfer in the bacterial oxidases, bo3 from E. coil, aa3 from Rhodobacter sphaeroides and ba3 from Thermus thermophilus will be investigated using a photoactivatable dye, thiouredopyrene-trisulfonate (TUPS), covalently linked to single reactive cysteine residues on the oxidases. Time-resolved optical absorption spectroscopy, in conjunction with SVD and global exponential fitting, will be used to determine the spectra of the intermediates present and the rate constants of individual electron transfer steps. By varying the distance between the labeled cysteine and the initial electron acceptor and by introducing breaks into presumed electron transfer pathways by site-directed mutagenesis, detailed information regarding intramolecular electron transfer pathways in heme-copper oxidases will be obtained. 4. We propose to make chemical analogs of the active site of cytochrome oxidase, including the His-Tyr cross-linked dipeptide and the cyclic pentapeptide (His-Pro-Glu-Val-Tyr) with and without Cu-ligands incorporated. The analogs will be studied using a multispectroscopic approach, including steady-state and time-resolved UV-Vis spectroscopy, FTIR and ESR.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Ikeda, Richard A
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University of California Santa Cruz
Schools of Arts and Sciences
Santa Cruz
United States
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Einarsdóttir, Olöf; McDonald, William; Funatogawa, Chie et al. (2015) The pathway of O?to the active site in heme-copper oxidases. Biochim Biophys Acta 1847:109-18
McDonald, William; Funatogawa, Chie; Li, Yang et al. (2013) Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases. Biochemistry 52:640-52
Szundi, Istvan; Funatogawa, Chie; Cassano, Jennifer et al. (2012) Spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and EQ(I-286) mutant of Rhodobacter sphaeroides cytochrome c oxidase. Biochemistry 51:9302-11
Einarsdóttir, Olöf; Funatogawa, Chie; Soulimane, Tewfik et al. (2012) Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers. Biochim Biophys Acta 1817:672-9
McDonald, William J; Einarsdóttir, Olöf (2010) Solvent effects on the physicochemical properties of the cross-linked histidine-tyrosine ligand of cytochrome c oxidase. J Phys Chem B 114:6409-25
Szundi, Istvan; Funatogawa, Chie; Fee, James A et al. (2010) CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus. Proc Natl Acad Sci U S A 107:21010-5
Mahoney, Maximillian E; Oliver, Allen; Einarsdottir, Olof et al. (2009) Synthesis of a cyclic pentapeptide mimic of the active site His-Tyr cofactor of cytochrome c oxidase. J Org Chem 74:8212-8
Paszek, Matthew J; Boettiger, David; Weaver, Valerie M et al. (2009) Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate. PLoS Comput Biol 5:e1000604
Offenbacher, Adam; White, Kimberly N; Sen, Indranil et al. (2009) A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase. J Phys Chem B 113:7407-17
McDonald, William J; Einarsdottir, Olof (2008) Solvent effects on the vibrational frequencies of the phenolate anion, the para-cresolate anion, and their radicals. J Phys Chem A 112:11400-13

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