The proposed research will focus on the mechanisms of electron transfer and proton pumping in respiration. Special emphasis will be on cytochrome oxidase, the primary site of coupling between the two processes. Our goals include the following: 1) The mechanism of the reduction of dixoygen to water by cytochrome oxidase will be studied under a variety of conditions with the flow-flash method, which uses the photolability of the CO complex to initiate the redox activity with O2. Time-resolved multichannel optical absorption spectroscopy will be used to follow the kinetics of electron and proton transfer on time scales of nanoseconds to milliseconds. Singular value decomposition and global exponential fitting methods will be applied to analyze the kinetics and determine the UV-Vis spectra of the transient intermediates. These studies should provide new insight into the mechanism of the dioxygen reduction by cytochrome oxidase. 2) Alternatives to CO photodissociation will be used to investigate the reaction of O2 with cytochrome oxidase. The reaction of unliganded reduced cytochrome oxidase with oxygen will be studied using a superfast direct mixing method, pulsed-accelerated-flow (PAF). The reaction of oxygen with the unliganded enzyme will also be investigated using O2 which is produced in situ on any relevant time scale by photodissociating synthetic dioxygen carriers such as dicobalt u-peroxo polyaine complexes. Both the PAF method and the photodissociation of the dicobalt u-peroxo and u-superoxo polyamine complexes represent new approaches to study the fast dioxygen reactions of cytochrome oxidase and both avoid the mechanistic ambiguities associated with the fate of photodissociated CO in transitional flow-flash experiments. 3) The mechanism of the redox-linked proton pump in cytochrome oxidase will be investigated. The kinetics of electron transfer and proton pumping upon flash-induced oxidation of cytochrome oxidase reconstituted into phospholipid vesicles will be monitored using time-resolved optical absorption spectroscopy. The proton pumping reactions will be probed by pH indicators located int he extra-vesicular space, trapped inside the vesicles, or covalently bound to the lipid or protein. These studies will allow us to correlate proton pumping events with individual steps in the dioxygen/cytochrome oxidase redox cycle and will provide a foundation for a structural model of the energy transduction mechanism in cytochrome oxidase.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of California Santa Cruz
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Santa Cruz
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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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