We focus on mechanisms that promote energy conserving electron and proton translocation through ubiquinol-cytochrome c oxidoreductase (bc1 complex). We shall make use of experimental systems that offer the means to activate the system by light flashes via a on a bacterial photochemical reaction center. Such systems can provide valuable information on a rapid timescale, of kinetics of oxidation/reduction and protonation/deprotonation of ratios of electron and proton movements and, in combination with redox potentiometry, of the fundamental properties of the active redox components. The first part is aimed at the mechanism of action of bc1 complexes in vivo and in vitro. The principal systems are: a) the native membranes from photosynthetic bacteria (Rhodopseudomonas sphaeroides and capsulata); b) hybrid constructions in vitro of well-characterized isolated reaction centers (Rps. sphaeroides), cytochrome c and bc1 complexes, (beef heart mitochondria). The in vivo photosynthetic bacterial membranes provide a coupled photon-electron-proton-phosphorylation membrane system that possesses the usual manipulative opportunities offered by bacteria, including growth/media manipulations and mutants blocked in the bc1, complex. The in vitro hybrid constructions provide wide experimental flexibility and provide the means of achieving more ordered, densely packed mono- and multilayer arrays of the hybrid systems on glass or electrodes, for structurally related spectrometric analysis and direct measurement and control of charge movements across the monolayer profile. The second part approaches the bc1 complex from a different direction: from the study of a key component--ubiquinone. Quinonoid compounds will be studied from the standpoint of their structure, their substituents, electrochemistry, and hydrophobic properties. Quinonoid compounds will be used to probe the bc1 complex catalytic sites of ubiquinone oxidation-reduction to determine the parameters that govern the association of molecule and reaction site(s). Further efforts will be made to develop a family of bc complexes, systematically altered in the electrochemistry of the replacement quinones. This approach should strongly augment features of the first theme of the proposal. Moreover, it will provide the basis for a much needed, alternate approach to obtaining kinetic and thermodynamic information from the systematically altering free energy gaps between reactants and add another dimension to the study of electric field effects on the bc1 complexes oriented as mono- and multilayers between electrode surfaces.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM027309-14
Application #
3484612
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1979-05-01
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
14
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Zheng, Zhong; Dutton, P Leslie; Gunner, M R (2010) The measured and calculated affinity of methyl- and methoxy-substituted benzoquinones for the Q(A) site of bacterial reaction centers. Proteins 78:2638-54
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Zhang, Haibo; Osyczka, Artur; Dutton, P Leslie et al. (2007) Exposing the complex III Qo semiquinone radical. Biochim Biophys Acta 1767:883-7
Osyczka, Artur; Zhang, Haibo; Mathe, Christelle et al. (2006) Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1. Biochemistry 45:10492-503
Zhang, Haibo; Osyczka, Artur; Moser, Christopher C et al. (2006) Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes. Biochemistry 45:14247-55
Moser, Christopher C; Farid, Tammer A; Chobot, Sarah E et al. (2006) Electron tunneling chains of mitochondria. Biochim Biophys Acta 1757:1096-109
Iwaki, Masayo; Yakovlev, Gregory; Hirst, Judy et al. (2005) Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy. Biochemistry 44:4230-7
Osyczka, Artur; Moser, Christopher C; Dutton, P Leslie (2005) Fixing the Q cycle. Trends Biochem Sci 30:176-82
Osyczka, Artur; Moser, Christopher C; Dutton, P Leslie (2004) Novel cyanide inhibition at cytochrome c1 of Rhodobacter capsulatus cytochrome bc1. Biochim Biophys Acta 1655:71-6
Iwaki, Masayo; Osyczka, Artur; Moser, Christopher C et al. (2004) ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex. Biochemistry 43:9477-86

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