In this proposal we will use high-resolution EPR spectroscopy to explore the catalytic domains of the bc1 complex trapped in states with paramagnetic intermediates bound. The cytochrome bc1 complex family plays an essential role in the energy metabolism of the biosphere. Three catalytic subunits, cyt b, cyt c1 and the Rieske iron sulfur protein (ISP), house the mechanism. Two catalytic sites in cyt b are involved in oxi-dation or reduction of ubiquinone. The integration of the oxidation and reduction reactions with the release or uptake of protons in the aqueous phases, allows the complex to pump protons across the membrane. To understand the mechanism, we need detailed information about the local reaction environment, including protein structure, hydrogen bonding, and distances, to provide the parameters that control rates, and partitioning of electrons to different pathways. Several partial reactions in the bc1 complex involve paramagnetic intermediates. The EPR approach is well suited to dissecting these, because pulsed-EPR techniques can probe interactions between the electron spin of the intermediate and local magnetic nuclei. They thus pro- vide direct information about spatial and electronic structure of the intermediate and the immediate protein and solvent environment. The species at the focus of the proposal are the reduced [2Fe-2S] cluster of the ISP participating in ubihydroquinone (quinol, QH2) oxidation at the Qo-site;and the semiquinone (SQ) involved in the reactions of the quinone-reducing Qi-site. The choreography of catalysis at these sites is largely controlled by the changes in local configuration needed to accommodate the binding requirements of the different quinone forms, and EPR of the SQ provides a direct tool. This work will be pursued in the context of separately funded studies of kinetic and mechanistic aspects, and an extensive inventory of biophysical, biochemical, and molecular engineering protocols allowing us to correlate data for paramagnetic species, and functional and structural changes in mutant strains. The main question to be addressed is that of how the protein environment modifies the spatial and electronic structure of the intermediates to fit the physiological function, a question of much wider interest in reaction mechanism theory. The central role of bc1 complex plays out in many medical scenarios in which defects lead to pathology, among them cellular death through ROS-mediated damage, mitochondrial myopathies, and apoptosis, and in drug targeting.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062954-09
Application #
7903120
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2001-01-01
Project End
2013-07-31
Budget Start
2010-08-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2010
Total Cost
$258,270
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Taguchi, Alexander T; O'Malley, Patrick J; Wraight, Colin A et al. (2014) Hyperfine and nuclear quadrupole tensors of nitrogen donors in the Q(A) site of bacterial reaction centers: correlation of the histidine N(?) tensors with hydrogen bond strength. J Phys Chem B 118:9225-37
Taguchi, Alexander T; O'Malley, Patrick J; Wraight, Colin A et al. (2014) Nuclear hyperfine and quadrupole tensor characterization of the nitrogen hydrogen bond donors to the semiquinone of the QB site in bacterial reaction centers: a combined X- and S-band (14,15)N ESEEM and DFT study. J Phys Chem B 118:1501-9
Hong, Sangjin; de Almeida, Wagner B; Taguchi, Alexander T et al. (2014) The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph. Biochemistry 53:6022-31
Crofts, Antony R; Hong, Sangjin; Wilson, Charles et al. (2013) The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex. Biochim Biophys Acta 1827:1362-77
Victoria, Doreen; Burton, Rodney; Crofts, Antony R (2013) Role of the -PEWY-glutamate in catalysis at the Q(o)-site of the Cyt bc(1) complex. Biochim Biophys Acta 1827:365-86
Taguchi, Alexander T; O'Malley, Patrick J; Wraight, Colin A et al. (2013) Conformational differences between the methoxy groups of QA and QB site ubisemiquinones in bacterial reaction centers: a key role for methoxy group orientation in modulating ubiquinone redox potential. Biochemistry 52:4648-55
Xue, Bo; Chow, Jeng Yeong; Baldansuren, Amgalanbaatar et al. (2013) Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase. Biochemistry 52:2359-70
Maklashina, Elena; Cecchini, Gary; Dikanov, Sergei A (2013) Defining a direction: electron transfer and catalysis in Escherichia coli complex II enzymes. Biochim Biophys Acta 1827:668-78
Dikanov, Sergei A; Liboiron, Barry D; Orvig, Chris (2013) VO(2+)-hydroxyapatite complexes as models for vanadyl coordination to phosphate in bone. Mol Phys 111:2967-2979
Taguchi, Alexander T; Mattis, Aidas J; O'Malley, Patrick J et al. (2013) Tuning cofactor redox potentials: the 2-methoxy dihedral angle generates a redox potential difference of >160 mV between the primary (Q(A)) and secondary (Q(B)) quinones of the bacterial photosynthetic reaction center. Biochemistry 52:7164-6

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