Enzymes of the bc1 complex family are at the core of respiratory and photosynthetic pathways, and are directly responsible for about 30% of the energy conversion of the biosphere. This central importance in biology provides an intrinsic interest, relating directly to our understanding of cellular physiology, energy conversion mechanisms, and maintenance. The essential role is highlighted by the importance of mitochondria in cellular aging, and apoptosis. The photosynthetic bacteria provide a model system for studying the important mitochondrial complex. The catalytic core of the bacterial bc1 complex (QH2:cyt c2 oxidoreductase) is highly conserved, and the reaction mechanism is essentially the same. In the bacterial system, the interplay between function and structure can be more easily studied because the system can be activated by illumination. After a short, bright, flash, the photochemical reaction center generates the substrates for the bc1 complex, initiating turnover in the 100 microsecond time scale, more than ten times faster than is possible in mixing experiments. In addition, the bacterial system is readily amenable to molecular engineering through specific mutagenesis. Under the projects supported by this grant, we take advantage of this experimental facility to explore the molecular mechanism in the context of structure, - the molecular architecture of the complex. The availability of crystallographic structures has stimulated much interest, and has provided strong support for the modified Q-cycle, which is generally accepted as the underlying mechanism. However, the structures have also provoked some interesting questions, mainly relating to unexpected dynamic features, including a large-scale domain movement, and evidence for additional more local dynamic features. In this proposal, we address some of the more controversial issues. We will tackle these through a combination of molecular engineering, biophysical assay, molecular spectroscopy, and structural studies. A key feature of direct medical interest is the production of reactive oxygen species that lead to DNA and protein damage, cellular aging, and eventually death. The reactions at the Q0-site of the complex generate superoxide as a direct result of the molecular mechanism, through an intermediate semiquinone. Our studies will be in part directed to an understanding of how evolution has designed the complex to minimize this harmful side reaction. Key features of the mechanism are the driving forces for the rate limiting step, and the conformational dynamics that protect the semiquinone from reaction with oxygen.

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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Preusch, Peter C
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University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
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Wilson, Charles A; Crofts, Antony R (2018) Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle. Biochim Biophys Acta Bioenerg 1859:531-543
Barragan, Angela M; Crofts, Antony R; Schulten, Klaus et al. (2015) Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex. J Phys Chem B 119:433-47
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
Hong, Sangjin; Victoria, Doreen; Crofts, Antony R (2012) Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc(1) complex. Biochim Biophys Acta 1817:1053-62
Samoilova, Rimma I; Crofts, Antony R; Dikanov, Sergei A (2011) Reaction of superoxide radical with quinone molecules. J Phys Chem A 115:11589-93
Lhee, Sangmoon; Kolling, Derrick R J; Nair, Satish K et al. (2010) Modifications of protein environment of the [2Fe-2S] cluster of the bc1 complex: effects on the biophysical properties of the rieske iron-sulfur protein and on the kinetics of the complex. J Biol Chem 285:9233-48
Dikanov, Sergei A; Samoilova, Rimma I; Kappl, Reinhard et al. (2009) The reduced [2Fe-2S] clusters in adrenodoxin and Arthrospira platensis ferredoxin share spin density with protein nitrogens, probed using 2D ESEEM. Phys Chem Chem Phys 11:6807-19
Kolling, Derrick R J; Samoilova, Rimma I; Shubin, Alexander A et al. (2009) Proton environment of reduced Rieske iron-sulfur cluster probed by two-dimensional ESEEM spectroscopy. J Phys Chem A 113:653-67
Crofts, Antony R; Holland, J Todd; Victoria, Doreen et al. (2008) The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex? Biochim Biophys Acta 1777:1001-19

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