Structure of reaction intermediates in the bc1 complex

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DESCRIPTION (provided by applicant): 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.