Regulation of Substrate Binding in the bc1 Complex

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Abstract Ubiquinol-cytochrome c oxidoreductase (bc1 complex, complex III) is a key membrane enzyme involved in respiration. It is known to be one of the major producers of reactive oxygen species (ROS) in mitochondria. Our overarching hypothesis is that there are at least three overlooked bc1 regulations mechanisms involving cytochrome (cyt) c1. Specifically, our Aim I is to use a combination of computational and experimental techniques to test anticooperative substrate binding in the bc1 complex. This effect was suggested based on available X-ray crystal structures but was not experimentally tested. Our preliminary molecular dynamics (MD) simulations provide us with a testable structural mechanism which we will test experimentally. Our Aim II is to establish the role of naturally occurring trimethylation of Lys-77 by a unique and specific cyt c lysine methylatransferase (Ctm1) in yeast. Our hypothesis that this posttranslation modification regulates the strength of cation-pi interaction between Lys-77 of cyt c and universally conserved in species with Ctm1p Phe-132 in cyt c1. Finally, our Aim III is focused on testing a hypothesis that lipid membrane composition and lipid charge can regulate substrate binding affinity in the bc1 complex. This project will use a multi-pronged approached combining computational and experimental techniques to predict molecular level bc1 regulation mechanisms and to test them experimentally. We will use long all-atom MD simulations of bc1 in different lipid environments to predict structural changes associated with different occupancy of the substrate binding sites and to guide our experimental work on detergent-solubilized and nanodisc-embedded bc1. We will use isothermal calorimetry (ITC) to test substrate binding in vitro, and to measure binding stoichiometries, association constants, and thermodynamic parameters as a function of ionic strength and lipid charge. We will use small-angle X-ray scattering (SAXS) to independently verify the ITC results, to confirm the locations of substrate binding sites, and to construct low-resolution solution-state structures of the enzyme-substrate complexes. Laser-induced time-resolved optical spectroscopy will be used to measure changes in the charge transfer rates as response to changes in lipid environment and substrate binding regulation. Finally, we will use kinetic spectroscopy to study the roles of lipid membranes and intermonomer interactions within the bc1 complex dimer on the catalytic turnover rates and the rate of ROS production. Overall, this interdisciplinary approach will advance understanding of cyt bc1 regulation and will test the three predicted regulation mechanisms. In addition, this project will directly support each year research training of 4 undergraduate students interested in pursuing biomedical careers.