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.