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Title:The interactions of cytochrome bo3 from Escherichia coli with its substrates - ubiquinone and oxygen
Author(s):Choi, Sylvia K
Director of Research:Gennis, Robert B
Doctoral Committee Chair(s):Gennis, Robert B
Doctoral Committee Member(s):Imlay, James A; Lu, Yi; Nair, Satish K
Department / Program:School of Molecular & Cell Biology
Discipline:Biophysics & Computional Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):cytochrome bo3
terminal oxidase
respiratory chain
heme copper oxygen reductase
cytochrome c oxidase
quinol oxidase
cytochrome aa3
E. coli
Escherichia coli
Rhodobacter sphaeroides
oxygen channel
oxygen binding
second order rate
site-directed mutagenesis
quinone binding
high affinity site
low affinity site
High-performance liquid chromatography (HPLC)
oxygen activity
cyt bo3
quinone extraction
Electron Paramagnetic Resonance (EPR)
methyl hyperfine coupling
Abstract:Heme-copper respiratory oxygen reductases reduce O2 to water and use the redox free energy to generate the proton motive force. Among the heme-copper oxygen reductases are the quinol oxidases, which catalyze the 2-electron oxidation of ubiquinol or menaquinol instead of cytochrome c. Escherichia coli (E. coli) cytochrome bo3 is the best characterized quinol oxidase. Depending on the detergent used to solubilize the enzyme, cyt bo3, preparations of this enzyme contain between 0 to 2 equivalents of ubiquinone-8. Studies using ubiquinol-1, a soluble quinol substrate, indicate one high affinity quinone binding site, which acts as a non-exchanging co-factor, and a second, low-affinity site which is the substrate site. The residues interacting with the quinone at the high affinity QH site are well defined through numerous mutagenesis and spectroscopic studies, but the identification of the low affinity QL site has remained difficult. To clarify whether the low affinity QL exists, we used structural models to guide us. First is based on the new structure of cytochrome bo3 we have obtained by collaboration with the Stowell group. It shows two ubiquinone molecules bound to the enzyme. The benzoquinone headgroup of one bound quinone is located at the expected QH site. The headgroup of the second bound quinone is, however, located on the opposite side of the molecule. Site-directed mutagenesis of residues located at the putative second site do not indicate that this second site is functionally important, leaving the question of the existence of the QL site open. The second model is based on bioinformatics. Recent publication from Bossis group used bioinformatics methods to identify a possible QL site. We have tested this model using site-directed mutagenesis but have found no evidence for disruption of a substrate binding site. Along with these two models, we have revisited the possibility of a QL site in subunit II with site-directed mutagenesis but did not find the QL site. A strong argument in favor of the existence of the QL site is the inability of the quinone bound at the QH site to exchange during enzyme turnover. These experiment were performed using the soluble quinone analogue, ubiquinone-1. This experiment has been repeated with long chain quinol substrates to test the possibility that these might behave differently. If these long chain quinones exchange into the QH site during turnover, there would be no need to postulate the existence of the second QL site. All of the previous attempts to exchange the quinone at the QH were done in the purified micelles-form of enzyme with short chain quinone analogue, UQ1 or inhibitors. In my work, I will demonstrate that, in the lipid bilayer this “tightly bound” quinone can not only dissociate, but also can exchange with an exogenous quinone. Cyt bo3 embedded liposomes allowed us to reconstitute a respiratory chain using with a monotopic membrane protein, NDH-2, and have a good turnover with hydrophobic long chain UQ10. Moreover, we show that the “tightly-bound” quinone can readily exchange with UQ2 even in micelles depending on incubation time and concentration of the substrate. Therefore, proving that this “cofactor-like” quinone can actually dissociate from the enzyme and exchange with exogenous quinone, allows us to argue that there is only one quinone binding site.
Issue Date:2015-11-02
Rights Information:Copyright 2015 Sylvia K. Choi
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12

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