Complex II superfamily members catalyze two separate reactions in respiration: interconversion of fumarate and succinate in the soluble milieu and interconversion of quinol and quinone in the membrane. Electrons liberated as a result of one reaction become substrates for the second, thermodynamically linking both sites and allowing complex II enzymes to catalyze electron entry to or exit from a respiratory chain. In anaerobic respiration, menaquinol:fumarate oxidoreductase (QFR) catalyzes the final step in the most widely used anaerobic respiratory pathway, fumarate reduction. This work analyzes structural and mechanistic details of function and inhibition at each of the two active sites.
The first part examines transition state formation at the active site responsible for fumarate and succinate interconversion, the dicarboxylate site. It was found that two threonine residues, Thr-234 and Thr-244, might play important roles in attaining and stabilizing the transition state. The second part of this work focuses on details of dicarboxylate site inhibition by substrate-like molecules and of substrate activation. It was found that all tight binding ligands or ligands transformed by the enzyme also induce large optical shifts in an FAD cofactor and align an activatable bond along the active C4a-N5 axis of FAD. This bond overlap was proposed to play a part of an orbital steering mechanism to guide substrate and activate it for catalysis. This alignment was found be conserved in a number of flavoenzymes that catalyze dehydrogenation reactions, even with completely unrelated substrates and structural folds.
The third part of this work focuses on the second active site, the quinone/quinol interconversion site. The E29L variant QFR was crystallized to study semiquinone formation. Preliminary results reveal that the substrate, menaquinone, may bind in a continuum of active site positions as the substrate undergoes catalysis. Another substrate, ubiquinone was shown to bring about the presence of strong electron density at a site located 13 angstroms away from menaquinone site. The forth and final part of this work examines the structural work done to isolate and stabilize complex formation between QFR and FliG, a part of the flagellar motor.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-02142011-170623 |
| Date | 21 February 2011 |
| Creators | Tomasiak, Thomas |
| Contributors | H. Alex Brown, Tina Iverson, Heidi Hamm, Hassane Mchaourab, Jens Meiler |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-02142011-170623/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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