The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
|Commitee:||Minh, David, Xiang, Jialing, Irving, Thomas|
|School:||Illinois Institute of Technology|
|School Location:||United States -- Illinois|
|Source:||DAI-B 81/2(E), Dissertation Abstracts International|
|Keywords:||HQNO, NQR, Proton pump, Pseudomonas aeruginosa, Ubiquinone, Vibrio cholerae|
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