Search results

(1 - 2 of 2)
Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Title
Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator
Raba, Daniel Alexander
Date
2019
Description
The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe 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.
Show less
IDENTIFICATION OF THE RIBOFLAVIN BINDING SITE IN VIBRIO CHOLERAE ION PUMPING NQR COMPLEX
Title
IDENTIFICATION OF THE RIBOFLAVIN BINDING SITE IN VIBRIO CHOLERAE ION PUMPING NQR COMPLEX
Creator
Lee, Chia-Hsing
Date
2021
Description
NQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain...
Show moreNQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain of many pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, Chlamydia trachomatis. Its electron transfer path requires three different flavin cofactors to facilitate: FAD, FMN, and riboflavin. The FMN in subunit B (FMNB) brings electrons to riboflavin and then transfers it to the final electron receptor UQ in subunit B, coupled with the Na+ pumping mechanism. NQR has a unique evolutionary history, and one of the pieces of evidence is that NQR has been reported as the only one flavoenzyme that uses riboflavin as its redox cofactor. However, the binding site of riboflavin has not been well understood. To gain insight into the electron transfer at this site in V.cholerae NQR, we generated mutants at the interface of subunits B, D, and E where the possible location of riboflavin is. To characterize these mutants, we assessed NQR properties with different approaches including enzyme kinetics and flavin radical profiling. We found that the mutagenesis surrounding the hydrophobic pocket disrupted the NQR activity, and cause the loss of neutral radical, but did not interfere with the binding affinity between the substrates and NQR. This study will help to understand electron transfer better in NQR and develop the drugs targeting the riboflavin binding site in the future.
Show less