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Title: Structure-function studies of the quinol dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans
Author: Gopalasingam, Chai
ISNI:       0000 0004 8506 2437
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Quinol dependent nitric oxide reductases (qNOR) are important enzymes of the denitrification pathway, which helps liberate nitrogen back into Earth's atmosphere, thereby playing a critical role in the recycling of nitrogen, an essential element of genetic material and amino acids. qNOR catalyses the reduction of Nitric Oxide (NO), an important signalling molecule, to Nitrous Oxide (N2O), one of the major causes of ozone layer destruction. qNORs contribute to energy conservation, as denitrification is an alternate form of respiration in some microorganisms under oxygen limiting environments. However, qNORs are commonly found in pathogenic bacteria, such as Neisseria meningitdis (Nm) (a cause of meningitis worldwide) and the opportunistic (and also denitrifying) pathogen Alcaligenes xylosoxidans (Ax), as a means to combat the host immune response. The mechanisms by which qNOR acquires and catalyses NO are still unknown, and current structural data comprise an inactive crystal structure of Geobacillus stearothermophilus (Gs) qNOR at 2.5 Å and a ~ 4.5 Å structure of the electrogenic NmqNOR, which lacks sufficient detail to delineate proton transfer pathways and the active site (heme and non-heme iron) arrangement, furthermore, both structures were reported to be monomeric. This thesis presents efforts to obtain the first high resolution structure of an active qNOR, which AxqNOR is a suitable candidate owing to confirmation of NO reduction and correct metal content/ratios. A crystallographic structure at 6.5 Å was obtained, which revealed a clear dimeric assembly, a previously unseen state. Efforts to improve the resolution were not successful, and analysis by cryogenic Electron Microscopy (cryo-EM) ensued. A 3D reconstruction of wildtype AxqNOR also showed a dimeric assembly and was resolved to 3.7 Å. The active site arrangement of an active qNOR was revealed, showing the non-heme iron to be ligated with three ligands, not four as in the related cytochrome c dependent NOR (cNOR). The dimer interface was maintained by a conserved helix in qNOR, which is not found in cNOR. Cryo-EM analysis of activity enhanced mutant at 3.2 Å enabled new features to be gleaned in the putative proton transfer channel. In addition, a catalytically inert mutant was solved to 4.5 Å, the latter of which was purified and solved as a monomer, showing significant helical rearrangement compared to dimeric structures. Site directed mutagenesis revealed residues close to the active were important for activity, but proton entry site is still unknown. The oligomeric state of GsqNOR was re-examined and found to be a dimer in the crystal, which prompted questions about NmqNOR oligomeric state. Cryo-EM analysis of the sample used to make crystals (crystallographic structure which is monomeric) surprisingly revealed a dimeric assembly, (reconstruction at 9 Å). Based on this work, the oligomeric status of qNOR is likely dimeric, with crystallisation possible disrupting the assembly in NmqNOR. The high-resolution structures of AxqNOR enable further work on the importance of dimerisation and allow the potential to obtain quinol-based inhibitor bound structures of an enzyme of environmental and medical importance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral