Spectroscopic, structural, mutational and biochemical characterisation of Quinol-dependent nitric oxide reductase of Neisseria meningitidis

Quinol-dependent nitric oxide reductase (qNOR) is a homodimeric, membrane-bound metalloenzyme with the catalytic function of reducing two molecules of nitric oxide to nitrous oxide. In bacteria, qNOR can serve either as a respiratory enzyme, or in pathogens as a defence mechanism against cytotoxic levels of nitric oxide radicals generated by the host immune response. One such pathogen is Neisseria meningitidis, a cause of meningitis and septicaemia worldwide. Various biochemical, biophysical, and computational techniques have been applied in study of the molecular mechanism underlying the reduction of nitric oxide by nitric oxide reductase, yet clearly defined mechanics of the enzymatic reaction have yet to be concluded. Understanding of the enzymatic mechanism requires a high-resolution crystal structure of the enzyme to provide a basis for computational calculations and directed biochemical/biophysical analysis. A crystal structure of the qNOR of Geobacillus stearothermophilus was solved to a resolution of 2.5 Å, but significantly in a catalytically inert form unsuitable for reliable characterisation of the active site. By this structure, molecular dynamics studies of potential solvent channels to the active site has led to the identification of a hydrophilic pathway from the cytoplasm for transfer of protons required for catalytic turnover. This would be the first nitric oxide reductase identified to take chemical protons from the cytoplasm instead of the periplasm (as occurs in the isofunctional cytochrome c-dependent nitric oxide reductase), a striking feature in respiratory terms as this membrane separation of proton/electron uptake would infer an electrogenic property to qNOR as yet unobserved in nitric oxide reductases. This thesis presents efforts to characterise the qNOR of Neisseria meningitidis, a highly active reductase that, from preliminary analyses, appears promising in its potential to retain a catalytically active form for crystallographic studies. This qNOR was heterologously over-expressed in a bacterial host and purified into a detergent micelle suspension. Spectroscopic techniques were employed to elucidate the electronic state of the active site of qNOR, and amperometric assays were used to study the nature of the nitric oxide reductase activity. N. meningitidis qNOR was found to have nitric oxide reductase activity significantly greater than that of other qNORs and largely independent to pH changes, and displayed Raman characteristics distinct from other qNORs. Crystallography of qNOR yielded X-ray diffraction to ~4.5 Å, and a crystal structure solved to a resolution of 5 Å. From this, significant structural homology to the high-resolution G. stearothermophilus qNOR crystal structure could be observed. Based on solvent networks identified in the crystal structure of G. stearothermophilus, a cytoplasmic pathway for chemical protons to the active site has been proposed. Research described in this thesis involves site-directed mutagenesis of residues believed to play a role in proton transfer. It was found that none of the mutant variants tested significantly affected the nitric oxide reductase capability of qNOR