Exploring the role of copper in novel ammonia-oxidising archaeal isolates and using dynamic structural biology tools to characterise copper monooxygenases

The oxidation of ammonium by ammonia oxidising archaea (AOA) is the first step of nitrification and is critical to the global nitrogen cycle. However, little is known about AOA physiology, especially their responses to bioavailable copper and copper enzyme mechanisms. Whilst AOA genome data is sparse, it does suggest that copper proteins could play a central role in nitrogen transformation pathways, and the biology of copper in soil and marine systems is central to this thesis. This thesis successfully enriched two soil AOA (C2, C4-2) relating to Nitrosocosmicus using a combination of liquid and solid cultivation methods. CARD-FISH confirmed AOA were predominant in both cultures. Physiological characterisation showed C4-2 was mesophilic, neutrophilic with optimum growth between 1-50 mM NH4+, 0-20 mM NO2- (Chapter 2) and bioavailable Cu(II) as low as (10-16 M) (Chapter 4). Amplicon and metagenomic sequencing (Chapter 3) showed that C2 and C4-2 were related to N. Franklandus, and N. hydrocola respectively. Metagenomic-assembled genomes (MAGs) identified key proteins involved in ammonia transport, carbon fixation and oxidative stress defence (Chapter 3). Several multicopper oxidases (MCOs) found in marine (Nitrosopumilus maritimus SCM1) and terrestrial AOA (Nitrosocosmicus franklandus C13), were also identified and selected for recombinant protein over-expression trials, however these proved unsuccessful (Chapter 5). Although not directly linked to the nitrogen cycle, soil dwelling copper containing two-domain lytic polysaccharide monooxygenases (LPMOs) from Streptomyces lividans were studied. The LPMO domain carries out oxidative chemistry creating chain breaks in polysaccharides and the carbohydrate binding domain facilitates the polysaccharide binding. Active site copper coordination geometries were studied using EPR spectroscopy and SEC-SAXS has been used to investigate the spatial arrangement of the two domains in solution. Finally, in Chapter 7 a LPMO domain was studied using serial femtosecond X-ray crystallography, and experiments to initiate molecular oxygen activation using an oxygen releasing photocage carried out.