Structural and spectroscopic characterisation of Cytochrome c’ and Cytochrome P460 from Methylococcus capsulatus (Bath)

Many Ammonia-oxidising nonlithotrophic bacterium (ANB) and Ammonia oxidising bacteria (AOB) have been shown to contain two phylogenetically related cytochromes: a cytochrome P460 and a cytochrome c’-β. Cytochrome P460s (so named due to their 460 nm peak in the ferrous state) are enzymes known to convert hydroxylamine to nitrous oxide, a key step in the metabolism of ammonia in bacteria which is considered to be one of the largest sources of nitrous oxide in the environment. Cytochromes c’-β are so called as they have spectral properties similar to the better studied c’-α but they are predicted to all have a highly beta sheet structure instead of the alpha helixes normally associated with a cytochrome c’. Whilst the role of Cytochrome c’s has not been definitively proved it has been proposed that they are involved in NO scavenging and protecting cells against nitrosoative stress. P460s have been well studied in AOB but less so in ANB, whilst very few members of the cytochrome c’-β have been characterised at all. This thesis focusses on the cytochrome P460 and c’ from Methylococcus capsulatus (Bath) characterising their structural and spectroscopic properties through the use of cryogenic single crystal X-ray crystallography and UV-visible and EPR spectra, along with kinetic studies and activity assays, on both the wt proteins and single point mutants, to investigate how structural differences in the distal heme pockets for two proteins with very similar overall protein folds can give rise to two very different functions.