%0 Thesis %A Perry, Ashlee. %D 2014 %T Structural studies of the two iron rubredoxin of Pseudomonas oleovorans %U https://figshare.le.ac.uk/articles/thesis/Structural_studies_of_the_two_iron_rubredoxin_of_Pseudomonas_oleovorans/10150844 %2 https://figshare.le.ac.uk/ndownloader/files/18293837 %K IR content %X Sequence alignment studies and biochemical investigations have suggested that the two iron rubredoxin of Pseudomonas oleovorans consists of two-rubredoxin type domains connected by a ~60 amino acid linker region. The rubredoxin gene (alk G) contained in the rubredoxin expression vector was targeted by site-directed mutagenesis to produce independent expression vectors for the N and C-terminal domains. The C-terminal vector directed the high-level production of the C-terminal domain and a simple procedure was used to purify the recombinant domain in the Fe-form. Attempts to directly express the N-terminal domain of the 2Fe-rubredoxin from a mini-gene were unsuccessful. However, the apo-form of the N-terminal domain was isolated through cleavage of an engineered 2Fe-rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The responsive proteins could readily be converted to the apoprotein, cadmium or iron forms following precipitation with trichloroacetic acid and resolubilisation in the presence or absence of cadmium chloride or ferrous ammonium sulphate, respectively. Both domains have been characterised by UV-visible, circular dichroism and 1D NMR spectroscopies. The spectra collected for the C-terminal domain are characteristic of typical rubredoxins. The spectra collected for the N-terminal domain are less characteristic of typical rubredoxins, likely reflecting poor structural stability, which is consistent with the properties of the full-length di-domain rubredoxin. In steady-state assays, both N and C-terminal domain rubredoxins were redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The solution structure of the C-terminal domain was determined using 2D NMR techniques. The structure is typical of rubredoxins and reveals that the linker region is structureless, which is consistent with limited proteolysis studies carried out on the C-terminal domain. The structures of the full-length rubredoxin and the C-terminal domain were investigated by small angle X-ray scattering methods. These analyses confirmed the finding that the linker region in the C-terminal domain is structureless, and demonstrated that the folded N and C-terminal domains of the full-length rubredoxin are associated, with their metal binding sites in close proximity.. %I University of Leicester