Octa-heme cytochrome c tetrathionate reductase (OTR) from Shewanella oneidensis MR-1 is a periplasmic protein and shows several extraordinary structural features around its active-site heme. OTR has been found able to catalyse the in vitro reduction of tetrathionate, nitrite, hydroxylamine and hydrogen peroxide. However the physiological function of this novel protein remains unknown. The subject of this thesis is the in vitro catalytic mechanism and the in vivo function of OTR. As OTR displays great similarity with bacterial penta-heme cytochrome c nitrite reductase (NrfA) in several aspects, it has been proposed that OTR might be physiologically involved in the metabolism of nitrite or other nitrogenous compounds. However kinetics assays and phenotypes studies carried out in this project suggest this is not the case. In vitro kinetic assays of the reduction of nitrite and hydroxylamine catalysed by OTR showed no significant difference in enzyme activities among the wild-type OTR and its mutant forms which have one active site residue replaced by alanine, namely OTR K153A, C64A, N61A and D150A. And the nitrite reductase activity of OTR (kcat/Km = 1.0×105 M-1•s-1) are much lower than that of NrfA (kcat/Km = ~108 M-1•s-1). These results indicate that OTR is not specifically adapted to reduce nitrite and it cannot compete for nitrite against NrfA in vivo. No phenotype difference was identified between the wild-type and the Δotr strain of Shewanella oneidensis MR-1 when nitrite or nitrate served as the sole electron acceptor. OTR appears not to be involved in the respiration or detoxification of nitrite, which is consistent with previous transcriptional and phenotype reports that involve OTR or its homologues. The in vitro tetrathionate reduction activity of OTR was unable to be reproduced in this project for unknown reasons. Although transcriptomic data from the literature suggest that OTR may be related to the metabolism of sulphur-containing compounds, kinetic and phenotype studies reveal that OTR does not directly participate in the respiration of thiosulfate, sulfite, tetrathionate, polysulfide or elemental sulphur. Cysteine 64 is a highly-conserved amino acid residue of OTR close to the active site and its side-chain sulphur atom is covalently bonded by either an oxygen or a sulphur atom as observed in the crystal structure. Such a modification is potentially important to the function of OTR. ESI mass spectroscopy results show that in native OTR the modified form is around 48 Da heavier than the unmodified form, and the MALDITOF peptide mass spectra show that the modified form could be converted into the unmodified form by reducing agent DTT. These results suggest that the modification could be a cysteine persulfide attaching an extra oxygen atom in the form of water or hydroxide anion.

Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.