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ItemMolecular characterization of copper tolerance and resistance proteins and enzymes in gram-negative bacteria( 2011)Copper is an essential trace element required by all living organisms. However, it is potentially toxic when in excess or in its “free” form due to its redox activity and highly competitive binding affinities. Specific metabolic pathways exist to safeguard its transport within the cells. A disruption at any point in these pathways in humans is manifested in several fatal diseases (eg. Wilson, Menkes) and contributes to symptoms of neurodegenerative diseases such as Alzheimer. Bacterial cells protect themselves from toxic environmental copper by expressing resistance defense, similar to antibiotic/drug resistance. The cue system in E. coli is part of the chromosomally encoded copper homeostatic system that enables the survival of bacterial cells at micromolar copper concentrations. This system involves two proteins: CopA, a P-type ATPase which removes excess CuI ions from the cytoplasm to the periplasm where they are oxidised by CueO to less toxic CuII ions. CueO is a multicopper oxidase (MCO). MCOs are a large family of enzymes capable of coupling four one-electron oxidation steps of substrates to the four-electron reduction of one molecule of dioxygen to water. Their active sites feature at least four copper atoms which are traditionally classified into three categories designated T1, T2 and binuclear T3 Cu sites. The T1 site catalyses substrate oxidation while a trinuclear cluster T2/T3 site catalyses the dioxygen reduction. However, CueO is unusual: it displays an extra methionine rich -helix insert which covers the T1 site and which accommodates a labile CuII binding site. This work demonstrates that this labile binding site is actually a specific CuI substrate docking/oxidation (CuI-SDO) site. As a cuprous oxidase, this site must be empty to allow catalytic turnover of CuI. As a phenol oxidase, this site must be occupied by a CuII ion to act as an electron-transfer mediator between the buried T1 site and substrates which are not able to dock directly at the specific CuI-SDO site. Using novel approaches, the affinities of this site for both CuI and CuII have been determined. The combined data provide compelling evidence that CueO is a cuprous oxidase in vivo and not a phenol oxidase. The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. The resistance to copper is attributed primarily to the presence of a large plasmid pMOL30 that includes a cop cluster composing of 21 genes. Expression of the three soluble periplasmic proteins CopK, CopC and CopA is highly induced by the presence of copper indicating their important roles in the copper resistance. This work has expressed and characterised these three proteins. CopK is unique to this system and exhibits intriguing copper binding chemistry. It is a weakly associated dimer that has little affinity for CuII (KD > 10-6 M), but can¬ bind CuI with modest affinity (KD = 2 x 10-11 M¬). However, CuI binding induces structural change that leads to dimer dissociation and generation of a high affinity CuII binding site (KD = 3 x 10-12 M) and the high affinity CuII binding in turn enhances the CuI¬ binding affinity by a factor of 102. Such strong binding cooperativity between CuI and CuII is unprecedented in copper binding proteins. The molecular basis for this unusual property has been explored and uncovered via biochemical studies (including site-specific mutagenesis) and structural approaches (including X-ray crystallography and NMR spectroscopy). CopC shares high sequence similarity with two well-characterised protein homologs, CopC from Pseudomonas syringae and PcoC from E. coli. All potential protein ligands for CuI and CuII are also highly conserved among these three proteins. The latter two proteins bind CuI and CuII specifically at two separate sites. Indeed, CopC from C. metallidurans expressed and isolated in this work features similar CuI and CuII binding chemistries. CopA has not been isolated and studied previously. Its primary sequence derived from the copA gene predicts it to be a MCO and to contain a Met-rich sequence motif in a position equivalent to that in CueO that accommodates the specific CuI-SDO site. Therefore, CopA is assumed to take a similar role as a cuprous oxidase in vivo. However, preliminary studies in this work indicate that CopA is not only a robust cuprous oxidase, but also a robust phenol oxidase. The former function is buffer-dependent, but in contrast to CueO, the reaction buffers have only minimum effect on the phenol oxidase activity of CopA. Preliminary data suggest that, relative to the enzyme functions of CueO, the new enzyme properties of CopA are arisen from the fact that CopA may feature a CuI-SDO site that can bind either CuI or CuII with higher affinities than those of the equivalent site in CueO. This work also demonstrates that CopA catalyses air-oxidation of CuICuII-CopC efficiently, but not of CuICuII-CopK. Although these two proteins bind CuI with similar affinities, their CuI binding sites are quite different in aspects such as solvent-exposure, ligand number and ligand composition. All these differences are likely to affect their specific interactions with the CopA enzyme and emphasise that specific molecular recognition and interactions are important in copper homeostasis in biology.