Copper is an essential biological metal. Many enzymes such as cytochrome C oxidase and superoxide dismutase use copper as a prosthetic group. However, free copper can be harmful by generating reactive oxygen species through Fenton reactions (Linder and Hazegh-Azam 1996). Therefore, copper is tightly regulated throughout the cell. Cu+ ATPases are transporters that regulate Cu+ homeostasis. Mutations to these Cu+ ATPases can cause diseases (Wilson's and Menkes diseases) (Ferenci 2005). CopA from Archaeoglobus fulgidus is a model Cu+ ATPase; it contains eight transmembrane helices and a cytosolic ATP binding domain. CopA is assisted by a Cu+ chaperone (CopZ) which we have shown delivers Cu+ to CopA via protein-protein interactions (Guerrero-Guerrero and Arguello 2008). CopZ has two domains which are both able to bind Cu+ (Guerrero-Guerrero and Arguello 2008). The N-domain has a mononuclear Zn2+ site, a [2Fe-2S] cluster and is extremely rich in cysteines (Sazinsky, LeMoine et al. 2007). To explore the role of cysteines and their Cu+ binding capacity, each cysteine was mutated to serine. All resulting proteins were able to activate CopA ATPase. However, mutation of two Cysteines (Cys4 and Cys38) led to binding of two additional Cu+ ions. Since these two Cys participate in Zn2+ coordination we hypothesized that the coordination of Cu+ might take place with the displacement of Zn2+. In order to explore this hypothesis, CopZ-WT was incubated with an excess amount of Cu+, followed by atomic absorption to measure the presence of Zn2+. These experiments demonstrate a displacement of Zn2+ by Cu+. Thus, our results show that the second Cu+ binding site in A. fulgidus CopZ is constituted by the same Cysteines participating in Zn2+ binding.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1718 |
| Date | 04 May 2009 |
| Creators | Guerrier, Jeanniffer Sabrina |
| Contributors | José M. Argüello, Advisor, , |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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