CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/194765 |
| Date | January 2009 |
| Creators | Singh, Satish Kumar |
| Contributors | Montfort, William R., Montfort, William R., McEvoy, Megan, Miranda, Katrina M., Cusanovich, Michael A., Visscher, Koen |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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