Watson, Cameron Misha Manson;
(2019)
Mechanistic insights into arsenite oxidase and implications for its use as a biosensor.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Arsenic is an environmental toxin which poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase (Aio) from various bacteria couples the oxidation of arsenite to the reduction of electron acceptors. The Aio from Rhizobium sp. str. NT-26 is in development as an arsenic biosensor. Aio consists of a large subunit (AioA), containing a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. The first objective was to identify the rate-limiting step of Aio catalysis to establish if the rate could be improved. The rate-limiting step was found to be electron transfer from the 2Fe-2S cluster to cytochrome c by using stopped-flow spectroscopy, steady state kinetics and isothermal titration calorimetry. An AioB mutant (F108A) specifically reduced activity with cytochrome c by affecting electron transfer. The AioB subunit was expressed alone and was able to weakly associate with cytochrome c suggesting that the AioA subunit is important in the cytochrome c interaction. Unfortunately, the AioA subunit was unstable alone so its cytochrome c interaction was not characterised. Most AioB possess a disulphide bridge proposed to be involved in electron acceptor selectivity. The NT-26 Aio does not possess a disulphide bridge while that of Alcaligenes faecalis does. Site-directed mutagenesis introduced and removed a disulphide bridge into the NT-26 and Alcaligenes faecalis Aio respectively. Presence of the disulphide bridge increased activity with azurin and decreased activity with cytochrome c. The oxidation of antimonite by Aio was examined to determine how the presence of antimony might affect biosensor performance and to assess if Aio could be used as an antimonite biosensor. Antimonite was found to be a potent, competitive inhibitor of Aio because the product of antimonite oxidation dissociates slowly from the active site. The impact of this on the biosensor’s viability is discussed.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Mechanistic insights into arsenite oxidase and implications for its use as a biosensor |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10066749 |
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