Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.771960
Title: Mechanistic insights into arsenite oxidase and implications for its use as a biosensor
Author: Watson, Cameron Misha Manson
ISNI:       0000 0004 7660 5292
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.771960  DOI: Not available
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