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Bioenergetics at extreme temperature: structural and functional studies of complexes II and IV of Thermus thermophilus

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dc.contributor.advisor Soulimane, Tewfik Noor, Mohamed R. 2018-09-17T10:38:43Z 2018-09-17T10:38:43Z 2012
dc.description peer-reviewed en_US
dc.description.abstract While abundantly evident that thermophilic organisms are adapted to grow at high temperatures, the exact thermoadaptation mechanisms are often very subtle. Various membrane-bound respiratory oxidases have been characterised and their structures determined but questions on their proton and electron transfer pathways still remain. In this study, two such protein complexes from the extreme thermophile Thermus thermophilus were investigated. Multiple variants of Complex II (succinate:quinone oxidoreductase) were recombinantly produced to homogeneity through a homologous expression system, including a novel synthetic monomer. For the first time, the monomeric Complex II was shown to be functional and proves the hypothesis of an intraprotomer electron transfer. One of the variants was also crystallised. With a diffraction to 3.8 Å, a preliminary crystallographic analysis is reported here. The second complex is caa3-oxidase, uniquely possessing a covalently-bound substrate cytochrome c and formed by ‘fused’ subunits. A recombinant protein expression and mutagenesis system is described along with circular dichroism and preliminary ultrafast spectroscopic studies. Together with analytical gel filtration chromatography, the wild type and mutant oxidases are shown to be homogeneous, well folded and suitable for future crystallisation trials. Preliminary femtosecond spectroscopy on wild type oxidase demonstrates a novel multiexponential NO binding kinetics at 140 and 930 ps. In addition, an oxidase subpopulation with a distinct spectrum is present after flash-photolysis of CO bound to the oxidase dinuclear centre that might represent molecules with an inaccessible CuB. Nonetheless, further mutagenesis studies, made possible by the expression system described here, would be required to confirm these findings. en_US
dc.language.iso eng en_US
dc.publisher University of Limerick en_US
dc.relation info:eu-repo/grantAgreement/EC/FP7/283570
dc.subject Thermus thermophilus en_US
dc.subject respiratory complex en_US
dc.subject bioenergetics en_US
dc.title Bioenergetics at extreme temperature: structural and functional studies of complexes II and IV of Thermus thermophilus en_US
dc.type info:eu-repo/semantics/doctoralThesis en_US
dc.type.supercollection all_ul_research en_US
dc.type.supercollection ul_published_reviewed en_US
dc.type.supercollection ul_theses_dissertations en_US
dc.contributor.sponsor SFI en_US
dc.contributor.sponsor IRC en_US
dc.contributor.sponsor ERC en_US
dc.relation.projectid BICF685 en_US
dc.relation.projectid 283570 en_US
dc.rights.accessrights info:eu-repo/semantics/openAccess en_US

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