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Respiratory complexes from Thermus thermophilus: Native and recombinant production of both complex II and caa3-type cytochrome c oxidase for structural studies

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posted on 2023-10-24, 11:52 authored by Sarah Rose O’Kane

Four membrane complexes constitute the respiratory chain of eukaryotes and prokaryotes. Two of these enzymes form the focus for this thesis - succinate:quinone reductase (Complex II; SQR) which oxidises succinate to fumarate as part of the Krebs (TCA) cycle while contributing electrons to the respiratory chain, and caa3-type cytochrome c oxidase (Complex IV) which is the terminal electron acceptor of the respiratory chain reducing dioxygen to water, while simultaneously pumping protons across the membrane to produce ATP. The source of these enzyme complexes is the extreme thermophilic bacterium Thermus thermophilus, which is used as the fundamental source of the native enzymes by large scale and extensive purification and manipulated as the expression strain for the production of the homologous recombinant enzymes. Our interest in these enzymes is from two perspectives; (i) the generation of the endogenous native enzymes to assist the investigation into the function of these protein complexes, in addition to supporting crystallisation trials for subsequent structural studies, and (ii) the generation of homologous recombinant enzymes to analyse the mechanism of action of these protein targets by site directed mutagenesis.

The native and recombinant SQRs have been purified and enzymes characterised by various biochemical and biophysical techniques. Crystals were obtained for the native and recombinant Complex II by the vapour diffusion, sitting drop method in the presence of the detergent n-dodecyl-β-D-maltoside.

The native and recombinant caa3-type cytochrome c oxidases have also been purified and characterised. The recombinant enzyme’s preliminary biochemical characterisation is comparable to that of the native enzyme. Furthermore, a strain deficient in the native enzyme (∆caa3) has been successfully produced by double homologous recombination, as determined by DNA sequencing, which indicates that the ba3-type cytochrome c oxidase enzyme replaces the cytochrome c oxidase activity under aerobic conditions when required.


History

Faculty

  • Faculty of Science and Engineering

Degree

  • Doctoral

First supervisor

Tewfik Soulimane

Other Funding information

I gratefully acknowledge the funding sources that made my PhD work possible: the SFI for funding my work during this thesis and also providing the travel grant for my stay in Germany. I am also grateful to the IRCSET-Ulysses travel grant, for funding my stay in Marseille for the EPR analysis

Department or School

  • Chemical Sciences

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