Noor_2012_Bioenergetics.pdf (5.5 MB)
Bioenergetics at extreme temperature: structural and functional studies of complexes II and IV of Thermus thermophilus
thesis
posted on 2023-01-19, 12:10 authored by Mohamed R. NoorWhile 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.
Funding
Study on Aerodynamic Characteristics Control of Slender Body Using Active Flow Control Technique
Japan Society for the Promotion of Science
Find out more...History
Faculty
- Faculty of Science and Engineering
Degree
- Doctoral
First supervisor
Soulimane, TewfikNote
peer-reviewedOther Funding information
SFI, IRC, ERCLanguage
EnglishDepartment or School
- Chemical Sciences
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