Loading...
Bioenergetics at extreme temperature: structural and functional studies of complexes II and IV of Thermus thermophilus
Date
2012
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.
Supervisor
Soulimane, Tewfik
Description
peer-reviewed
Publisher
Citation
Collections
Files
ULRR Identifiers
Funding code
Funding Information
Science Foundation Ireland (SFI), Irish Research Council (IRC), European Research Council (ERC)