- Enzymmechanismus (1) (remove)
- Structure-based theoretical characterisation of the redox-dependent titration behaviour of cytochrome bc1 (2006)
- Cytochrome bc1 is a coenzyme-Q-cytochrome-c-oxidoreductase that represents complex III of the mitochondrial respiratory chain. It spans the inner mitochondrial membrane and uses the free energy of electron transfer from coenzyme Q (CoQ) to cytochrome c to shift protons across the membrane. The chemical energy of reduced CoQ is thus converted into the energy of a proton motive force. The coupling between electron transfer and proton translocation is based on the Q-cycle mechanism. This mechanism comprises two CoQ-binding active sites, that catalyse the oxidation/deprotonation and reduction/protonation of CoQ, respectively. The two sites are located at opposite sides of the membrane. Their intricate chemistry is a matter of ongoing debate. This thesis describes a structure-based theoretical approach to characterise redox-linked protonation state changes in cytochrome bc1, that are at the heart of its catalytic mechanism. The analysis of the titration behaviour of cytochrome bc1 is however complicated by its membrane environment, its high number of titratable sites, their interaction with each other and with redox-active groups, and the conformational variability of the CoQ oxidation site. A series of four studies has prepared the grounds to approach this challenging system. The first article analyses the effect of conformational variability and electrostatic interaction on the titration behaviour of simple model systems (Manuscript A). Based on this study, the coupling between conformational and protonation state changes has been analysed in a relatively simple soluble protein (Manuscript B). The effect of pH on the position of CoQ in a CoQ-reducing transmembrane protein has been quantified as described in Manuscript C. Manuscript D presents a study of the coupling between redox and protonation reactions of the Rieske iron-sulphur cluster, that is one of the prosthetic groups of cytochrome bc1. Based on crystal structures of cytochrome bc1 from Saccharomyces cerevisiae, the protonation probabilities of all titratable groups in the protein have then been calculated, once for its completely oxidised state and once for its completely reduced state. The results allow to identify individual residues that undergo redox-linked protonation state changes. They are consistent with the results of Fourier transform infra-red spectroscopy, and aid in the often complicated interpretation of these experimental data. The calculation results reveal a modified path for proton uptake to the CoQ reduction site (ManuscriptE). In the CoQ oxidation site (Manuscript F), the population of protonation and conformational states is consistent with a previously proposed gating mechanism of the catalytic reaction, that may help to prevent harmful bypass reactions. Coupling between the reduction and protonation of both the Rieske cluster and haem bL highlight the importance of these cofactors in the combined oxidation and deprotonation of CoQ.