Co-reconstitution of respiratory chain enzymes and characterisation of Escherichia coli cytochrome b561

Biner, Olivier Felix (2018). Co-reconstitution of respiratory chain enzymes and characterisation of Escherichia coli cytochrome b561 (Unpublished). (Dissertation, University of Bern, Faculty of Science of the University of Bern)

Text (PhD thesis Olivier Biner) PhD Thesis_150518_small.pdf - Submitted Version Restricted to registered users only Available under License BORIS Standard License. Download (7MB) | Request a copy

Oxidative phosphorylation is the central energy transforming metabolic process that ultimately leads to production of ATP, the universal energy currency of the cell. In mitochondria, four membrane-bound and redox active enzymes (complexes I to IV) interact to convert electron transfer reactions into an electrochemical proton gradient, which is dissipated by the ATP synthase to form ATP. Although the structures and mechanisms of all respiratory enzymes are well understood, it is not possible to carry out the whole reaction series in vitro using purified proteins reconstituted into a model membrane system. One of the aims of this PhD thesis is to develop methodologies to construct an artificial respiratory chain. It has been shown that using a detergent-mediated approach, it is possible to co-reconstitute two membrane proteins. However, it is unlikely that a one-pot method works for the co-reconstitution of all five respiratory enzymes. Therefore, we set out to first purify and reconstitute the respiratory enzymes into single liposome populations and then to fuse the corresponding proteoliposomes. Fusion of proteoliposomes was achieved by incorporating oppositely charged lipids into separate liposome populations followed by mixing of the two populations, which led to spontaneous membrane protein co-reconstitution. This approach is about five times faster and simpler than the SNARE-mediated fusion methods described earlier. Charge-mediated fusion could further be applied to insert membrane proteins into both giant unilamellar vesicles and inverted membrane vesicles. Our fusion strategy should allow for construction of an artificial respiratory chain in follow up projects, a hallmark towards a synthetic mitochondrion.
In a second project, the role of the protein cytochrome b561 in the Escherichia coli aerobic respiratory chain was explored. First studies about cytochrome b561 were conducted already in 1984, which hypothesized that cytochrome b561 is a respiratory enzyme, but its biochemical function has never been established. Using detergent-solubilized cytochrome b561, we observed that the protein can transfer electrons from superoxide to ubiquinone in a diffusion-limited reaction, thus eliminating this deleterious reactive oxygen species. Measurements in proteoliposomes and inverted membrane vesicles showed that the protein mainly reacts with superoxide produced at the membrane. Our measurements indicate that there might exist distinct superoxide pools in a cell, one in the cytoplasm and another at the membrane. This important finding is of general interest to the cellular reactive oxygen species metabolism and deserves further investigation. In summary, cytochrome b561 is the first ever reported membrane-bound superoxide scavenger and the first known enzyme that transfers electrons directly from superoxide to quinones. We have classified this new family of enzymes as superoxide oxidases. While we now know this reaction takes place in vitro, the in vivo function of cytochrome b561 has yet to be validated and future work will focus on understanding the physiological role of this enzyme.

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