Graf, Simone; von Ballmoos, Christoph; Brzezinksi, Peter (4 June 2017). Proton pumping bo3 oxidase from Vitreoscilla (Unpublished). In: Gordon Research Conference Bioenergetics. Proctor Academy Andover NH. 04.06.2017-09.06.2017.
Membrane bound heme-copper oxidases reduce oxygen to water, using the released energy for vectorial proton transport across the membrane. Both reactions contribute to the generation of the proton motive force by electrogenic proton and electron transfer reactions across the membrane. These reactions are tightly coupled and precisely orchestrated to ensure energy conservation and suppression of Reactive Oxygen Species (ROS) production. Experimental discrimination between protons required for oxygen reduction and pumped protons is typically not possible. Therefore, a terminal oxidase pumping a different ion than protons would provide an elegant system to study mechanistic aspects of these intricate pumps. The group of Webster described a sodium pumping quinol oxidase in the bacterium Vitreoscilla, but no further studies have been described with this enzyme.
Our aim was thus to isolate, purify and analyse the properties of the Vitriocilla quinol oxidase to see, if it could be used a model system to elucidate the pumping mechanism using single turnover measurements with the flow-flash technique. We successfully cloned, heterologously expressed and purified the enzyme in E. coli. To our disappointment, we could neither observe sodium dependency of the enzymatic activity nor sodium pumping of the protein in inverted membrane vesicles, purified protein in detergent, or the enzyme reconstituted into proteoliposomes. Instead, we conducted several experiments that directly show proton pumping by the Vitreoscilla cytochrome bo, as compared with the related enzyme of E. coli.
Item Type: |
Conference or Workshop Item (Poster) |
|---|---|
Creator: |
Graf, Simone |
Division/Institute: |
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
Graduate School: |
Graduate School for Cellular and Biomedical Sciences (GCB) |
UniBE Contributor: |
Graf, Simone Sandra, von Ballmoos, Christoph |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
Language: |
English |
Submitter: |
Simone Graf |
Date Deposited: |
02 Aug 2017 13:41 |
Last Modified: |
02 Mar 2023 23:29 |
URI: |
https://boris.unibe.ch/id/eprint/101348 |
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