Graf, Simone Sandra; Hong, Sangjin; Müller, Philipp; Gennis, Robert; von Ballmoos, Christoph (2021). Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli. Scientific reports, 11(1), p. 21234. Springer Nature 10.1038/s41598-021-00651-6
|
Text
s41598-021-00651-6.pdf - Published Version Available under License Creative Commons: Attribution (CC-BY). Download (1MB) | Preview |
Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.
Item Type: |
Journal Article (Original Article) |
|---|---|
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, Müller, Philipp Isaak, von Ballmoos, Christoph |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
ISSN: |
2045-2322 |
Publisher: |
Springer Nature |
Language: |
English |
Submitter: |
Christina Schüpbach |
Date Deposited: |
01 Feb 2022 14:19 |
Last Modified: |
05 Dec 2022 16:03 |
Publisher DOI: |
10.1038/s41598-021-00651-6 |
PubMed ID: |
34707181 |
BORIS DOI: |
10.48350/164337 |
URI: |
https://boris.unibe.ch/id/eprint/164337 |
Actions (login required)
 |
Edit item |