Wilkening, S., Schmitt, F. J., Lenz, O. et al. (3 more authors) (2019) Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor. Biochimica et Biophysica Acta - Bioenergetics. 148062. ISSN 0005-2728
Abstract
The hydrogen-oxidizing “Knallgas” bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 Elsevier B.V. All rights reserved. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. |
Keywords: | Fluorescence sensor protein,Frex,Light-driven bio-hydrogen production,NAD,NADH,R. eutropha,Redox sensing,Soluble hydrogenase |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) |
Depositing User: | Pure (York) |
Date Deposited: | 16 Sep 2019 08:00 |
Last Modified: | 16 Oct 2024 15:59 |
Published Version: | https://doi.org/10.1016/j.bbabio.2019.148062 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1016/j.bbabio.2019.148062 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:150819 |
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