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Investigation of the NADH/NAD(+) ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox
Zitatschlüssel ISI:000390626500010
Autor Tejwani, Vijay and Schmitt, Franz-Josef and Wilkening, Svea and Zebger, Ingo and Horch, Marius and Lenz, Oliver and Friedrich, Thomas
Seiten 86-94
Jahr 2017
ISSN 0005-2728
DOI 10.1016/j.bbabio.2016.11.001
Adresse PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS
Journal BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Jahrgang 1858
Nummer 1
Monat JAN
Verlag ELSEVIER SCIENCE BV
Zusammenfassung Ralstonia eutropha is a hydrogen-oxidizing (''Knallgas'') bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H-2-driven production of biodegradable polymers and hydrocarbons. H-2 oxidation by R. eutropha takes place in the presence of O-2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H-2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H-2 oxidation with the reduction of NAD(+) to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD(+) pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD(+)] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD(+)] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD(+)] ratios represents a novel and sensitive tool to determine the redox state of cells. (C) 2016 Elsevier B.V. All rights reserved.
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