Investigation of the NADH/NAD+ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.
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 H2-driven production of biodegradable polymers and hydrocarbons. H2oxidation by R. eutropha takes place in the presence of O2and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2utilization. The so-called soluble hydrogenase (SH) couples reversibly H2oxidation 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.
SUNY Polytechnic Institute, College of Nanoscale Science and Engineering, 257 Fuller Road, Albany, NY, 12203, U.S.A.
Vijay
V
Tejwani
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Franz-Josef
FJ
Schmitt
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Svea
S
Wilkening
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Ingo
I
Zebger
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Marius
M
Horch
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Oliver
O
Lenz
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany. Electronic address: friedrich@chem.tu-berlin.de.
Thomas
T
Friedrich
D015374:Biosensing Techniques / Q000379:methods; D020118:Cupriavidus necator / Q000378:metabolism*; D005453:Fluorescence; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction
D009243:NAD; D006859:Hydrogen; D006864:Hydrogenase
Decay-associated spectra; Fluorescence lifetime; Fluorescence sensor protein; Hydrogenase; NADH:NAD(+) ratio; Ralstonia eutropha