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Biochim Biophys Acta Bioenerg. 2017 Jan;1858(1):86-94. doi: 10.1016/j.bbabio.2016.11.001. Epub 2016 Nov 3.

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

Author information

1
SUNY Polytechnic Institute, College of Nanoscale Science and Engineering, 257 Fuller Road, Albany, NY, 12203, U.S.A.
2
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
3
Technische Universität Berlin, Institut für Chemie, PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany. Electronic address: friedrich@chem.tu-berlin.de.

Abstract

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. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 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.

KEYWORDS:

Decay-associated spectra; Fluorescence lifetime; Fluorescence sensor protein; Hydrogenase; NADH:NAD(+) ratio; Ralstonia eutropha

PMID:
27816420
DOI:
10.1016/j.bbabio.2016.11.001
[Indexed for MEDLINE]
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