Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor

Publication date: Available online 13 August 2019Source: Biochimica et Biophysica Acta (BBA) - BioenergeticsAuthor(s): S. Wilkening, F.-J. Schmitt, O. Lenz, I. Zebger, M. Horch, T. FriedrichAbstractThe 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 Peredo...
Source: Biochimica et Biophysica Acta (BBA) Bioenergetics - Category: Biochemistry Source Type: research