Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420

Department of Microbiology, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands Correspondence Jan T. Keltjens J.Keltjens{at}science.ru.nl Coenzyme F 420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F 420 -dependent hydrogenase. The standard free-energy change at pH 7 of F 420 reduction was determined to be –15 kJ mol –1 , irrespective of the temperature (25–65 °C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F 420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F 420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F 420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F 420 is a useful probe to assess in situ hydrogen concentrations in H 2 -metabolizing methanogens. Abbreviations: , hydrogen partial pressure; pH i , intracellular pH Present address: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.