Hydrogen formation from glycolate driven by reversed electron transport in membrane vesicles of a syntrophic glycolate‐oxidizing bacterium

Oxidation of glycolate to 2 CO 2 and 3 H 2 (Δ G °′=+36 kJ/mol glycolate) by the proton‐reducing, glycolate‐fermenting partner bacterium of a syntrophic coculture (strain FlGlyM) depends on a low hydrogen partial pressure ( p H2 ). The first reaction, glycolate oxidation to glyoxylate ( E °′=–92 mV) with protons as electron acceptors ( E °′=–414 mV), is in equilibrium only at a p H2 of 1 μPa which cannot be maintained by the syntrophic partner bacterium Methanospirillum hungatei ; energy therefore needs to be spent to drive this reaction. Glycolate dehydrogenase activity (0.3–0.96 U · mg protein −1 ) was detected which reduced various artificial electron acceptors such as benzyl viologen, methylene blue, dichloroindophenol, K 3 [Fe(CN) 6 ], and water‐soluble quinones. Fractionation of crude cell extract of the glycolate‐fermenting bacterium revealed that glycolate dehydrogenase, hydrogenase, and proton‐translocating ATPase were membrane‐bound. Menaquinones were found as potential electron carriers. Everted membrane vesicles of the glycolate‐fermenting bacterium catalyzed ATP‐dependent H 2 formation from glycolate (30–307 nmol H 2 · min −1 · mg protein −1 ). Protonophores, inhibitors of proton‐translocating ATPase, and the quinone analog antimycin A inhibited H 2 formation from glycolate, indicating the involvement of proton‐motive force to drive the endergonic oxidation of glycolate to glyoxylate with concomitant H 2 release. This is the first demonstration of a reversed electron transport in syntrophic interspecies hydrogen transfer.