Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Abstract

Fermentative hydrogen production by enterobacteria derives from the activity of the formate hydrogenlyase (FHL) complex, which couples formate oxidation to H2 production. The molybdenum-containing formate dehydrogenase and type-4 [NiFe]-hydrogenase together with three iron-sulfur proteins form the soluble domain, which is attached to the membrane by two integral membrane subunits. The FHL complex is phylogenetically related to respiratory complex I, and it is suspected that it has a role in energy conservation similar to the proton-pumping activity of complex I. We monitored the H2-producing activity of FHL in the presence of different concentrations of the protonophore CCCP. We found an inhibition with an apparent EC50 of 31 µM CCCP in the presence of glucose, a higher tolerance towards CCCP when only the oxidizing hydrogenase Hyd-1 was present, but a higher sensitivity when only Hyd-2 was present. The presence of 200 mM monovalent cations reduced the FHL activity by more than 20%. The Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) combined with CCCP completely inhibited H2 production. These results indicate a coupling not only between Na+ transport activity and H2 production activity, but also between the FHL reaction, proton import and cation export.

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli

“…An energy-conserving character of FHL is suggested by sequence homology 19 , and due to the fact that the ATP synthase inhibitor DCCD substantially reduces gas production by E. coli , suggesting a link of FHL to the membrane potential 20 . Studies using inverted membrane vesicles derived from anaerobically grown E. coli reported formate-dependent formation of a transmembrane proton gradient that was eliminated by knockout of hyc genes 21 , although more recent studies have variably supported or questioned an energy-conserving character for FHL 10 , 12 , 22 .…”

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli

“…An energy-conserving character of FHL was proposed on the basis of sequence homology 16,17 , and due to the fact that the ATP synthase inhibitor DCCD substantially reduces gas production by E. coli , suggesting a link of FHL to the membrane potential 18 . Studies in inverted membrane vesicles of anaerobically grown E. coli showed formate-dependent formation of a transmembrane proton gradient that was eliminated by knockout of hyc genes 19 , although more recent studies have variably supported or questioned an energy-conserving character for FHL 11,20,21 .…”