Escherichia coli is able to encode four [Ni-Fe] hydrogenases (Hyd); three of which are described and characterized well. All Hyd enzymes are reversible and can work in H2 uptake (oxidizing) or producing mode depending on pH and carbon source . In the present study we propose that H2 cycling and the proton motive force (pmf) are linked together. This is possible through Hyd enzymes which form H2 cycling and via the proton F0F1-ATPase, main generator of pmf during fermentation. This relationship is pH dependent, and it is working mainly at pH 7.5 and pH 5.5.
To confirm, H2 producing or uptake activity by Hyd enzymes, H+ transport and pmf generation by whole cells and ATPase activity of membrane vesicles were determined. Importantly, H+ cycling through F0 to H2 cycling by Hyd enzymes or its reverse is forming redox path to neutralize the fermentation acids and to equilibrate the cytoplasmatic pH. If H2 cycling is disturbed by deletions of genes for Hyd enzymes, a change in pmf generation is detected, especially at extreme of pHs. The findings of pmf generation and ATPase activity with hypF mutant, lacked all Hyd enzymes, point out the the role of Hyd enzymes.These results suggest that E. coli Hyd enzymes forming H2 cycling have a vital role in generating the overall pmf besides the F0F1-ATPase.