Electrochemical study of proton translocation function of hydrogenase 3

The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied. The extent of acidification was dependent on the amount of formate added. An average H+/formate ratio of 1.3 was obtained. The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone. Inverted vesicles of E. coli were found to translocate protons upon oxidation of formate at pH 6.5. The extent of alkalization was also dependent on the amount of formate added. The maximum H+/formate ratio for this reaction was close to 0.6. Formate oxidation by inverted vesicles from E. coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. It was supposed that the hydrogenase 3 (hyc) component of E. coli formate hydrogen lyase is responsible for the translocation of protons at low pH.