Hydrogen oxidation by Fdh-N and Fdh-O is dependent on the accessory proteins FdhD and FdhE The fdoGHI operon encoding Fdh-O is flanked by fdhD and fdhE, both of which encode accessory enzymes required for the synthesis

of active Fdh enzymes [22, 23]. To demonstrate the dependence of the H2-oxidizing activities of both Fdhs on FdhD and FdhE, individual mutants lacking either the fdhD or the fdhE gene were analyzed under the same conditions as described above for the wild type and fdoG and fdnG mutants. All three activities were absolutely dependent on both FdhD and FdhE (Figure 4). Complementation experiments revealed that while FdhD on a plasmid fully complemented the fdhD mutation, plasmid-encoded FdhE only partially complemented

the fdhE mutation. Discussion We demonstrate here Selumetinib in vivo that both of the respiratory formate dehydrogenases Fdh-N and Fdh-O have hydrogen-oxidizing enzyme activity. Together with the three characterized [NiFe]-hydrogenases, these are the only two enzymes in E. coli crude extracts that had this activity. These results suggest that the Fdh-N and Fdh-O enzymes show a degree of non-specificity with regard to the electron donor they can use. Notably, formate and dihydrogen (CO2/formate, Eo’ = -432 mV [24]) and (H+/hydrogen, Eo’ = -414 mV) are both strong reductants. Previous studies have demonstrated that E. coli can couple hydrogen oxidation Rucaparib mouse to nitrate reduction and Hyd-1 and Hyd-2 participate in this process [25]. However, attempts to demonstrate significant hydrogen-dependent nitrate reduction in the absence of Hyd-1 and Hyd-2 did not deliver reproducible Selonsertib in vivo levels of hydrogen oxidation, presumably due to the limited

hydrogen-oxidizing Staurosporine datasheet activity of Fdh-N and Fdh-O. Nevertheless, the findings reported here might have physiological relevance in other microorganisms. For example, enzymes with subunits orthologous to FdnG are found in the obligate dehalorespiring and hydrogen-oxidizing Dehalococcoides spp., e.g. strain CBDB1, and have an associated subunit with similarity to hydrogenase membrane-anchoring subunits [26]. Rather than having a selenocysteinyl residue in their presumptive active site they have a seryl residue. It is established that in E. coli replacement of selenocysteine with serine abolishes the formate-oxidizing activity of Fdh-H [27]. Moreover, it is also clear that Dehalococcoides strain CBDB1 cannot use formate as a substrate, suggesting that this formate dehydrogenase-like enzyme might have another function. One possibility based on the findings presented here might be that it accepts H2 as substrate. As both Fdh enzymes are selenium-dependent, impaired co-translational insertion of selenocysteine prevented synthesis of either enzyme and concomitantly abolished the [NiFe]-hydrogenase-independent H2: BV oxidoreductase activity.