Identification and characterization of Formate dehydrogenases (FDHs) from Bacillus subtilis

The Mo/W-bisPGD Formate Dehydrogenases (FDHs) family interplays in several metabolic pathways ranging from carbon fixation to energy harvesting owing to their reaction with a wide variety of redox partners. Here, we have unveiled two non-canonical FDHs in Bacillus subtilis which are organized into two-subunit complexes, as shown by mass spectrometry, with unique features, ForCE1 and ForCE2. The ForC catalytic subunit interacts with an unprecedented partner subunit ForE and its amino acid sequence within the active site deviates from the consensus residues typically associated to FDH activity, with an histidine residue naturally substituted by a glutamine. The ForE essential subunit mediates the utilization of menaquinone as electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency EPR experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature consistent with the characteristic Mo-bisPGD cofactor of this enzyme family. Altogether, this work enlarges our representation of the FDH family by deciphering a non-canonical FDH, which differs both in terms of architecture, amino acid conservation around the Mo cofactor and reactivity.