F: Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein

Abstract Escherichia coli can perform two modes of for-mate metabolism. Under respiratory conditions, two peri-plasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmem-brane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO2 and H2. The respira-tory formate dehydrogenases are redox enzymes that com-prise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transfer-ring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine trans-port (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. PuriW-cation of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation