The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.Hydrogen and formate are electron donors frequently used by anaerobic microorganisms. Metabolism of hydrogen and formate is often highly interlinked in many bacteria that can oxidize both compounds. This is exemplified in the fermentative metabolism of the enterobacterium Escherichia coli where up to one third of the carbon from glucose is converted to formate; formate is then disproportionated to H2 and CO2 [1-3]. Formate can be metabolized by three membrane-associated, molybd