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

Abstract

BackgroundEscherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.ResultsHere 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.ConclusionsThe 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.

A-Type Carrier Protein ErpA Is Essential for Formation of an Active Formate-Nitrate Respiratory Pathway in Escherichia coli K-12

“…The activities of the highly similar Fdh-N and Fdh-O enzymes cannot be readily distinguished using standard enzyme assays; however, by specific staining using formate and PMS-NBT after nondenaturing PAGE, the Fdh-O enzyme complex can be readily visualized (44). A single active enzyme complex present in extracts from fdnG, narG, sufA, and fnr mutants could be clearly distin- guished and had a similar intensity to that seen in extracts from strain MC4100 (Fig.…”

“…2B). Expression of the fdoGHI operon is independent of the FNR regulator (1,44). In contrast to Nar, however, but like Fdh-N, maturation of Fdh-O is completely dependent on a functional TAT translocon (49), as no enzyme activity could be detected in extracts derived from the tatC mutant JW3815 (Fig.…”

“…Initially, we examined the effects of sufA and iscA mutations individually on Fdh-N and Nar enzyme activities in anaerobically respiring E. coli. Under the assay conditions, Fdh-O contributes only a small percentage to the overall activity, and therefore activity will be referred to as Fdh-N only (44). The sufA mutation (strain CP1223) caused an approximately 5-fold increase in both Nar and Fdh-N activity compared with the activity measured in extracts from strains MC4100 and MG1655 (Table 2).…”

“…A further aerobic enzyme, Fdh-O, highly similar to Fdh-N with regard to physiological function, is also synthesized in the presence of nitrate (1,44). Making use of endogenously generated formate as an electron donor and coupling this to nitrate reduction allow the bacterium to generate a proton gradient using a classical redox-loop chemiosmotic mechanism (8, 21-23, 32, 37) (Fig.…”

Ecology and Biotechnology of Selenium-Respiring Bacteria

“…Acetate oxidation to hydrogen and subsequent conversion of hydrogen and CO 2 to methane through the syntrophic association between acetate-oxidizing bacteria and hydrogenotrophic methanogens form a dominant methanogenic pathway in anaerobic digesters, particularly in the presence of high concentrations of ammonia and volatile fatty acids (17)(18)(19). Selenium in the form of selenocysteine has been identified in the active site of formate dehydrogenases and is important for formate oxidation (20,21). Accumulation of formate, an intermediate of propionate oxidation, triggers a feedback inhibition resulting in propionate accumulation (22), which may lead to process failure in anaerobic digesters.…”