Characterization of the reduction of selenate and tellurite by nitrate reductases

Abstract

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V(max) and K(m) were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 micromol x min(-1) x mg(-1) and 0.12 mM were obtained for V(max) and K(m), respectively, whereas the V(max) values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.

FIG. 1

Nondenaturing electrophoresis of soluble extracts and membranes of R. sphaeroides f. sp. denitrificans wild type and nap mutant MS523, P. denitrificans, P. pantotrophus, and R. eutropha. Gels were stained for nitrate reductase (lanes A), tellurite reductase (lanes B), or selenate reductase (lanes C) activity with dithionite-reduced methyl viologen or benzyl viologen.

FIG. 2

(A) Silver-stained sodium dodecyl sulfate-polyacrylamide gel. Lane 1, periplasmic extract of R. sphaeroides (8 μg of protein); lane 2, His-tagged purified Nap (0.5 μg of protein). MW, molecular weight. (B) Nondenaturing electrophoresis of His-tagged purified Nap. Gels were stained for reductase activity with dithionite-reduced methyl viologen. Lane 1, nitrate reductase activity (1 μg of protein); lane 2, tellurite reductase activity (25 μg of protein); lane 3, selenate reductase activity (25 μg of protein); lane 4, gel stained with Coomassie brillant blue (5 μg of protein).

FIG. 3

Lineweaver-Burk plots: kinetics of reduction of nitrate, tellurite, and selenate by purified Nap when methyl viologen is the electron donor. V_max is expressed in micromoles of substrate reduced per minute per milligram of enzyme. The correlation coefficients for the nitrate, tellurite, and selenate plots are 0.92, 0.88, and 0.88, respectively.