The structure of the respiratory nitrate reductase (NapAB) from Rhodobacter sphaeroides, the periplasmic heterodimeric enzyme responsible for the first step in the denitrification process, has been determined at a resolution of 3.2 A. The di-heme electron transfer small subunit NapB binds to the large subunit with heme II in close proximity to the [4Fe-4S] cluster of NapA. A total of 57 residues at the N- and C-terminal extremities of NapB adopt an extended conformation, embracing the NapA subunit and largely contributing to the total area of 5,900 A(2) buried in the complex. Complex formation was studied further by measuring the variation of the redox potentials of all the cofactors upon binding. The marked effects observed are interpreted in light of the three-dimensional structure and depict a plasticity that contributes to an efficient electron transfer in the complex from the heme I of NapB to the molybdenum catalytic site of NapA.

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 mol ⅐ min ؊1 ⅐ 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.Selenium is part of the amino acid selenocysteine present in numerous enzymes and is essential to all living cells (42). Furthermore, selenium can help prevent cancer and other diseases (11). However, at high concentrations this compound, predominantly in the form of selenate and selenite oxyanions, is toxic and can cause some environmental problems; for example, in the San Joaquin Valley in central California, bird malformations due to selenium have been reported (28). Tellurium is not an essential element and is relatively rare in the environment, but it can be found at high concentrations near waste discharge sites. It is also extremely toxic, and the MIC for Escherichia coli is approximately 2 g of potassium tellurite per ml (3). Nevertheless, some gram-negative organisms are resistant to potassium tellurite (24). Different explanations for resistance have been proposed; these include exclusion, increased efflux, and reduction to the less toxic metallic form. Several genetic determinants have been shown to confer tellurite resistance (15,27,44,45,48,50). Although physiological functions can be attributed to some of these determinants (for example, tpm and tehB, which exhibit homology with methyl transferases [1,8,19] and arsRDABC, which encodes an oxyanion efflux transporter [45]), most of them exhibit no similarity to each other or to the locus encoding any enzyme whose function is known. Therefore, the mechanisms which allow these loci to confer resistance remain largely unknown (46). When reduction occurs, intracellular deposition of tellurium can be observed, and bacteria form black colonies (20, 24). Resistance to selenium oxides is also partially attributed to reduction and accumulation of the red amorphous Se 0 form in the cell (12, 14; M. Bébien, J.-P. Chauvin, J.-M. Adriano, S.Grosse, and A. Verméglio, submitted for publication). For instance, the photosynthetic bacterium Rhodobacte...

BackgroundMembrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.Methodology/Principal FindingsThe expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.Conclusions/SignificanceMost of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.