Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Resistance to arsenate conferred on Escherichia coii by the are operon of piasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenite. A genetic anaiysis was performed to identify the source of reducing potentiai in vivo, in addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and giutaredoxin, respectiveiy, had no effect on arsenate resistance. Aithough resistance required the arsC gene, the rate of reduction of arsenate to arsenite was nearly the same in ceiis iacking the ars operon. in strains deficient in giutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When giutathione was supplied exogenously to such mutants, resistance was restored only to ceiis expressing the ars operon, and oniy such ceiis had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high ievei resistance, physicai coupiing of the ArsC reaction to efflux of the resuiting arsenite is hypothesised.

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The properties of human erythrocyte S-adenosyl-L-methionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6) were studied with respect to the role of S-adenosylmethionine in transmethylation reactions. Kinetic values obtained with both a cytosolic and a 350-fold purified preparation of enzyme were compared with measured intracellular concentrations of substrates and products. This analysis revealed that effective regulation of enzyme activity and product concentration can occur through feedback inhibition by S-adenosylmethionine (Ki = 2.0-2.9 microM; the endogenous concentration is 3.5 microM). This enzyme can be distinguished from S-adenosylmethionine synthetases found in other tissues and appears to be specialized for its role in erythrocyte methyl group metabolism, especially with regard to protein carboxyl methyl-transfer reactions.

Cytochrome d terminal oxidase mutants were isolated by using hydroxylamine mutagenesis of pNG2, a pBR322-derived plasmid containing the wild-type cyd operon. The mutagenized plasmid was transformed into a cyo cyd recA strain, and the transformants were screened for the inability to confer aerobic growth on nonfermentable carbon sources. Western blot analysis and visible-light spectroscopy were performed to characterize three independent mutants grown both aerobically and anaerobically. The mutational variants of the cytochrome d complex were stabilized under anaerobic growth conditions. All three mutations perturb the b595 and d heme components of the complex. These mutations were mapped and sequenced and are shown to be located in the N-terminal third of subunit II of the cytochrome d complex. It is proposed that the N terminus of subunit II may interact with subunit I to form an interface that binds the b595 and d heme centers.

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