Thioredoxin constitutes the prototype of the thiol‐disulfide oxidoreductase family. These enzymes contain an active‐site disulfide bridge with the consensus sequence Cys‐Xaa‐Xaa‐Cys. The more N‐terminal active‐site cysteine is generally a strong nucleophile with an abnormal low pK a value. In contrast, the more C‐terminal cysteine is buried and only little is known about its effective pK a during catalysis of disulfide exchange reactions. Here we have analyzed the pK a values of the active‐site thiols in wild type thioredoxin and a 400‐fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively 13Cβ‐Cys‐labeled proteins. We find that the effective pK a of the buried cysteine (pK b) of the variant is increased, while the pK a of the more N‐terminal cysteine (pK N) is decreased relative to the corresponding pK a values in the wild type. We propose two empirical models which exclusively require the knowledge of pK N to predict the redox properties of thiol‐disulfide oxidoreductases with reasonable accuracy.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a‐domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild‐type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a‐domain could complement DsbA deficiency in a DsbB‐dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.

The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa1-Xaa2-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for thein vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (−270 to −195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivocharacterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys‐Xaa‐Xaa‐Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000‐fold difference in the equilibrium constant (Keq) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa‐Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild‐type, with the order wild‐type < PDI‐type < DsbA‐type < glutaredoxin‐type. The most oxidizing, glutaredoxin‐like variant has a 420‐fold decreased value of Keq, corresponding to an increase in redox potential by 75 mV. While oxidized wild‐type thioredoxin is more stable than the reduced form (ΔΔGox/red = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH‐dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active‐site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild‐type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin‐like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured ΔΔGox/red values and Cys32 pKa values was only qualitative.

New measurements on (CaCl2 + rH2O) in the temperature range 329 to 559 K have revealed the existence of a hydrate having r = 13. This hydrate was characterized as to composition, X-ray diffraction pattern, and enthalpy of solution in water. For comparison, enthalpies of solution of the monohydrate, dihydrate, and anhydrous CaCl2 were determined.

For ecological as well as economic optimization of highway deicing operations, one must know the relative efficiencies of the two agents most commonly used--calcium chloride and rock salt. Because data on this subject are in conflict, new measurements have been made of the extent to which anhydrous calcium chloride pellets and typical Michigan rock salt undercut a sheet of 0.3175-cm-thick (1/8-in.-thick) ice bonded to a concrete block. The extent to which undercutting (the breaking of the bond between ice and concrete) occurred as a function of time and temperature was followed by adding a dye to the chemical, which caused the brine formed by melting ice to flouresce under ultraviolet light. Commercial anhydrous calcium chloride pellets have a relatively narrow particle size distribution and are very uniform in action; reproducible data could be obtained. However, the action of rock salt was found to be strongly affected by wide variability in size and purity of individual particles. After the action of two particle sizes of pure fused sodium chloride was measured, a reasonable estimate could be made of the average action of a typical commercial Michigan rock salt. The results are presented as a table of the quantity of chemical per unit area required to completely undercut the ice sheet as a function of time and temperature. Rock salt is about equal to calcium chloride above -3.89 deg C (25 deg F) for 1 h. As temperatures are lowered and times are shortened, increasingly larger relative quantities of rock salt are required. This must be considered an ecological concern.