The Involvement of the Thioredoxin System in the Reduction of Methionine Sulfoxide and Sulfate*

Abstract Earlier work by others had established the occurrence of two complex enzyme systems in yeast which catalyze the reductions of methionine sulfoxide and sulfate, respectively. We now show that thioredoxin and thioredoxin reductase from yeast can function as hydrogen carriers in both types of reactions and it is suggested that they form part of both enzyme systems.


DEAE-cellulose
was prepared according to Peterson and Sober (7). Sephadex of different grades was purchased from Pharma-* This work wits supported by grants to P. Reichard cia, Sweden. Glucose 6-phosphate dehydrogenase from yeast was a product of British Drug Houses, Ltd., England.
%Sodium sulfate (10 mCi per mmole) was purchased from the Radiochemical Centre, Amersham, England. It was diluted with nonlabeled Na2S04 to give the desired specific activity. All other reagents were of analytical grade and were obtained from commercial sources. Tris buffers were prepared by neutralization of the free base with HCl.
%S-PAPS' was prepared enzymatically essentially as described by Wilson et al. (2) with the sulfate-activating enzyme complex (Fraction III) of Robbins and Lipman (8). Isolation of PAPS was carried out after adsorption to charcoal.
For this purpose 10 g of charcoal (Norite) were added to the solution after the removal of KC104 (cf. Reference 8). After 1 hour, the charcoal was recovered by filtration and washed with water. The adsorbed nucleotides were then eluted with 150 ml of ethanolic ammonia (9), the solution was evaporated to dryness in a vacuum, and the residue was dissolved in a small volume of 0.01 M Tris, pH 7.5-0.001 M EDTA.
This solution contained a total of 390 pmoles of adenine compounds, as judged from its ultraviolet absorption, and a total of 22 pmoles of 35S-PAPS.
The latter value was calculated from the s5S present in the solution.
Essentially all isotope was present in PAPS, as judged from paper electrophoresis.
This solution was used directly in our experiments, since the presence of other adenine nucleotides did not influence the interpretation of the results. Thioredoxin (10) and thioredoxin reductase (11) from Escherichia coli were preparations available in this laboratory. The corresponding proteins from yeast were prepared as described in the preceding paper (6). Yeast thioredoxin was a mixture of thioredoxin I and II with an estimated purity of about 90%. A few crucial experiments were done with the separated thioredoxins, which showed no appreciable differences in their activities. Yeast thioredoxin reductase was the purest preparation available.
Methionine sulfoxide reductase (enzyme III) was purified from an extract of baker's yeast as described by Black et al. (1)  (2 x 120 cm), equilibrated with the same buffer. The main protein peak appearing at the void volume contained all Enzyme III activity.
This fraction was precipitated with solid ammonium sulfate (610 mg per ml). The precipitate was dissolved in a small volume of 0.05 M Tris, pH 7.5-0.001 M EDTA and filtered through a short column of Sephadex G-25, equilibrated with the same buffer. The final product (17 mg) had a specific activity (1) of 0.032 unit per mg.
PAPS reductase (Enzyme B) was purified from acetone powder extracts of yeast as described by Wilson et al. (2), including the alcohol precipitation step. The material (1800 mg) was then dissolved in 49 ml of 0.02 M phosphate buffer, pH 7.0, and the solution was adsorbed to a column of DEAE-cellulose (11 x 5 cm) equilibrated with 0.02 M phosphate buffer, pH 7.0. The column was first eluted with 900 ml of 0.15 M phosphate buffer, pH 7.0. The active enzyme, free of thioredoxin and thioredoxin reductase, was then eluted with 230 ml of 0.5 M phosphate buffer, pH 7.0. An aliquot of this solution (30 ml) was precipitated with solid ammonium sulfate (472 mg per ml), dissolved in a small volume of 0.05 M Tris buffer (pH 7.0), and freed from ammonium sulfate by filtration through a short column of Sephadex G-50, equilibrated with the same buffer. This solution was the source of the purified PAPS reductase used in the experiments.
Assay of Sulfate Reduction-The method of Wilson et al.
(2) was used. The substrate was either %S-sulfate (crude extracts) or %-PAPS (purified enzyme). In both cases the reaction was carried out under argon (pressure, 25 Torr) in the main compartment of a Warburg vessel in the presence of carrier sulfite (2). After 60.min incubation at 37" the reaction was stopped by the addition of acid from the side arm and volatile % and carrier sulfite were recovered in NaOH in the center well. Aliquots were used for the determination of radioactivity and sulfite (12). The recovery of carrier sulfite was i n most cases better than 80%. The detailed conditions for the incubations are given in the legends to Figs. 1 to 4.
Assay of Methionine Sulfoxide Reduction-The reaction was followed by measuring the decrease in the absorbance at 340 mp due to the oxidation of NADPH during the reduction of methionine sulfoxide (1). In some cases the formation of methionine was measured directly with a Beckman Model 120B amino acid analyzer.
For further details the legend to Fig. 5 should be consulted.
Protein Determinations-During the early stages of purification of Enzyme III and Enzyme B a turbidimetric method (13) was used. With the purified enzymes we assumed a value of 1.0 for the absorbance at 280 mp of a solution containing 1 mg of protein per ml.

Enzymatic Reduction of Sulfate
Crude Extract-When the reduction of sulfate was studied with increasing amounts of crude yeast extract the S-shaped curve of Fig. 1 was observed.
The nonlinearity indicated the operation of a multienzyme system, confirming the findings of earlier workers (2, 3).
The addition of increasing amounts of yeast thioredoxin strongly stimulated the formation of sulfite from sulfate, while addition of thioredoxin reductase showed little effect (Fig. 2). These experiments gave the first indication that thioredoxin might participate as a hydrogen donor during the reduction of sulfate.
During purification, the sulfate-activating enzymes had been removed (2) and %S-labeled PAPS was therefore used as substrate.
Part A of Fig. 3 shows the dependence of sulfite formation on the amount of PAPS reductase added. In these experiments, thioredoxin and thioredoxin reductase were added to the incubation mixtures.
Part B of Fig. 3 shows the dependence of sulfite formation on the amount of thioredoxin added (in the presence of thioredoxin reductase) and Part C gives the results with increasing amounts of thioredoxin reductase (in the presence of thioredoxin).
With the purified PAPS reductasa the enzymatic formation of sulfite was clearly completely dependent on the simultaneous presence of thioredoxin and its reductase.
In these experiments the amount of sulfite formed during the At different time intervals 0.2 ml was removed from the incubation mixture and Itsed for the assays.

2-hour incubation
period did not exceed the amount of thioredoxin added to the system. In Fig. 4  were defined by their capacity to substitute for the E. coli thioredoxin system during ribonucleotide reduction. The results presented in this paper clearly establish that thioredoxin and its reductase can function as hydrogen carriers in two further processes: the reduction of sulfate and that of methionine sulfoxide.
In both cases, earlier work (l-3) had implicated the participation of proteins with properties similar to thioredoxin and thioredoxin reductase in the reaction sequences.
It appears that the thioredoxin system may have a quite general function in several reductions. This is schematically depicted in Fig. 6. There, reduced thioredoxin serves as the hydrogen donor in the reduction of an oxidized substrate which may be a ribonucleotide, sulfate, or methionine sulfoxide; each reaction is catalyzed by a specific reductase.
In a formal sense, the three reactions involve the removal of an oxygen from the substrate and the thioredoxin system functions by making available the reducing power of NADPH for this purpose.
It seems possible that other reductases than those implicated above may be coupled to the thioredoxin system. The capacity of thioredoxin-(SH)2 to reduce different disulfides (1, 5) should also be recalled. This latter reaction is nonenzymatic.
Finally, we want to point out that our experiments do not definitely establish that the thioredoxin system in viva participates during the reduction of sulfate or methionine sulfoxide.
Decisive proof for this would require a detailed characterization of mutants blocked in the reaction sequences. With respect to the reduction of sulfate, such mutants appear already to be available (14).