Human Mitochondrial Glutaredoxin Reduces S-Glutathionylated Proteins with High Affinity Accepting Electrons from Either Glutathione or Thioredoxin Reductase*

Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (kcat/Km). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower kcat. Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress.

Thiol groups are central to most redox-sensitive processes in the cell, and their redox state controls cellular processes like growth, differentiation, and apoptosis. The intracellular thiol homeostasis is maintained by the thioredoxin and glutaredoxin systems, which both utilize reducing equivalents from NADPH to reduce protein and low molecular weight disulfides (1, 2). The thioredoxin system is composed of thioredoxin reductase (TrxR), 1 in mammalian cells a selenocysteine-containing, dimeric nicotinamide nucleotide disulfide oxidoreductase (3), which reduces thioredoxin (Trx) (Reaction 1), the major protein disulfide reductase with a large number of functions in mammalian cells (4).
TrxϪ(SH) 2 ϩNADP ϩ REACTION 1 The glutaredoxin system consists of glutathione reductase, a dimeric enzyme with similarities to TrxR, which catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), which in turn reduces glutaredoxin (Grx) (Reactions 2 and 3) (5). Reduced glutaredoxin acts as a disulfide reductase but is active in particular in the reduction of S-glutathionylated substrates (6,7).
Grx-S 2 ϩ 2 GSH 3 GrxϪ(SH) 2 ϩGSSG REACTION 3 Thioredoxins and glutaredoxins share a similar three-dimensional structure, known as the thioredoxin fold (8), and both use two redox-active cysteines in a conserved CXXC active site motif to reduce protein disulfides and low molecular weight disulfides. These reactions are initiated by a nucleophilic attack of the N-terminal CXXC thiolate on the disulfide substrate, resulting in a mixed disulfide intermediate, which subsequently is reduced by the C-terminal CXXC cysteine, releasing the reduced substrate (4). Grx-catalyzed reduction of S-glutathionylated substrates, also called glutathione-mixed disulfides, proceeds by a monothiol pathway, requiring only the N-terminal active site cysteine (Reaction 4) (9). In these reactions a glutathione-glutaredoxin intermediate is formed that is reduced by a second molecule of GSH.

Grx
RSH ϩ GSSG REACTION 4 Complete Trx and the Grx systems in mammalian cells are present both in the cytosol and the mitochondria (10 -13). Whereas the cytosolic TrxR and Trx display high similarity to their mitochondrial counterparts (14 -17), there are surprisingly large differences between the cytosolic and mitochondrial glutaredoxins (12,13). The two Grxs show only 34% sequence identity, and the proteins differ both in size and active site sequence. The 12-kDa cytosolic Grx1 has been extensively studied (18 -29), and three-dimensional structures have been determined for the reduced form and the glutathione-mixed disulfide intermediate (27,30,31). Grx1 is an electron donor for ribonucleotide reductase (5,6,24) and is involved in many different cellular processes like dehydroascorbate reduction (32), actin polymerization (33,34), protection against oxidative stress (35,36), apoptosis after acute cadmium exposure (37), and cellular differentiation (38). The second Grx (Grx2) is due to an alternative splicing mechanism targeted either to the nucleus or to mitochondria. Grx2 has a predicted size of 18 kDa and contains a CSYC active site motif that differs from the Grx consensus CPYC (12,13). The structure of Grx2 has not been determined, but a structural prediction (see Fig. 1) reveals conservation of all major structural features found in Grx1 (12,13). Although not characterized in detail, Grx2 has been shown to be active in a standard assay for glutaredoxins using the GSH-coupled reduction of hydroxyethyl disulfide (HED) or dehydroascorbate with 10-fold lower specific activity than Grx1 (12).
In the present study we characterized Grx2 and revealed enzymatic properties linked to the non-conserved dipeptide sequence in the active site of Grx2. Furthermore Grx2, unlike Grx1 and other Grxs previously characterized, is a substrate for thioredoxin reductase. The unusual properties of the mitochondrial glutaredoxin may reflect an important role in the defense against oxidative stress in an environment with large production of reactive oxygen species and a key role in apoptosis.
Construction of Grx1P23S, Grx2C40S, and Grx2S38P Mutants-The Grx1P23S, Grx2C40S, and Grx2S38P mutants were prepared using the QuikChange site-directed mutagenesis kit according to the manufacturer's recommendations using Pfu polymerase and DpnI. The cDNAs for Grx1 and Grx2 (exons II-IV), subcloned into the pGEM-T vector, were used as templates with two complementary primers containing the desired mutation (Grx1P23S, 5Ј-CATCAAGCCCACCTGCTCTTAC-TGCAGGAGG-3Ј; Grx2C40S, 5Ј-CATCCTGTTCTTACTCTACAATGG-CAAAAAGC-3Ј; Grx2S38P, 5Ј-CTCAAAAACATCCTGTCCGTACTGT-ACAATGGC-3Ј). The constructs were verified by DNA sequence analysis by KI-seq, the core facility unit at the Karolinska Institute, before they were cloned into the NdeI-BamHI sites of the pET15b vector for protein expression.
Purification of TrxR1 and TrxR2-Bovine TrxR1 was purified from calf liver essentially as described earlier (39,40). The mitochondrial TrxR2 was a preparation available in the laboratory, purified from calf liver mitochondria by a procedure similar to that for TrxR1.
Preparation of Glutathionylated RNase (RNase-SG) and Glutathionylated BSA (BSA-SG)-RNase-SG was prepared essentially as described previously (42). BSA-SG was prepared by reduction of the protein for 30 min at 37°C with 5 mM dithiothreitol followed by desalting on a NAP-5 column. The reduced protein was incubated overnight with a 100-fold molar excess of GSSG followed by desalting on a NAP-5 column and dialysis against 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 200 mM NaCl. The amount of bound glutathione was 6 -8 molecules per molecule of RNase and 0.7 molecules per molecule of BSA, quantified by complete reduction of the glutathionylated proteins as described previously (42).
Reducing Activities Using Electrons from Glutathione-Grx activities in the HED assay were determined as described previously (18). Briefly 0.7 mM HED was added to a freshly prepared mixture of 100 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1 mg/ml BSA, 200 M NADPH, 1 mM GSH, 6 g/ml glutathione reductase. After 3 min of preincubation, glutaredoxin was added to the sample cuvettes, and buffer was added to the reference cuvette. The decrease in absorbance at 340 nm was followed using a Shimadzu UV-2100 spectrophotometer. Activity was expressed as mol of NADPH oxidized/min using a molar extinction coefficient of 6200 M Ϫ1 cm Ϫ1 . One unit was defined as the oxidation of one mol of NADPH/min.
The K m value for glutathione was determined with the procedure described above using 0.5-4 mM GSH and 1.25 nM Grx1, 10 nM Grx2, or 5 nM Grx2C40S. Activity and substrate specificity toward glutathionylated substrates were determined using either 0.1-3 mM HED, 0.25-8 M RNase-SG, or 3.1-45 M BSA-SG in a mixture of 100 mM potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM GSH, 0.1 mg/ml BSA, 240 M NADPH, and 6 g/ml yeast glutathione reductase. The reaction was started by the addition of either 20 or 40 nM glutaredoxin to the sample cuvette and an equal amount of buffer to the reference cuvette. Grx activity was determined from the decrease in absorbance at 340 nm. Three independent experiments were performed at each substrate concentration, and the apparent K m and k cat values were calculated by non-linear regression using the program GRAFIT.
Grx2 as Substrate for Thioredoxin Reductase-NADPH-dependent reduction of Grx2 by TrxR was performed in a mixture containing 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 mg/ml BSA, 200 M NADPH, and 10 M Grx2. The reaction was started by addition of either 48 nM bovine TrxR1 or 77 nM bovine TrxR2. The amount of NADPH oxidized was determined from the decreased absorbance at 340 nm. The reaction was compared with the reduction of Grx2 by GSH, performed in the same mixture as above, replacing TrxR with 0.125 mM GSH and 6 g/ml glutathione reductase.
The K m and k cat values of TrxR for Grx2 were determined in a mixture of 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 mg/ml BSA, 200 M NADPH, and 11.2 M RNase-SG (corresponding to 67-90 M glutathione-mixed disulfides). Grx2 (1.2-21 M) was added to the sample cuvettes, and buffer was added to the reference cuvette. The reaction was initiated by addition of 9.2 nM TrxR1 both to the sample and the reference cuvettes, and the activity was determined from the decrease in absorbance at 340 nm. The apparent K m and k cat values were calculated by non-linear regression using the program GRAFIT.
In separate experiments, 3.7 M Grx2 or Grx2C40S was reduced by 47-228 nM TrxR in a mixture containing 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 mg/ml BSA, and 200 M NADPH. The reaction was started by addition of 555 M Cys-SG as substrate, and activity was calculated from the decrease in absorbance at 340 nm.
Reducing Activity of Grx2 Using Electrons from TrxR-Reduction of low molecular weight disulfides by Grx2, independent of GSH, was analyzed in a mixture of 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 mg/ml BSA, 200 M NADPH, and 47 nM TrxR1. Grx2 (6 M) was added to the sample cuvette, and buffer was added to the reference cuvette. Then 250 -1000 M HED or 200 -400 M CoA-disulfide was added to both reference and sample cuvettes. Activity was determined from the decrease in absorbance at 340 nm. Reduction of glutathione-mixed disulfides was analyzed by the same procedure using 0.5 M Grx2, 380 nM TrxR, and 13 M CoA-SG, Cys-SG, or GSSG.

Reduction of Glutathionylated Substrates by Human Grx1
and Grx2-Grx2 displayed a 10-fold lower specific activity compared with Grx1 (12) as determined in the standard glutaredoxin activity assay with HED as a substrate (18). In this "HED assay," glutaredoxin activity is followed as the oxidation of NADPH at 340 nm in a coupled system with 1 mM GSH and glutathione reductase; HED and GSH in the reaction mixture give rise to ␤-ME-SG-mixed disulfide during an initial preincubation for 3 min (18). Grx catalyzes the reduction of ␤-ME-SG by GSH, which is the rate-limiting reaction. To increase sensitivity, reactions are run at a high pH (8.0) with excess HED (0.7 mM). We have analyzed the kinetic properties of Grx1 and Grx2 using two glutathionylated proteins, RNase-SG and BSA-SG, as well as ␤-ME-SG as substrates in the presence of 1 mM GSH (18). The reactions followed the same pH dependence for both Grx1 and Grx2 with a maximum rate at pH 8.5 (data not shown). This is in agreement with the proposed mechanism with the rate-limiting step being the reduction of the Grx-SG intermediate by a second molecule of GSH (6). The kinetic parameters were determined at pH 7 to reduce the spontaneous background reaction in the absence of Grx. The k cat and K m values were determined using non-linear regression, yielding 2 values lower than 10 Ϫ7 . The results of these experiments (summarized in Table I) demonstrate that, although Grx2 catalyzed the reduction of these substrates with a lower rate than Grx1, the apparent affinities of Grx2 were much higher (lower K m ), resulting in slightly higher catalytic efficiencies (k cat /K m ). The highest efficiency was determined for RNase-SG followed by BSA-SG and ␤-ME-SG. Grx2 showed almost the same affinity for BSA-SG and RNase-SG-mixed disulfides (taking into account that RNase contained 6 -8 GSH moieties/molecule), and both K m values were very much lower than those for HED, representing an approximation of the levels of ␤-ME-SG. These results demonstrated that Grx2 is highly specific for glutathione-mixed disulfides and in particular S-glutathionylated proteins.
The apparent K m value for GSH, determined by varying the amount of GSH in the HED assay, was higher for Grx2 compared with Grx1 (Table II) but in the same range as the K m values determined for other glutaredoxins, which are all in the millimolar range (43)(44)(45)(46).
Characterization of Active Site Mutants of Glutaredoxins-To explore the importance of the Pro to Ser exchange in the active sites of Grx2 and Grx1 (Fig. 1), we constructed an active site mutant of each protein, swapping their active sites. We also replaced the C-terminal active site Cys residue of Grx2 by a Ser. All three mutant proteins were purified to homogeneity and characterized.
The Grx2S38P mutant, mimicking the active site in Grx1, significantly increased the specific activity of Grx2 in the HED assay. The specific activity was 2.3 times (76 units/mg of protein) higher compared with the wild type enzyme but still 5 times lower compared with wild type Grx1. The Grx2S38P mutant had lower affinity for both RNase-SG and ␤-ME-SG (higher K m ) but exhibited higher turnover numbers compared with the wild type protein (Table I). Remarkably the corresponding mutant of Grx1, Grx1P23S mimicking the active site in Grx2, showed almost the same low specific activity (35 units/mg of protein) and catalytic properties as Grx2 (Table II). This mutant revealed increased affinity and decreased turnover for RNase-SG and ␤-ME-SG compared with wild type Grx1. These data demonstrate that a Pro to Ser exchange in the active site dipeptide of Grx2 is the major determinant for the affinity for glutathionylated substrates as well as for the k cat .
Replacement of the more C-terminal active site cysteine in Grx2 with a serine residue increased the specific activity in the HED assay by 70%. Although this mutant showed higher turnover numbers in the reduction of glutathionylated substrates, the concomitant increase in K m resulted in an overall lower catalytic efficiency (k cat /K m ) compared with the wild type enzyme. These results demonstrate that Grx2, similar to Grx1, requires only the active site thiol closer to the N terminus to reduce glutathionylated substrates. The K m for GSH was lower for this mutant compared with the wild type enzyme (Table I), also similar to previous observations for monothiol mutants of Grx1 (21,27).
Human Grx2 Is a Substrate for Thioredoxin Reductase-The significant differences between Grx1 and Grx2 in structure and catalytic properties prompted us to analyze whether oxidized Grx2 is substrate for thioredoxin reductase. Since Grx2, like other glutaredoxins, contains a disulfide in the active site after purification and storage (data not shown), we measured the amount of NADPH oxidized in single turnover experiments (Reaction 5).
Grx2Ϫ(SH) 2 ϩNADP ϩ REACTION 5 Surprisingly both the cytosolic and the mitochondrial isoforms of mammalian TrxR were able to reduce human Grx2 (Fig. 2), whereas oxidized human Grx1 was not a substrate for either of the TrxR isoforms, confirming previous results (5). The reduction of Grx2 by TrxR was also coupled to the reduc-

Reduction of glutathionylated substrates by human glutaredoxins and active site mutants
The reaction was performed at pH 7.0 in a mixture containing glutathione reductase, NADPH, GSH, and either RNase-SG, BSA-SG, or HED. The reaction was started by addition of Grx, and the activity was determined from the decrease in absorbance at 340 nm. Three independent experiments were performed at each substrate concentration, and the apparent K m and k cat values were calculated by non-linear regression. For more details see "Experimental Procedures." ND, not determined. tion of RNase-SG (Fig. 3). The efficient reduction of RNase-SG by Grx2 in this reaction demonstrated that Grx2 used reducing equivalents directly from NADPH via TrxR to reduce glutathionylated substrates. We analyzed the kinetics of this reaction, and the K m of TrxR for Grx2 was estimated to be 22 M, and the k cat was estimated to be 822/min, representing about one-third of the k cat of TrxR for the previously established substrate Trx (40).
To investigate the catalytic mechanism of the reduction of Grx2 by TrxR we used the monothiol active site mutant Grx2C40S in a coupled reaction with either RNase-SG or Cys-SG. Surprisingly Grx2C40S was also a substrate for TrxR, yielding higher activity compared with wild type Grx2 (Fig. 4). Together these results demonstrate that TrxR can reduce both the active site disulfide in human Grx2 and the mixed disulfide formed between glutathione and Grx2 (Fig. 5, reactions 3a and  3b).
Reduction of Low Molecular Weight Disulfides Independent of GSH-The new electron transfer pathway via TrxR allowed us to investigate substrates for Grx2 in a non-glutathione-dependent manner. The reduction of Grx2 by TrxR was coupled to the reduction of the physiologically relevant CoA-disulfide, CoA-SG, and GSSG as well as the artificial model disulfide HED. By adjusting the concentration of reagents, apparent second order rate conditions were created under which Grx2 slowly reduced both the CoA-disulfide and HED. However, the second order rate constant for reactions involving the glutathione-mixed disulfides was almost 1000-fold higher (Table III). These results demonstrate that reduced Grx2 can reduce low molecular weight disulfides as expected, although the capacity to reduce S-glutathionylated substrates via a GSH-binding site is the hallmark of a glutaredoxin. They also demonstrated that Grx2 might use reducing equivalents from TrxR to efficiently reduce GSSG creating a potentially important rescue system in mitochondria.

DISCUSSION
The major differences in primary structure between cytosolic Grx1 and mitochondrial Grx2 are also reflected in their catalytic activities as revealed in this study. Thus, mitochondrial Grx2 has a high affinity for S-glutathionylated substrates, and this is to a large extent dependent on the substitution of the Pro residue in the classical CPYC active site sequence by a Ser residue. Quite unexpected was the discovery that Grx2 is a direct substrate for thioredoxin reductase and thus acts as a thioredoxin, being able to accept electrons from both TrxR1 and TrxR2 as well as from GSH. This is a first example of a cellular glutaredoxin being able to cross-react this way. Previous studies have shown that in E. coli, yeast, or mammalian cells there is no cross-reactivity. How can these results be reconciled in the light of the localization of the Grx2 isoforms in mitochondria or the nucleus?
Our results clearly demonstrate that Grx2 reduced glutathionylated substrates with lower rates than Grx1 and E. coli glutaredoxins (42,45,47), but the higher affinity results in the same or even slightly higher catalytic efficiency (k cat /K m ). Recognizing S-glutathionylated proteins in the mitochondria or at the nuclear membrane with high affinity may be important. The Grx2C40S mutant, unable to form an intramolecular disulfide in the active site, confirmed that Grx2 followed the usual glutaredoxin catalytic mechanism in that only the active site thiol closer to the N terminus was required for reduction of S-glutathionylated substrates. The higher k cat observed for this mutant, compared with the wild type enzyme, is similar to earlier observations for the corresponding mutant proteins of Grx1 from both human and pig (21,27) but in contrast to the monothiol mutants of E. coli Grx1 and -3 and phage T4 Grx1, which exhibited significantly decreased activity (9,44,48). The higher turnover has been explained by the absence of the intramolecular side reaction in which the Grx forms an intramolecular disulfide bond releasing GSH (27), although it is not possible to predict this behavior today a priori. Furthermore the active site cysteines have been suggested to form a thiol-thiolate hydrogen bond network, which stabilizes the more N-terminal active site thiolate in the reduced form of the proteins (48 -51). Thus, the higher turnover for the Cys to Ser monothiol mutants could be due to a stronger thiolate-hydroxyl hydrogen bond, which decreases the pK a of the thiol closer to the N terminus, increasing its leaving group ability, a key factor of the rate-limiting step in the reduction of proteinglutathione-mixed disulfides (6, 51). The amino acid sequences were aligned using ClustalX (80). The secondary structure of Grx1 (extracted from the three-dimensional NMR structure, Protein Data Bank code 1JHB) (81) and the predicted secondary structure (ss) of Grx2 are shown above and below the sequences, respectively. The numbering of Grx2 starts at the second Met, representing the common sequence of all known Grx2 isoforms (exons II-IV). The active site sequence is boxed, and identical residues are highlighted.
The dipeptide sequence within the CXXC motif is characteristic for individual members of the thioredoxin superfamily, and its importance has been studied earlier in several mutant variants (51)(52)(53)(54)(55). Previous studies indicate that the proline residue in the CPYC motif favors formation of hydrogen bonds between the more N-terminal cysteine thiolate and the amide protons of the tyrosine and the C-terminal cysteine, which stabilize the thiolate and decrease its pK a value (51). Replacement of this Pro with Ser, as in the active site of Grx2, most likely affects these interactions, changing the pK a of the thiol and thereby the redox properties of the protein (53,55,56). The Grx1P23S and the Grx2S38P mutants clearly demonstrate that a Pro to Ser exchange decreases the activity of the glutaredoxin but increases their affinity for glutathionylated substrates.
Obviously the fact that Grx2 is a substrate for thioredoxin reductase should make it particularly suited to remove and control the level of S-glutathionylated proteins and low molecular weight disulfides following overproduction of reactive oxygen species during oxidative stress. The standard redox potential for GSH/GSSG is Ϫ0.24 V (57), and the actual potential within the cell depends upon the intracellular or intraorganellar concentrations of GSH and GSSG. At sufficiently oxidizing conditions, either by efflux of GSH (58) or consumption, the active site in glutaredoxin will not be reduced by GSH, and if this takes place in mitochondria the electron transport chain may be blocked, leading to apoptosis (59). The direct reduction via thioredoxin reductase may then rescue the redox balance. The only previous example of this interaction comes from virusinfected E. coli where bacteriophage-coded T4 Grx1 (4) is an equally good substrate for E. coli TrxR1 as the host Trx1. The specificity of the electron transport to the virus ribonucleotide reductase suggested that the mechanism aided in making the virus production of deoxynucleotides maximally effective (60). The K m value of TrxR1 for Grx2 was higher than the K m determined for Trx (2.5 M) but lower than the K m reported for protein disulfide isomerase (35 M) (40,61). Since TrxR1 and TrxR2 from various species reveal almost identical kinetic parameters (14 -16), one can expect human mitochondrial TrxR2 to reduce Grx2 with an efficiency similar to that of the bovine TrxR1. Activity with the monothiol active site mutant Grx2C40S, in a coupled assay with Cys-SG, demonstrated further that TrxR can support both monothiol and dithiol reactions catalyzed by Grx2. This suggests that the glutaredoxin-GSH-mixed disulfide is attacked by the selenolate in TrxR, forming a mixed disulfide intermediate, releasing the glutathione moiety (Fig. 5, reaction 3b), revealing a new glutathioneindependent path of electron flow from NADPH to various Grx substrates. This electron pathway is similar to the one suggested for the thioredoxin glutathione reductase enzymes where the monothiol or dithiol Grx domain accepts electrons from either the TrxR domain or from GSH (62-64). Using TrxR as an electron donor, we found that Grx2 could reduce both hydroxyethyl disulfide and CoA-disulfide apart from their glutathione-mixed disulfides. Although reduction of the intramolecular disulfides was less efficient, it clearly demonstrates that Grx2 is able to reduce low molecular weight disulfides independently of GSH. CoA, which preferentially is compartmentalized to mitochondria and known to undergo thiol/disulfide exchange reactions with GSH and GSSG both in vitro and in vivo, could be a potential substrate for Grx2 in vivo (65)(66)(67). The redox state of CoA certainly affects many fundamental processes such as the oxidation and activation of fatty acids, suggested to have key roles in the mitochondrial membrane permeability transition (68), and the activity of the pyruvate and ␣-ketoglutarate dehydrogenase complexes. The relevance of glutathionylation/deglutathionylation reactions has been intensively discussed in the last years (69 -73). It has been suggested to be not only a way to protect critical thiols from irreversible oxidation but also as a common mechanism for redox regulation (74 -76). Recent studies imply an important role also in mitochondria since the mitochondrial redox status modulates the activity of the ␣-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH-ubiquinone oxidoreductase (complex I) (59,77,78). The fact that cytosolic Grx1 facilitates reactivation of the inactivated enzymes suggests a central role for glutaredoxins during oxidative conditions. Under normal conditions the reduction of Grx by GSH is fast. However, the rate of reduction falls substantially as the [GSH]/ [GSSG] decreases as discussed above or when the intracellular pH falls (43). During these conditions, the importance of alternative pathways for reduction of Grx becomes evident. The feature of Grx2 to use reducing equivalents from TrxR, whose activity is not sensitive for either changes in the glutathione redox buffer or decreases in the intracellular pH (79), facilitates an efficient backup system that enables reactivation of inactivated enzymes and reduction of GSSG to restore the redox equilibrium (Fig. 6). Together these results suggest an important role for Grx2 in protection and recovery from oxidative stress. In fact, Grx2 may be a key regulator of apoptosis via the mitochondrial checkpoint.

TABLE III
Reduction of low molecular weight disulfides by Grx2 using electrons from TrxR and NADPH The reaction was performed at pH 7.4 in a mixture containing NADPH, TrxR1, and Grx2 and started by addition of the various low molecular weight disulfides. Activity was determined from the decrease in absorbance at 340 nm, and the second order rate constants were determined from three independent experiments. For further details see "Experimental Procedures."