Gluta redoxin (Grx) was discovered as a small protein that contains two thiol groups and acts as a glutathione (GSH)-dependent hydrogen donor for synthesizing deoxyribonucleotides by Escherichia coli ribonucleotide reductase (1). Glutaredoxin in the oxidized form has a redox-active disulfide bond (Grx-S2) and is reduced to the dithiol form (Grx-SH2) by two molecules of GSH in a thioldisulfide exchange reaction via a GSH mixed-disulfide intermediate (2, 3). Glutaredoxin is also a general GSH-disulfide oxidoreductase that reduces either disulfide bonds by a dithiol mechanism or GSH-mixed disulfides by a monothiol mechanism (2, 3). The glutaredoxin system comprises NADPH, GSH, glutathione reductase, and glutaredoxin. It catalyzes the NADPH- and GSHdependent reduction of disulfides and keeps the inside of the cell reduced.
Glutaredoxins exist in all prokaryotic and eukaryotic cells that contain glutathione, and they are also encoded by the genes of the larger DNA viruses. Glutaredoxins generally have a mass of 9 to 12 kDa, and the active site has the consensus sequence -Cys-Pro-Tyr-Cys- (4). Glutaredoxin in mammalian cells is identical to GSH-homocystine transhydrogenase or thioltransferase. The primary structures of glutaredoxin show only limited sequence similarity to those of thioredoxins, but the three-dimensional structure of glutaredoxin shows that the active site dithiol/disulfide is located at the end of a beta-strand and at the beginning of an alpha-helix, characteristic of the thioredoxin fold. One hallmark of glutaredoxin is a binding site for GSH that is used in both the reduction of Grx-S2 by GSH and for substrate recognition and reduction of GSH-mixed disulfides by Grx-(SH)2 .
Glutaredoxins have numerous functions, such as acting as hydrogen donors for reductive enzymes and transmitting changes in the oxidation/reduction potential of the GSH/GSSG (glutathione disulfide) system for protein activity during thiol redox regulation and signal transduction, or in restoring oxidatively damaged proteins after oxidative stress.
1. Glutaredoxin Functions
Glutaredoxins have been isolated from E. coli (1, 5), yeast (6), plants (7), and mammals (8, 9) and are encoded by T4 bacteriophage and vaccinia virus (10-12). E. coli contains three different glutaredoxins: Grx1, Grx2, and Grx3 (5). Grx1 is a highly efficient hydrogen donor for ribonucleotide reductase, either in the presence of 1 mM DTT or with the more physiological system of 4 mM GSH, NADPH, and excess glutathione reductase, when the apparent Km (Michaelis constant) for Grx1 is 0.13 |iM (2, 3). The apparent turnover number of glutaredoxin as a dithiol hydrogen donor for ribonucleotide reductase is tenfold higher than that of thioredoxin. Glutaredoxin was discovered in an E. coli mutant that lacks thioredoxin (1), and double mutants that lack thioredoxin and glutaredoxin are viable (13). This includes E. coli cells that lack glutathione (gshA-). In combination with a thioredoxin mutation (gshA-, trxA-), such cells very strongly induce (up to 55-fold) Grx1 (14). Grx3 in E. coli has the same active site structure as Grx1, but only 6% of the V-max with ribonucleotide reductase and a 35 mV higher redox potential (E’q) (15, 16). The function of at least Grx1 strongly depends on the GSH-concentration and the GSH:GSSG ratio, because the E’q of the redox-active disulfide bond in glutaredoxin determines whether it is be reduced to the active dithiol (16).
Glutaredoxin functions reducing both sulfate and methionine sulfoxide in E. coli and yeast (17). Synthesis of Grx1 in E. coli is induced via the oxyR transcription factor, and Grx1 controls its activity by reducing a disulfide bond that can be formed in the protein (18). The two glutaredoxins in yeast control the defense against superoxide and hydrogen peroxide stress, respectively, in addition to their roles in deoxyribonucleotide synthesis by ribonucleotide reductase (6).
Phage T4 induces a ribonucleotide reductase upon infection of E. coli, and a T4 glutaredoxin (originally called thioredoxin) is also a specific hydrogen donor for the enzyme that works with GSH as a reducant (19), and a substrate for thioredoxin reductase. T4 ribonucleotide reductase uses E. coli Grx1 as a hydrogen donor, but not thioredoxin (19). However, T4 glutaredoxin is reduced by thioredoxin via thioredoxin reductase, favoring phage-specific DNA synthesis (20).
The transcription factor activity of nuclear factor I is regulated by glutaredoxin in vitro, involving formation of mixed disulfides (21). Glutaredoxin is a direct target of oncogenic Jun protein (22). Other roles for glutaredoxin involve reduction of dehydroascorbic acid (23) and involvement in repairing proteins that have with mixed disulfides after oxidative stress (24) or in arsenite reduction (25).
2. Amino Acid Sequences
With few exceptions [eg, see Grx2 (26)], the glutaredoxins from bacterial viruses to humans are 9- to 12-kDa proteins that have both sequence and structural homology to the members of the thioredoxin superfamily of proteins. Alignment of the amino acid sequences from diverse organisms (Fig. 1) gives a good impression of the sequence variability of these proteins. Unlike the thioredoxins, whose sequences align without gaps of more than one residue, numerous gaps of up to 5 residues are needed, in addition to the relatively long (up to 20 amino acid residues) N- and/or C-terminal extensions in some members. Several residues are strictly conserved among the glutaredoxins. The active site sequence -Cys11-Pro12-Tyr13-Cys14 (E. coli numbering) is the hallmark of the glutaredoxins. The tripeptide fragment that contains the proline residue with the cis peptide bond (Thr58-Val59-Pro60) and the residues Ile69-Gly70-Gly71-Tyr72-Thr73-Asp74 are also strictly conserved. Mammalian glutaredoxins are highly homologous to one another and, in addition to containing both N- and C-terminal extensions, contain additional non-structural cysteine residues that, analogous to the mammalian thioredoxins, might function in regulating glutaredoxin activity (9).
Figure 1. Amino acid sequence alignment of selected glutaredoxins. Sequences were taken from a(9, 32); 6(33); c(34); d( acid sequences were aligned based on the three-dimensional structures of pig (29), T4 (28) and E. coli (Grx1) glutaredox.
3. Mechanisms of Disulfide Reduction
As outlined in Fig. 2, glutaredoxin participates in reducing disulfide bonds in substrates by a dithiol mechanism that involves both thiol groups and a disulfide intermediate. Glutaredoxin also catalyzes monothiol reductions of GSH-mixed disulfides.
Figure 2. Mechanisms of glutaredoxin in GSH-disulfide oxidoreductions. The monothiol mechanism requires only the accessible N-terminal active-site Cys residue. The dithiol mechanism of disulfide reduction (like that in ribonucleotide reductase) requires both Cys residues (27, 35).
4. E. coli Grx Structure
Three-dimensional protein structures of oxidized and reduced E. coli Grx-1 (27), oxidized T4 Grx (28), and oxidized pig Grx (29) have been determined. In addition, the secondary structures and fold topology of reduced E. coli Grx-3 (15) and reduced human Grx (30) have also been reported. These studies confirm the presence of the thioredoxin fold that consists of a central four-stranded mixed b-sheet flanked by one a-helix on each face (Fig. 3). In addition to this core fold, the N- and C-terminal extensions observed in sequence comparisons contain two additional helical segments.
Figure 3. Representative NMR solution strucutre of E. coli Grx1 (C14 S mutant) in a mixed disulfide with GSH via Cys11 (27, 31). (a), Cartoon of the polypeptide backbone with the side chains of Cys11 and Ser14 displayed as sticks. The glutathione ligand is also displayed as a stick model whose bonds connect non-hydrogen atoms. ( b) The molecular surface colored according to the electrostatic potential (dark gray: positive, medium gray: negative, and light gray: uncharged. Residues that interact with GSH are labeled at their approximate positions.
5. Glutaredoxin Mixed-Disulfide Complex with GSH
The intermolecular mixed-disulfide complex between E. coli Grx1 and GSH has been determined by NMR techniques (27). Mutation of the C-terminal active site Cys14 residue in E. coli Grx1 abolishes its activity as a dithiol reductant for ribonucleotide reductase (31), but retains its activity in the monothiol mechanism (Fig. 2). Overall, the polypeptide backbone in the complex is similar to that observed in the structures of the oxidized and reduced forms, also determined by NMR. The covalently attached glutathione is found in a largely extended conformation in a cleft formed by residues from three discontinuous regions of the polypeptide chain (Fig. 3). The intimate relationship between the Grx and glutathione is also confirmed by the fact that the 15 N Tlr relaxation rates of the amide nitrogen atoms of Cys and Gly of the glutathione are similar to those observed for the Grx backbone. The floor of the cleft is made up largely of contributions from Val 59, Pro 60, and Gly 71. The sides of the cleft are formed from Thr 58 on one side and Tyr 13 and Thr 73 on the other.
