Glutaredoxins : structure, function and mechanism

Abstract

Thioredoxin (Trx) and glutaredoxin (Grx) are small (9-12 kDa) intracellular disulfide reducing enzymes. They were originally described as hydrogen donors for ribonucleotide reductase (RR), the enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. A fundamental difference between Trx and Grx is that Trx is reduced by a specific flavoenzyme, whereas Grx is reduced by the ubiquitous tripeptide glutathione (GSH). In addition to being a hydrogen donor for RR, Grx has a general GSH-mixed disulfide oxidoreductase activity. A third hydrogen donor system RR must be present in E.coli, since mutants lacking both Trx and Grx are viable. Characterization of such mutants showed a large (25-fold) increase of RR levels, and the presence of a glutathione-dependent hydrogen donor system. Two novel glutaredoxins (Grx2 and Grx3) were purified to homogeneity. Grx3 was shown to be an active, albeit inefficient hydrogen donor for RR, whereas Grx2 was inactive. The combination of the increased levels of RR and the hydrogen donor activity of Grx3, provides a credible explanation for the viability of the double mutant. Grx2 and Grx3 showed active sites typical of glutaredoxins, Cys-Pro-Tyr-Cys, but Grx2 had an atypical molecular size of 27 kDa.

The primary structure of Grx3 was determined by amino acid sequence analysis. Cloning and overexpression of the gene for Grx3 enabled preparation of protein for a determination of the secondary structure by NMR. This established that Grxl and Grx3 are closely related 9 kDa proteins with 33% sequence identity and similar folds. The two proteins shared similar activities as reductants of GSH-mixed disulfides, but Grx3 had a much lower ability than Grxl to serve as a reductant of regular disulfide substrates, e.g., RRor insulin disulfides. The reduction of GSH-mixed disulfide substrates was found to involve only the N-terminal of the two active site cysteine residues, as determined by the construction of active site mutants (CPYS) of Grx1 and Grx3.

The reduced forms of both Grxl and Grx3 had similar propensities to form a mixed disulfide with glutathione, described by the similar values for the equilibrium constant K1 - a result which is compatible with the similar activities of the two enzymes as GSH-mixed disulfide reductants. The subsequent formation of the active site disulfide (K2) is much more favoured in Grxl, and provides an explanation for the different abilities to serve as disulfide reductants. This also suggested a difference in redox potential between Grxl and Grx3, resulting from a difference in the relative stabilities of the oxidized forms of the two proteins.

A novel method for determinations of redox potentials was developed. Application of this technique allowed the determination of the standard state redox potentials for Grx1 and Grx3 to -198 and -233, respectively. This difference of 35 mV, could be further corroborated by applying the linkage between the stability of the disulfide bond (i.e., redox potential) and the difference in conformational stability between the oxidized and reduced forms of the proteins. Grxl, the better disulfide reductant, was more stable (1.0 kcal/mol) in its oxidized form than its reduced form. The reverse situation was the case for Grx3, where the reduced form was more stable (0.78 kcal/mol).

Several studies have shown that the sequence of the active site tetrapeptide is an important determinant of the redox potential for several members of the thioredoxin superfamily. The 35 mV difference in redox potential between Grx1 and Grx3 demonstrates the importance of other factors. In the case of Grxl and Grx3, the difference in redox potential originates from a difference in the relative stabilities of the oxidized forms of the proteins, relative to the reduced forms (which have similar stabilities).