Thioredoxin (Trx) and glutaredoxin (Grx) are small (9-12 kDa) intracellular disulfidereducing enzymes. They were orginally described as hydrogen donors for ribonucleotidereductase (RR), the enzyme that catalyzes the conversion of ribonucleotides todeoxyribonucleotides. A fundamental difference between Trx and Grx is that Trx is reducedby 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-mixeddisulfide 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 mutantsshowed a large (25-fold) increase of RR levels, and the presence of a glutathione-dependenthydrogen donor system. Two novel glutaredoxins (Grx2 and Grx3) were purified tohomogeneity. 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 thehydrogen donor activity of Grx3, provides a credible explanation for the viability of thedouble 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 adetermination of the secondary structure by NMR. This established that Grxl and Grx3are closely related 9 kDa proteins with 33% sequence identity and similar folds. The twoproteins shared similar activities as reductants of GSH-mixed disulfides, but Grx3 had amuch 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 involveonly the N-terminal of the two active site cysteine residues, as determined by theconstruction of active site mutants (CPYS) of Grx1 and Grx3. The reduced forms of both Grxl and Grx3 had similar propensities to form a mixeddisulfide 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-mixeddisulfide reductants. The subsequent formation of the active site disulfide (K2) is muchmore favoured in Grxl, and provides an explanation for the different abilities to serve asdisulfide reductants. This also suggested a difference in redox potential between Grxl andGrx3, resulting from a difference in the relative stabilities of the oxidized forms of the twoproteins. A novel method for determinations of redox potentials was developed. Applicationof this technique allowed the determination of the standard state redox potentials for Grx1and Grx3 to -198 and -233, respectively. This difference of 35 mV, could be furthercorroborated by applying the linkage between the stability of the disulfide bond (i.e., redoxpotential) and the difference in conformational stability between the oxidized and reducedforms 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 animportant determinant of the redox potential for several members of the thioredoxinsuperfamily. The 35 mV difference in redox potential between Grx1 and Grx3 demonstratesthe importance of other factors. In the case of Grxl and Grx3, the difference in redoxpotential originates from a difference in the relative stabilities of the oxidized forms of theproteins, relative to the reduced forms (which have similar stabilities).Stockholm 1996 ISBN 91-628-2094-
