Escherichia coli glutaredoxin (MW 9700) catalyzes intracellular redox reactions utilizing a disulfide/dithiol enzymatic mechanism involving the active-site residues -Cys-Pro-Tyr-Cys-. It is functionally related to the thioredoxin family and is expected to share similar three-dimensional structure [Eklund, H., Cambillau, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We constructed an overexpression system in which production of glutaredoxin is controlled by temperature-sensitive expression of the phage T7 promoter. In addition to glutaredoxin, a second gene product is observed; this species, which we call glutaredoxin N, is glutaredoxin extended by the sequence Met-Arg-Arg-Glu-Ile- at the N terminus. We have begun characterization of the structure and stability of the oxidized and reduced forms of glutaredoxin (grx-S2 and grx-(SH)2, respectively). Secondary structure calculated from CD data agrees with that predicted from the three-dimensional model of Eklund et al. The cooperative denaturation reactions of oxidized and reduced glutaredoxin were measured in temperature-induced and guanidine hydrochloride induced unfolding experiments. Surprisingly, oxidized and reduced glutaredoxins are very similar in stability. In heat-induced denaturation, monitored by CD, Tm is 55 and 57 degrees C for oxidized and reduced, respectively. In GuHCl denaturation, monitored by fluorescence, the midpoint denaturant concentrations are 2 M for both oxidized and reduced. It follows that the redox potentials of the disulfide bond are similar in unfolded and folded glutaredoxin. This is unexpected because in E. coli thioredoxin the oxidized form is far more stable than the reduced [Kelley, R.F., Shalongo, W., Jagannadham, M.V., & Stellwagen, E. (1987) Biochemistry 26, 1406-1411] and the redox potential of folded thioredoxin is significantly more negative than that of unfolded thioredoxin [Lin, T.-Y., & Kim, P. (1989) Biochemistry 28, 5282-5287].