REDOX POTENTIALS OF GLUTAREDOXINS AND OTHER THIOL-DISULFIDE OXIDOREDUCTASES OF THE THIOREDOXIN SUPERFAMILY DETERMINED BY DIRECT PROTEIN-PROTEIN REDOX EQUILIBRIA

Glutaredoxins belong to the thioredoxin superfamily of structurally si milar thiol-disulfide oxidoreductases catalyzing thiol-disulfide excha nge reactions via reversible oxidation of two active-site cysteine res idues separated by two amino acids (CX1X2C). Standard state redox pote ntial (E-o') values for glutaredoxins are presently unknown, and use o f glutathione/glutathione disulfide (GSH/GSSG) redox buffers for deter mining E-o' resulted in variable levels of GSH mixed disulfides. To ov ercome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduce d forms present in the thiol-disulfide exchange reaction at equilibriu m after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pairwise comparisons of the reducing capacitie s of the proteins and mutant forms. Equilibrium constants from pair-wi se reaction with thioredoxin or its P34H mutant, which have accurately determined E-o' values from their redox equilibrium with NADPH cataly zed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox pote ntials for the Escherichia coli glutaredoxins 1 and 3, which contain i dentical active site sequences CPYC, mere found to be E-o' = -233 and -198 mV, respectively. These values were confirmed independently by us ing the thermodynamic linkage between the stability of the disulfide b ond and the stability of the protein to denaturation, Comparison of ca lculated E-o' values from a number of proteins ranging from -270 mV fo r E, coli Trx to -124 mV for DsbA obtained using this method with thos e determined using glutathione redox buffers provides independent conf irmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equili bria is of general interest as it overcomes errors in determining redo x potentials calculated from large equilibrium constants with the stro ngly reducing NADPH or by accumulating mixed disulfides with GSH.