Redox Potentials for Yeast, Escherichia c oli and Human Glutathione Reductase Relative to the NAD+/NADH Redox Couple:  Enzyme Forms Active in Catalysis

The flavoenzyme glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide, yielding two molecules of glutathione. The oxidation−reduction potentials, Eox/EH2 (two-electron reduced enzyme), for yeast, Escherichia coli, and human glutathione reductase have been determined between pH 6.0 and 9.8 relative to the nonphysiological substrate couple NAD+/NADH and were found to be −237, −243, and −227 mV (±5 mV) at pH 7.0 and 20 °C, respectively. The potential as a function of pH demonstrated slopes of −51, −45, and −42 mV/pH unit, respectively, at low pH and −37, −31, and −34 mV/pH unit, respectively, at high pH. The change in slope indicated pK a values of 7.4, 8.5, and 7.6, respectively. The slopes indicate that two protons are associated with the two-electron reduction of Eox at low pH and that only one proton is involved with the two-electron reduction of Eox at high pH, provided that the effects of nearby titratable residues are considered in the data analysis. The influence of four such groups, Cys50, Cys45, His456‘, and either Tyr107 or the flavin-(N3), has been included (residue numbering refers to the yeast sequence). The enzyme loses activity upon deprotonation of the acid−base catalyst at high pH. Since the pK a ascribed to the EH2-to-EH- ionization is lower than the pK a of the acid−base catalyst, both the EH2 and EH- forms of glutathione reductase must be catalytically active, in contrast to the closely related enzyme lipoamide dehydrogenase, for which only EH2 is active.