Mixed Disulfide with Glutathione as an Intermediate in the Reaction Catalyzed by Glutathione Reductase from Yeast and as a Major Form of the Enzyme in the Cell

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH. The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction. The nascent dithiol of two-electron reduced enzyme (EH2) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction. In addition to the nascent dithiol composed of Cys45 and Cys50, the enzyme contains an acid catalyst, His456, having a pK a of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reoxidation of EH2 by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfide with EH2 is stoichiometric in the absence of an excess of glutathione. The equilibrium position among Eox, MDS, and EH2 is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5. The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (Eox) at pH 6.2. Its spectrum can be distinguished from that of EH2 by a slightly lower thiolate (Cys50)−FAD charge-transfer absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.