Reduction potentials of protein disulfides and catalysis of glutathionylation and deglutathionylation by glutaredoxin enzymes

Abstract

Glutaredoxins (Grxs) are a class of GSH (glutathione)-dependent thiol–disulfide oxidoreductase enzymes. They use the cellular redox buffer GSSG (glutathione disulfide)/GSH directly to catalyze these exchange reactions. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely dithiol Grx(SH)2, mixed disulfide Grx(SH)(SSG) and oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential . The values for the redox couple Grx(SS)/Grx(SH)2 are available, but a recent estimate differs by over 100 mV from the literature values. No estimates are available for for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH)2 + GSH). This work determined both and for two representative Grx enzymes, Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. The classic method of acid ‘quenching’ was demonstrated to shift the thiol–disulfide redox equilibria. Both enzymes exhibit an (vs. SHE) at a pH of 7.0. Their values (−213 and −230 mV for EcGrx1 and HsGrx1, respectively) are slightly less negative than that () of the redox buffer GSSG/2GSH. Both and vary with log [GSH], but the former more sensitively by a factor of 2. This confers dual catalytic functions to a Grx enzyme as either an oxidase at low [GSH] or as a reductase at high [GSH]. Consequently, these enzymes can participate efficiently in either glutathionylation or deglutathionylation. The catalysis is demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site.