NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: Implications for the enzymatic mechanism

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein dis ulfide groups and glutathione-containing mixed disulfide groups via an acti ve site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solu tion structure of the Escherichia coli Grx3 mixed disulfide with glutathion e (Grx3-SG) was determined using a C14S mutant which traps this intermediat e in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular be ta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specifi city of reduction observed in Grxs. Molecular details of the structure in r elation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of t he thioredoxin superfamily is presented, which illustrates the highly conse rved intermolecular interactions in this protein family. (C) 1999 Academic Press.