Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two r edox active cysteine residues in the sequence -C-11-P-Y-C-14-H-. The H-1 NM R resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is obser ved at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cvs-ll t hiolate, which is shown to have an abnormally low pK(a) value. A hydrogen b ond would also agree,vith activity data of Grx3 active site mutants. Furthe rmore, the activity is reduced in a Grx3 H15V mutant, indicating electrosta tic contributions to the stabilization of the Cys-ll thiolate, (C) 1999 Fed eration of European Biochemical Societies.