Effect of glutaredoxin and protein disulfide isomerase on the glutathione-dependent folding of ribonuclease A.

Abstract

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase A (rd-RNase A) and mixed disulfide between glutathione and reduced RNase A derivative (GS-RNase A) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ESMS) time-course analysis and recovery of the native catalytic activity. The ESMS analysis permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding of rd-RNase A and GS-RNase A was also performed in the presence of glutaredoxin (Grx) and/or protein disulfide isomerase (PDI). All the analyses indicate a pathway of sequential reactions in the formation of native RNase A. First, the reduced protein reacts with a single glutathione molecule to form a mixed disulfide which then evolves to an intramolecular S-S bond via thiol-disulfide exchange. Only at this stage, the intermediate containing one intramolecular S-S reacts with a further glutathione molecule, reiterating the process. An analogous mechanism occurs in the refolding of GS-RNase A. The structural analysis of the intermediates formed during the refolding of RNase A showed for the first time that Grx is actually able to catalyze both formation and reduction of mixed disulfides involving glutatione. In both refolding processes, starting from either rd-RNase A or GS-RNase A, Grx displays a significant catalysis at the early stages of the process. Addition of PDI led to a net catalysis of the entire process without appearing to alter the refolding pathway. In the presence of both Grx and PDI, the two enzymes showed a synergistic activity either starting from rd-RNase A, as previously reported [Lundström, J., and Holmgren, A. (1995) J. Biol. Chem. 270, 7822-7828], or starting from GS-RNase A. Present data suggest that the synergistic effect can be explained assuming that Grx actually facilitates PDI action by catalyzing formation or reduction of mixed disulfides. The mixed disulfides are then rapidly converted into intramolecular disulfides in the presence of PDI. These steps are repeated sequentially throughout the whole refolding, resulting in an immediate formation of fully oxidized species even at the very beginning of the reaction. Finally, a Grx mutant, C14S Grx, in which one of the active site cysteine residues (Cys14) had been replaced by serine, had a similar effect on the distribution of folding intermediates, compared to the wild-type protein, thus demonstrating that Grx acts by a monothiol mechanism either in the reduction or in the oxidation step.