GLUTAREDOXIN ACCELERATES GLUTATHIONE-DEPENDENT FOLDING OF REDUCED RIBONUCLEASE-A TOGETHER WITH PROTEIN DISULFIDE-ISOMERASE

Glutaredoxin (Grx) contains a redox-active disulfide and catalyzed thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 mu M reduced and denatured ribonuclease RNase-(SH)(8) in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S-2)(4), a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 mu M) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'(0) of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO32-), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.