Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction

The reduction equivalents necessary for the ribonucleotide reductase (RNR)- catalyzed production of deoxyribonucleotides are provided by glutaredoxin ( Grx) or thioredoxin (Trx). The initial location for transfer of reducing eq uivalents to RNR is located at the C terminus of the B1 subunit and involve s the reduction of a disulfide between Cys754 and Cys759. We have used a 25 -mer peptide corresponding to residues 737-761 of RNR B1 (C754-->S) to synt hesize a stable mixed disulfide with Escherichia coli Grx-1 (C14 --> S) res embling the structure of an intermediate in the reaction. The high-resoluti on solution structure of the mixed disulfide has been obtained by NMR with an RMSD of 0.56 Angstrom for all the backbone atoms of the protein and the well-defined portion of the peptide. The binding interactions responsible f or specificity have been identified demonstrating the importance of electro static interactions in this system and providing a rationale for the specif icity of the Grx-RNR interaction. The disulfide is buried in this complex, implying a solely intra-molecular mechanism of reduction in contrast to the previously determined structure of the glutathione complex where the disul fide was exposed; mutagenesis studies have shown the relevance of intermole cular reduction processes. Substantial conformational changes in the helice s of the protein are associated with peptide binding which have significant mechanistic implications for protein disulfide reduction by glutaredoxins. (C) 1999 Academic Press.