Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.