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 C(41)XXC(44), C(155)XXC(158), C(173)XXXXC(178). To get information on the role played by these cross-links in determining the structural and functional properties of the protein, we performed site-directed mutagenesis on Cys residues and investigated the changes in folding, stability and functional features of the mutants and analysed the results with computational analysis. The reductase activity of SsPDO and its mutants was evaluated by insulin and thioredoxin reductase assays also coupled with peroxiredoxin Bcp1 of S. solfataricus. The three-dimensional model of SsPDO was constructed and correlated with circular dichroism data and functional results. Biochemical analysis indicated a key function for the redox site constituted by Cys155 and Cys158. To discriminate between the role of the two cysteine residues, each cysteine was mutagenized and the behaviour of the single mutants was investigated elucidating the basis of the electron-shuffling mechanism for SsPDO. Finally, cysteine pK values were calculated and the accessible surface for the cysteine side chains in the reduced form was measured, showing higher reactivity and solvent exposure for Cys155.
Limauro
Danila
D
Dipartamento Biologia Strutturale e Funzionale, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cinthia, 80126 Naples.
Saviano
Michele
M
Galdi
Ilaria
I
Rossi
Mosè
M
Bartolucci
Simonetta
S
Pedone
Emilia
E
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D011490:Protein Disulfide Reductase (Glutathione); D003545:Cysteine