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PEDS Advance Access published online on November 6, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn061
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Danila Limauro1, Michele Saviano2, Ilaria Galdi1, Mosè Rossi1, Simonetta Bartolucci1,3 and Emilia Pedone2,3

1Dip. Biologia Strutturale e Funzionale, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cinthia, 80126 Naples 2Istituto di Biostrutture e Bioimmagini, C.N.R., Via Mezzocannone 16, 80134 Naples, Italy

3 To whom correspondence should be addressed. E-mail: empedone{at}unina.it (E.P.)/bartoluc{at}unina.it (S.B.)

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO) contains three disulphide bridges linking residues C41XXC44, C155XXC158, C173XXXXC178. 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 mutagenised 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.

Keywords: Sulfolobus solfataricus/protein disulphide oxidoreductase/redox sites/thioredoxin fold/thioredoxin system

Received July 17, 2008; revised October 2, 2008; accepted October 6, 2008.


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