Mutations of noncatalytic sulfhydryl groups influence the stability, folding, and oxidative susceptibility of rhodanese

David M. Miller-Martini, John M. Chirgwin, Paul M. Horowitz

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Mutants of rhodanese (EC 2.8.1.1) which substitute serine residues for each of the 4 cysteine residues have been studied to determine the roles of cysteines in the structure and function of the enzyme. The proteins compared in these studies were: the wild-type, C63S, C247S, C254S, C263S, C254S/C263S, and C63S/C254S/C263S. These current studies show that cysteine 247 is the only cysteine required for the activity of the enzyme. Although the other sulfhydryl groups do not participate in sulfur transfer, mutations of the noncatalytic cysteines result in the destabilization of the native structure of the enzyme. All the active proteins had similar kinetic parameters. Mutants substituting cysteine 254, compared with the other species, were: (a) more resistant than wild-type to inactivation by dithiothreitol, (b) more readily reactivated following oxidative inactivation, and (c) found to adopt conformations that show increased exposure of hydrophobic surfaces following removal of the transferable sulfur. On the other hand, cysteine to serine substitutions had very little effect on: (a) the rates of oxidative inactivation, (b) the increased fluorescence following the removal of transferable sulfur, or (c) the effectiveness of spontaneous refolding after urea denaturation. Forms of rhodanese that were formerly considered to be irreversibly oxidized can be reactivated if the protein is denatured in urea before reductants are used. It is proposed that these forms differ from reversibly oxidized states due to the inaccessibility of intramolecular disulfides to reductants and not to the formation of higher oxidation states of the protein.

Original languageEnglish (US)
Pages (from-to)3423-3428
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number5
StatePublished - Feb 4 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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