Mutation of cysteine 254 facilitates the conformational changes accompanying the interconversion of persulfide-substituted and persulfide- free rhodanese

T. A. Islam, D. M. Miller-Martini, P. M. Horowitz

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15 Scopus citations

Abstract

Mutants of rhodanese (EC 2.8.1.1) that substitute Ser for Cys-254 and/or Cys-263, i.e. C254S, C263S, and the double mutant C254/263S, were compared with the wild-type enzyme to test the hypothesis that the persulfide formation to give sulfur-substituted rhodanese (ES) during catalysis can exert a stabilizing influence on the global structure of the protein. All the ES forms were quite refractory toward limited tryptic digestion. Extensive digestion was observed only in the persulfide-free forms (E) of mutants having substitutions of Ser for Cys-254 (C254/263S and C254S). Limited proteolysis of C254/263S produced a species on SDS gels at approximately 21 kDA that was proteolyzed further to give a slightly smaller product. Amino acid sequencing, together with mass spectrometry, indicated that these two components had the same N-terminal sequence as wild-type rhodanese and that they resulted from cleavage in the C-terminal domain, first at residues Arg- 186 or Arg-182 and subsequently at Arg-175 or Lys-174. In addition to increased proteolytic susceptibility, the conversion from ES to E of C254/263S was accompanied by increased hydrophobic exposure and increased quenching of the intrinsic protein fluorescence by acrylamide. The substitution of Ser for Cys-254, within the helix suggested to be involved in stabilizing the persulfide bond in ES, seems to promote the formation of flexible conformations of sulfur-free rhodanese. In keeping with this model, C254/263S shows a marked increase in the hysteresis that is observed in rhodanese catalysis at pH 7 and high ionic strength. These results can be understood in terms of a model, suggesting that there is mutual stabilization of the protein structure and the persulfide bond in the ES form of rhodanese.

Original languageEnglish (US)
Pages (from-to)7908-7913
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number11
StatePublished - 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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