Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif

Walter H. Watson, Jan Pohl, William "Bill" Montfort, Olga Stuchlik, Matthew S. Reed, Garth Powis, Dean P. Jones

Research output: Contribution to journalArticle

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Abstract

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E 0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an α helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trxl activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.

Original languageEnglish (US)
Pages (from-to)33408-33415
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number35
DOIs
StatePublished - Aug 29 2003

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Thioredoxins
Disulfides
Oxidation-Reduction
Catalytic Domain
Thioredoxin-Disulfide Reductase
Mass spectrometry
Cysteine
Diamide
Oxidation
Oxidative stress
Ionization
Desorption
Proteins
Tandem Mass Spectrometry
dithiol
Lasers
Oxidative Stress
Western Blotting
Substrates

ASJC Scopus subject areas

  • Biochemistry

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Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. / Watson, Walter H.; Pohl, Jan; Montfort, William "Bill"; Stuchlik, Olga; Reed, Matthew S.; Powis, Garth; Jones, Dean P.

In: Journal of Biological Chemistry, Vol. 278, No. 35, 29.08.2003, p. 33408-33415.

Research output: Contribution to journalArticle

Watson, Walter H. ; Pohl, Jan ; Montfort, William "Bill" ; Stuchlik, Olga ; Reed, Matthew S. ; Powis, Garth ; Jones, Dean P. / Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 35. pp. 33408-33415.
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