Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX

Sang Gon Kim, Yong Hun Chi, Jong Sun Lee, Sara Rae Schlesinger, Masoud Zabet-Moghaddam, Jung Sung Chung, David B. Knaff, Sun Tae Kim, Sang Yeol Lee, Sung Kun Kim

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10 Scopus citations

Abstract

AtTDX is an enzyme present in Arabidopsis thaliana which is composed of two domains, a thioredoxin (Trx)-motif containing domain and a tetratricopeptide (TPR)-repeat domain. This enzyme has been shown to function as both a thioredoxin and a chaperone. The midpoint potential (Em) of AtTDX was determined by redox titrations using the thiol-specific modifiers, monobromobimane (mBBr) and mal-PEG. A NADPH/Trx reductase (NTR) system was used both to validate these Em determination methods and to demonstrate that AtTDX is an electron-accepting substrate for NTR. Titrations of full-length AtTDX revealed the presence of a single two-electron couple with an Em value of approximately -260mV at pH 7.0. The two cysteines present in a typical, conserved Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis. These replacements (i.e., C304S, C307S, and C304S/C307S) resulted in a complete loss of the redox process detected using either the mBBr or mal-PEG method to monitor disulfide/dithiol redox couples. This result supports the conclusion that the couple with an Em value of -260mV is a disulfide/dithiol couple involving Cys304 and Cys307. Redox titrations for the separately-expressed Trx-motif containing C-domain also revealed the presence of a single two-electron couple with an Em value of approximately -260mV at 20°C. The fact that these two Em values are identical, provides additional support for assignment of the redox couple to a disulfide/dithiol involving C304 and C307. It was found that, while the disulfide/dithiol redox chemistry of AtTDX was not affected by increasing the temperature to 40°C, no redox transitions were observed at 50°C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60°C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays.

Original languageEnglish
Pages (from-to)2213-2221
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1804
Issue number12
DOIs
StatePublished - Dec 2010

Keywords

  • Methoxyl PEG maleimide
  • Redox titrations
  • Tetratricopeptide-repeating domain
  • Thioredoxin-motif

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    Kim, S. G., Chi, Y. H., Lee, J. S., Schlesinger, S. R., Zabet-Moghaddam, M., Chung, J. S., Knaff, D. B., Kim, S. T., Lee, S. Y., & Kim, S. K. (2010). Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX. Biochimica et Biophysica Acta - Proteins and Proteomics, 1804(12), 2213-2221. https://doi.org/10.1016/j.bbapap.2010.09.005