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Biochim Biophys Acta. 2010 Dec;1804(12):2213-21. doi: 10.1016/j.bbapap.2010.09.005. Epub 2010 Sep 16.

Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX.

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Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798-7348, USA; Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.


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 (E(m)) 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 E(m) 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 E(m) value of approximately -260 mV 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Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis.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 E(m) value of -260 mV 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 E(m) value of approximately -260 mV at 20°C. The fact that these two E(m) 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.

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