Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX

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Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis.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 degrees 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 degrees C, no redox transitions were observed at 50 degrees C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60 degrees C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays. (C) 2010 Elsevier B.V. All rights reserved.