TY - JOUR
T1 - Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX
AU - Kim, Sang Gon
AU - Chi, Yong Hun
AU - Lee, Jong Sun
AU - Schlesinger, Sara Rae
AU - Zabet-Moghaddam, Masoud
AU - Chung, Jung Sung
AU - Knaff, David B.
AU - Kim, Sun Tae
AU - Lee, Sang Yeol
AU - Kim, Sung Kun
N1 - Funding Information:
This work was funded by grants from the Young Investigator Development Program at Baylor University (to S.-K.K), from Ministry of Education, Science, and Technology/Korea Science and Engineering Foundation Grant R15-2003-012-01001-0 for the Environmental Biotechnology National Core Research Center , and from the Robert A. Welch Foundation ( D-0710 to D.B.K). We are grateful to Prof. Jean-Pierre Jacquot at Université Henri Poincaré for donation of AtNTRB.
PY - 2010/12
Y1 - 2010/12
N2 - 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Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis.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.
AB - 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.
KW - Methoxyl PEG maleimide
KW - Redox titrations
KW - Tetratricopeptide-repeating domain
KW - Thioredoxin-motif
UR - http://www.scopus.com/inward/record.url?scp=77957965148&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2010.09.005
DO - 10.1016/j.bbapap.2010.09.005
M3 - Article
C2 - 20849982
AN - SCOPUS:77957965148
SN - 1570-9639
VL - 1804
SP - 2213
EP - 2221
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 12
ER -