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Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis.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°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.
Kim
Sang Gon
SG
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.
Chi
Yong Hun
YH
Lee
Jong-Sun
JS
Schlesinger
Sara Rae
SR
Zabet-Moghaddam
Masoud
M
Chung
Jung-Sung
JS
Knaff
David B
DB
Kim
Sun Tae
ST
Lee
Sang Yeol
SY
Kim
Sung-Kun
SK
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism
D029681:Arabidopsis Proteins; C504764:AtTDX protein, Arabidopsis; D004220:Disulfides; D014050:Toluene; D012694:Serine; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine; C004848:dithiol