Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif.
Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.
Watson
Walter H
WH
Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA. wwatson@emory.edu
Pohl
Jan
J
Montfort
William R
WR
Stuchlik
Olga
O
Reed
Matthew S
MS
Powis
Garth
G
Jones
Dean P
DP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001643:Bridged Bicyclo Compounds; D004220:Disulfides; D008565:Membrane Proteins; D011994:Recombinant Proteins; C491203:TXN protein, human; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase; D014357:Trypsin; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; C028381:monobromobimane