Direct electrochemical analyses of a thermophilic thioredoxin reductase: interplay between conformational change and redox chemistry.
Thioredoxin reductases (TrxRs) are flavin-containing dithioloxidoreductases that couple reduction equivalents from the soluble NAD(P)H pool to the soluble protein thioredoxin (Trx). Previous crystallographic studies of the Escherichia coli enzyme ( ecTrxR) have shown that low molecular weight TrxRs can adopt two distinct conformations: the first (FO) is required for the oxidation of the flavin cofactor and the generation of reduced Trx; the second (FR) is adopted for the reduction of the flavin by NAD(P)H. Here, protein electrochemistry has been used to interrogate the equilibrium between the oxidized and reduced conformations of the ecTrxR and a novel, low molecular weight TrxR from the thermophilic archaeon Thermoplasma acidophilum ( taTrxR) that is characterized structurally and biochemically in the accompanying paper [Hernandez et al. (2008) Biochemistry 47, 9728-9737]. A reversible electrochemical response is observed that reveals a dynamic behavior dependent upon the temperature of the experiment. At low temperatures (283 K) a broad, quasi-reversible electrochemical envelope is observed centered at a value of approximately -300 mV and displaying a peak width of over 150 mV. The voltammetric response sharpens dramatically as the temperature increases, becoming much more reversible (as determined by peak separation and peak width). The overall potential and shape of the voltammetric data indicate that the flavin (FAD/FADH 2) and disulfide/dithiol couples are very close in thermodynamic potentials, and the data are interpreted in terms of the model of two-state conformational change between flavin reducing (FR) and flavin oxidizing (FO) states, where the difference in potential for the flavin and disulfide cofactors must be within 40 mV of one another. In this model, the low temperature peak broadening is interpreted as an indication of a heterogeneous population of TrxR conformations that exist at low temperature; at higher temperatures, FO and FR conformers can rapidly interconvert, and voltammetry reports upon an average potential of the conformations.
Hamill
Michael J
MJ
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Chobot
Sarah E
SE
Hernandez
Hector H
HH
Drennan
Catherine L
CL
Elliott
Sean J
SJ
D004563:Electrochemistry; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D005415:Flavins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase