ARCHIVES

OF BIOCHEMISTRY

Vol. 299, No. 1, November

AND

BIOPHYSICS

15, pp. 1933198, 1992

Direct Electrochemistry of Thioredoxins at a Lipid Bilayer-Modified Electrode’ Z. Salamon, Department

F. K. Gleason,’ of Biochemistry,

and Glutathione

and G. Tollin”

University

of Arizona,

Tucson, Arizona 85721

Received June 22, 1992, and in revised form August 5, 1992

By using direct electrochemical analysis we have escoli thioretablished that the reduction of Escherichia doxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 + 0.01, -0.07 + 0.01, and -0.06 -t 0.01 V, whereas the second one has values of -0.48 f 0.01, -0.39 f 0.01, and -0.45 + 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the oneelectron steps, one can calculate a thermodynamic halfwave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with m&allOprOteinS. 62 is92 Academic PEES, h.

i This work was supported in part st,itutes of Health (DK15057 to G.T.) (DCB-9008136-01 to F.K.G.). ’ Permanent address: Department Minnesota, St. Paul, MN 55108. 3 To whom correspondence should

by grants from the National Inand National Science Foundation of Plant

Biology,

be addressed.

0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

University

Thioredoxins are small proteins (M, approximately 12,000) found in all living cells. Their active sites are highly conserved and contain two redox-active cysteine residues with the sequence Cys-Gly-Pro-Cys. The oxidized form (thioredoxin-Sz) contains a disulfide bridge which is reduced to the dithiol form by NADPH and the FAD-containing flavoprotein, thioredoxin reductase. The reduced form (thioredoxin-(SH)J can catalyze reduction of protein disulfide bonds or serve as a reducing agent for ribonucleotide reductase and sulfate and methionine sulfoxide reductions (for reviews, see (l-3)). Both thioredoxin and glutathione can function in a variety of biological systems involving regulation of protein activity by thiol redox control (4). coli The three-dimensional structure of Escherichia thioredoxin-Sz (EcT)~ has been determined by X-ray crystallography to 2.8 A (5) and recently refined to 1.68 A resolution (6). The crystallographic structure shows the active site disulfide ring (comprising the residues CysZ2Gly-Pro-Cysa,) located on the surface of the protein but on the solvent-shielded side of an a-helix. Cys-32 is exposed to solvent, whereas Cys-35 is recessed and interacts with residues in the interior of the molecule. The surface around the active site is flat and contains a hydrophobic region which has been suggested as a main interaction site for other proteins (7, 8). Thioredoxins are distinguished from glutaredoxins (which are also small redox-active proteins with one active site dithiol per molecule) by the way in which they are reduced. Whereas thioredoxins are reduced only by thioredoxin reductase, glutaredoxins are reduced by glutathione (9). When E. coli is infected by bacteriophage T4, a phage-encoded thioredoxin (T4T) and ribonucleotide reductase are produced (10, 11). There is relatively little sequence homology between T4T and EcT. Although the active site is located at the amino-terminal end of a helix in both cases, T4T differs from EcT in its disulfide loop

of 4 Abbreviations used: EcT, E. coli thioredoxin; T4T, thioredoxin bacteriophage T4; GSSG, glutathione; PC, phosphatidylcholine.

from 193

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sequence, which is Cys-Val-Tyr-Cys (12,13). Functionally, T4T is a hybrid of EcT and E. coli glutaredoxin in that it can be reduced by both glutathione and thioredoxin reductase (14). Structurally, the T4 protein resembles other glutaredoxins and thioltransferases and it has been proposed that it be renamed T4 glutaredoxin (15). The two-electron redox potentials of the disulfideldithiol couple for both T4T and EcT have been calculated from equilibrium measurements of the thioredoxin reductase reaction using the value for the NADPH/NADP+ couple; values of -0.23 and -0.26 V, respectively, have been obtained (16, 17). The two-electron redox potential of glutathione (GSSG/2GSH) has been determined to be -0.25 V, also by an equilibrium method using the NADP+/ NADPH couple (18). We have measured the redox potentials of these three disulfides using cyclic voltammetry at a self-assembled lipid bilayer-modified gold electrode. It is well known that proteins generally adsorb strongly from aqueous solution onto metal electrode surfaces, and that the adsorbed protein film can play an important role in the electrode reactions of these materials (19, 20). Adsorbed proteins usually do not exhibit reversible electrochemical behavior, and in many cases the redox reactions of proteins in the solution phase are inhibited (21). These considerations become especially important when proteins with disulfide and thiol redox-active groups are involved. Organosulfur derivatives are known to coordinate strongly to many metal surfaces (22). This is the main reason that direct electrochemical measurements of organic disulfides are often complex; in addition, they may be influenced by chemical reactions of the substrate or reduction products with the electrode material. This is very pronounced at mercury electrodes, e.g., in classical polarography, but is also evident at other metal electrodes. Measurements with glassy carbon electrodes are the least complicated, but even with these, the rate constant for heterogeneous electron transfer is low, and the average values of cathodic and anodic peak potentials in cyclic voltammetry are not the thermodynamic redox potentials (23). In order to establish reversible electron transfer reactions at the electrode-solution interface, surface modifiers are often used (24). In previous studies from this laboratory, we have shown that a gold electrode modified with a self-assembled bilayer lipid membrane can serve as a good, biocompatible, electrochemical system promoting rapid and reversible electron transfer between metalloproteins and metal electrodes. The detailed study of the electrochemistry of mitochondrial cytochrome c, plant ferredoxins and spinach plastocyanin at such electrodes has clearly demonstrated that these systems can be effective and at the same time very specific for different types of metalloproteins. The interaction forces between such modified electrodes and redox proteins can readily be controlled by the lipid (generally phosphatidylcholine, PC) concentration, the concentration of added charged

AND

TOLLIN

surfactants, and the ionic strength of the supporting electrolyte, thus providing specificity in their electrochemical properties (25-27). In the present experiments we have extended these investigations to thioredoxins and to glutathione. Whereas a bare gold electrode does not show any measurable electrochemical response with both EcT and T4T, a lipid bilayer-modified electrode is capable of interacting with these two enzymes to produce well-defined, quasi-reversible, diffusion-controlled, cyclic voltammograms with two separate waves. Similar two-wave cyclic voltammograms have also been obtained with glutathione. We attribute these results to the occurrence of two separate one-electron reaction steps. The two-electron redox potentials, calculated from the average of the potentials at which these two one-electron processes occur, are in very good agreement with those determined by equilibrium methods (16-18). As far as we are aware, this is the first report of direct electrochemistry of disulfide/dithiol proteins. MATERIALS

AND

METHODS

Lipid bilayer membranes were deposited on a gold electrode from the following membrane-forming solutions: 2.5,50, or 150 mg/ml egg lecithin (Sigma Chemical Co.) in squalene (Fluka Chemie AG)/hutanol (1:3 v/ v) as previously described (28). A gold wire of 0.5 mm diameter (Aldrich Chemical Co.) with a Teflon sleeve of 0.52 mm inner diameter was used as a working electrode. The reference electrode was a saturated Ag/ AgCl electrode. The supporting electrolyte used was either 0.04 M NaClO, in 0.02 M phosphate buffer (pH 7.0) for E. coli thioredoxin and glutathione, or 0.04 M NaClO, in 0.01 M Tris buffer (pH 7.0) for T4 thioredoxin. Solutions were degassed with high purity humidified argon before use. The cyclic voltammetric experiments were performed as described previously, using a custom-made function generator with a current interface (25). Potential and current outputs were digitized by a HeathZenith SD 4850 digital oscilloscope and the waveforms were transferred to the hard disk of an XT computer. A BASIC program performed all data analysis and background subtractions. To improve the signal/noise ratio, the current data were subjected to 5-10 point moving average smoothing routine, which with the present data density gives a ratio of the smoothing interval width to the full width at half maximum of

Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode.

By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and gluta...
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