Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one‐electron oxidation

Abstract

To test thioredoxin resistance to oxidizing free radicals, we have studied the one-electron oxidation of wild-type thioredoxin and of two forms with the point mutations D30A and W35A, using azide radicals generated by c-ray or pulse radiolysis. The oxidation patterns of wild-type thioredoxin and D30A are similar. In these forms, Trp35 is the primary target and is ÔrepairedÕ by one-electron reduction; first by intramolecular electron transfer from tyrosine, and then from other residues. Conversely, during oxidation of W35A, Trp13 is poorly reactive. For all proteins, activity is conserved showing an unusual resistance toward oxidation.Keywords: thioredoxin; one-electron oxidation; radiolysis; tryptophan35 oxidation.Thioredoxins (Trx) are ubiquitous small proteins (100-120 amino acids) found in all living organisms from bacteria to vertebrates [1]. These proteins, whose active site contains the amino acid sequence -Cys-Gly-Pro-Cys-, exist either in an oxidized form with an intramolecular disulfide bond (Trx-S 2 ) or in a reduced form with two thiol functions [Trx-(SH) 2 ]. They are involved in the reduction of disufide bonds and play a major role in the control of intracellular reduction potential and defense against oxidative stress. In addition, these proteins control the release of transcription factors NFKB and AP-1, and thus their oxidation state is important in gene expression.During aerobic life, amino acid residues in proteins are subject to one-electron oxidation by reactive oxygen species, in such a way that the efficiency of cell defense against oxidative stress relies on the resistance of Trx to oxidation. Recently, Watson and Jones [2] showed that in cells both nuclear and cytoplasmic type 1 thioredoxins (Trx1) are relatively protected against oxidation and that the redox state of the cysteine residues in Trx1 was a good marker of oxidative stress. However, in addition to the cysteine residues of the active site, other amino acids can be oxidized by free radical processes, which may induce modifications of the enzymatic properties of Trx. In proteins, one-electron oxidation is known to affect primarily Met, Tyr and Trp residues [3]. The major degradation products resulting from such radical attack are dityrosine for tyrosine, N-formylkynurenin for tryptophan and methionine sulfoxide for methionine. Any of these transformations may affect the function of the enzyme and thus the redox homeostasy.The aim of this work was to determine the sensitivity of Trx toward one-electron oxidation. Therefore we studied the effect of overoxidation on Trx in its disulfide oxidized form (Trx-S 2 ) by azide radicals (N 3_ ) using pulse and gamma radiolysis, and by measuring its enzymatic activity. Pulse and gamma radiolysis are complementary techniques. The first allows identification of transient radicals formed with their absorption spectra, and the second is used to oxidize protein solutions in greater quantity to perform analysis of the degradation products. With radiolysis, very specific radicals are generated quan...

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