Báo cáo khoa học: Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one-electron oxidation potx

Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one-electron oxidation Ce´cile Sicard-Roselli1, Ste´phane Lemaire2, Jean-Pierre Jacquot3, Vincent Favau

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

Ce´cile Sicard-Roselli1, Ste´phane Lemaire2, Jean-Pierre Jacquot3, Vincent Favaudon4, Christophe Marchand5 and Chantal Houe´e-Levin1

1

Laboratoire de Chimie Physique and2Institut de Biotechnologie des Plantes, Universite´ Paris XI, Orsay, France;3UMR 1136 Interaction Arbres Microorganismes INRA UHP, Universite´ de Nancy I, Vandoeuvre, France;4U 612 INSERM, Institut Curie, Centre Universitaire, Orsay, France;5Institut de Biochimie et Biophysique Mole´culaire et Cellulaire, CNRS UMR8619 and IFR46, Universite´ Paris XI, Orsay, France

To test thioredoxin resistance to oxidizing free radicals, we

have studied the one-electron oxidation of wild-type

thio-redoxin 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
repaired 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 con-served 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-S2) 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-formyl-kynurenin 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-S2) by azide radicals (N3_) 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 quantitatively Azide radicals are powerful one-electron acceptors formed by the reaction

of N3 with the OH

radicals produced during irradiation of water solutions under N2O atmosphere [4]:

N2Oþ eaq! OH

N3 þ OH ! N

The reduction potential of N3_is lower than that of OH•

, and the values are 1.3 V and 1.8 V vs normal hydrogen electrode at neutral pH, respectively Nevetheless, N3_ is more selective and provides a simpler model of oxidation than OH

radicals allowing the determination of the main process of oxidation of OH•

radicals, without all of the side-effects They are known to react first with aromatic residues and most rapidly with tryptophan (Reaction 3) [5,6] Thus a well-known kinetic scheme is expected for the one-electron oxidation of proteins containing aromatic residues:

N

3þ HTrp-XX-TyrOH ! Trp

-XX-TyrOHþ N3 þ Hþ

ð3Þ Trp

-XX-TyrOH! HTrp-XX-TyrO

ð4Þ

CNRS UMR 8000, Baˆt 350, Universite´ Paris XI, F-91405 Orsay

Cedex, France Fax: +33 1 69 15 30 53, Tel.: +33 1 69 15 55 49,

E-mail: cecile.sicard@lcp.u-psud.fr

Abbreviations: Trx, thioredoxin; Trx1, type 1 thioredoxin; TyrOH,

, tyrosinyl radical; WT, wild-type.

(Received 6 May 2004, revised 24 June 2004,

accepted 7 July 2004)

-XX-TyrOH! HTrp-XX

The tryptophan free radical Trp•

resulting from Reaction 3

is then reduced by a tyrosine residue (Reaction 4) or by

various amino acids (Reaction 5) by an intramolecular

reaction Thus, the reaction ends up with dityrosine, among

other final compounds

Trx possesses two tryptophan residues (positions 13 and

35) One of them (Trp35) is part of the conserved active

site sequence, Trp-Cys-Gly-(Ala/Pro)-Pro-Cys-(Lys/Arg)

Mutation of this residue affects the environment of the

two Cys residues [7] and the protein biochemical activity [8]

Two tyrosine (positions 53 and 85) and two methionine

residues (positions 41 and 79) are also present Their role in

enzymatic activity is not known It is particularly interesting

to evaluate the sensitivity of Trp35 towards free radicals;

therefore in addition to the wild-type thioredoxin h from

the green alga Chlamydomonas reinhardtii, we oxidized the

mutant form W35A The aspartic acid residue at position

30, which is highly conserved in Trx from different species

[9], is also crucial for general acid–base catalysis in the

reductive opening of the disulfide oxidized thioredoxin

[10,11], yet NMR studies have shown that the D30A mutant

has the same global fold as the wild-type (WT) protein In

order to evaluate the importance of this residue in oxidative

processes we also determined the reaction of the mutant

D30A with the N3_radical

Materials and methods

Proteins

Recombinant Trx h from the green alga C reinhardtii was

purified from E coli as described previously [12] D30A and

W35A mutants were prepared, also as described previously

[13] Samples for irradiation were dialyzed several times

against phosphate buffer (final buffer: 20 mM phosphate,

100 mMNaN3, pH 7) Concentrations of the three forms of

Trx were adjusted to 77 lM(unless otherwise stated) using

absorbance and e278¼ 14 500M )1Æcm)1 for the WT and

D30A mutant, and e278¼ 8900M )1Æcm)1for W35A

Tryptophan

Tryptophan solutions (500 lM) were prepared in 20 mM

phosphate, pH 7, 500 mMNaN3buffer Tryptophan

solu-tions containing tert-butanol contained the same buffer

components, with the addition of 500 mMtert-butanol

Gamma and pulse radiolysis experiments

Gamma radiolysis experiments were performed using a

panoramic 60Co source (IL60PL, Cis-Biointernational,

Saclay, France) A Fricke dosimeter [4] was used to

determine the dose rate

Pulse radiolysis was performed using the linear electron

accelerator of the Curie Institute in Orsay [14] The doses

per pulse (200 ns duration, 5–15 Gy) were calibrated from

the absorption of the thiocyanate radical (SCNÞ


2 obtained

by radiolysis of thiocyanate ion solution in N2O-saturated

phosphate buffer {10 mMKSCN, 10 mMphosphate, pH 7,

G[(SCNÞ
]¼ 0.55 lmolÆJ)1, e ¼ 7580M )1Æcm)1} [15]

All protein samples were prepared in 20 mMphosphate buffer, pH 7, containing 100 mMNaN3and saturated with

N2O by flushing N2O gas for 1 h over the samples, avoiding bubbling in the solution

Absorption and fluorescence Absorption spectra were recorded at room temperature with a PerkinElmer (k9) spectrophotometer Fluorescence spectra were recorded on a FL111 Spex fluorimeter Electrophoresis

SDS/PAGE was performed using a 12% (w/v) acrylamide/ bisacrylamide gel with a Tris/Tricine buffer [16] Reductive conditions were obtained by adding 2-mercaptoethanol to the protein Proteins bands were stained with Coomassie blue R-250

HPLC analysis HPLC was performed on a Beckman Gold 168 (Beckman Coulter, Aulnay, France) with diode array detection The analytical column was a C4 reverse-phase column (150· 4.6 mm, 5 lm) The mobile phase eluants used were: (A) 0.1% (v/v) trifluoroacetic acid in water; (B) 0.1% (v/v) trifluoroacetic acid and 70% (v/v) CH3CN Gradient elution used was 40–60% of B in 20 min, 1 mLÆmin)1flow

at room temperature

Mass spectrometry All spectra were acquired in positive-ion mode on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Bio-systems, Courtaboeuf, France) equipped with a 337 nm nitrogen laser Determination of the molecular masses of irradiated proteins was performed in linear mode (acceler-ating voltage 25 kV, grid voltage 93%, guide wire 0.3%, delay 600 ns) with external calibration

Freeze-dried fractions obtained from HPLC purification

of irradiated Trx were diluted with 15 lL of 30% (v/v)

CH3CN, 0.3% (v/v) trifluoroacetic acid One millilitre of the solution was mixed with 4 lL of a saturated solution of sinapinic acid in 30% (v/v) CH3CN, 0.3% (v/v) trifluoro-acetic acid Finally, 1.5 lL of this premix was deposited onto the sample plate and allowed to dry at room temperature Activity measurements

The activity of Trx was measured using the reduction of insulin [17] One millilitre of the following solution was prepared: 100 mM phosphate, pH 7.1, 130 lM human insulin (zinc form), 2 mM EDTA; to which 30 lL of a Trx solution (77 lMTrx, 20 mM phosphate buffer, pH 7,

100 mMNaN3) was added to obtain a final concentration of 2.5 lMof the protein The experiment was started imme-diately after the addition of 500 lMdithiothreitol and the activity was monitored using the change in absorbance at

650 nm A blank was made using the same conditions without adding Trx All these experiments were carried out

at 27C

Transients

Aqueous solutions of Trx (77 lM WT, W35A or D30A,

in 20 mM phosphate buffer, 100 mM NaN3 pH 7) were

irradiated by pulse radiolysis in the presence of NaN3under

an atmosphere of N2O Under these conditions the N3_

radical was the only oxidant species generated by radiolysis

The absorption spectra of protein free radicals are shown

in Fig 1 Comparison of Fig 1A and B (WT and D30A)

shows that both proteins yield a similar behavior The

spec-trum obtained 50 ls after the pulse exhibited a broad

absorption band at 510 nm, characteristic of the

tryptophanyl radical (Trp•

) and a narrow peak at 410 nm reminiscent of a tyrosinyl radical (TyrO•

) [18] The second-order rate constants of formation of the Trp•

radical, determined under pseudo first-order conditions, were of the

same order of magnitude for both proteins (Table 1) The

intensity of the 410 nm peak subsequently increased at the

expense of the 510 nm band and reached a pseudo-plateau

after approximately 300 ls The rate constants for this reaction measured at 510 and 410 nm were very close to each other, suggesting quantitative oxidation of TyrOH by Trp•

, as for other proteins However, the intramolecular rate constants of charge transfer differed by a factor of two between WT Trx and the D30A mutant (Table 1) The Trp•

and TyrO•

yields were estimated from the magnitudes of the absorbance changes at 510 and 410 nm, assuming that the extinction coefficients of protein bound radicals are the same as those for free amino acids or peptides, namely, for Trp•

1800 and 300 mol)1ÆLÆcm)1at

510 and 410 nm, respectively; for TyrO•

, 70 and 2600 mol)1ÆLÆcm)1at 510 and 410 nm respectively [18,19] Thus, the stoichiometry N


3 /Trp•

was estimated to be equal to

1 : 1 and the percentage of transfer to around 60% in both compounds This transfer is not total, as some tryptophan radical persists at the end of the reaction (approximately

300 ls after the pulse)

Figure 1C shows the absorption spectrum of the W35A mutant form of Trx obtained 50 and 300 ls after the pulse Among the transients that were formed by oxidation of W35A, the Trp•

510 nm broad band was not detected anymore; instead, a peak at 420 nm and weak bands at 390 and 480 nm appeared The peak at 420 nm indicates that tyrosine could be oxidized directly by N

3 radicals The rate constant of formation was determined under pseudo first-order conditions (Table 1) and was substantially lower than

in other proteins

The bands at 390 and 480 nm might belong to a methionyl residue In general, N

3 radicals are unable to perform oxidization of methionine because the one-electron reduction potential of methionine is higher than that of N

3 However it was shown that interaction with other residues and particularly carbonyl groups, may lower the methionine redox potential considerably [20] Here the methionine radical would appear as an S-O complex with an absorption spectrum peaking around 390 nm [21] and/or interacting with another sulfur atom, giving an absorption spectrum with a maximum at 480 nm Both types of radicals can exist simultaneously in the same molecule [20] Alternatively the

480 nm band might be assigned to a Trp•

radical according

to Joshi and Mukherjee [22,23] These authors oxidized tryptophan by CCl3O2radicals and observed a blue-shift from 510 nm to 480 nm, which they ascribed to a change in polarity of the environment However, this blue-shift was formerly interpreted by Packer et al as an adduct of

Cl3COO•

on the C2 or C3 of the indole ring of tryptophan [24] In order to properly assign the 480 nm band, we investigated the effect of the solvent polarity on the absorption of a Trp radical N

3-induced pulse radiolysis oxidation of tryptophan was performed in phosphate buffer solution in the presence of tert-butanol (500 mM NaN3,

500 mMtert-butanol) The presence of tert-butanol induced

a broadening and a red-shift of the absorption from 510 nm

to 540 nm (not shown) Therefore, assigning the 480 nm band to Trp•

appears to be unlikely

Analysis of final compounds Three analytical methods were used to gain insight into the nature of the oxidized forms of Trx generated by c-ray radiolysis up to 900 Gy

Fig 1 Differential absorption spectra of WT, D30A and W35 Trx

•

after

2 cm (A) WT Trx; (B) D30A; (C) W35A The dose was 6.5 Gy.

Firstly, reducing and nonreducing SDS/PAGE analysis

was carried out on the three forms of oxidized Trx WT Trx

(Fig 2) and W35A exhibit a single band corresponding to

the mass (12 kDa) expected from intact Trx For D30A,

two new higher molecular mass bands are generated in a

dose-dependent manner, suggesting the formation of

aggre-gates

Secondly, UV absorption spectra of the three forms of

Trx were recorded before and after one-electron oxidation,

as tyrosine dimers can be evidenced with an absorption at

315 nm coupled to a fluorescence band at 410 nm A new

315 nm absorption band increased with dose for the W35A

mutant only In addition to absorption, fluorescence

analysis of the three forms of Trx after radiolytic oxidation

was performed Excitation at 315 nm induced a broad

fluorescence band at 410 nm for the W35A mutant (Fig 3)

while no new signal could be seen for WT and D30A Trx

Thirdly, liquid chromatography was performed to isolate

the degradation products of WT Trx, D30A and W35A

after oxidation with a dose of 100 Gy For each form, the

chromatograms were very similar and showed the

forma-tion of a single major product The yield of formaforma-tion of this

product (W35A, 47 nmolÆJ)1; WT, 35 nmolÆJ)1; D30A,

40 nmolÆJ)1) was calculated using the area of each peak

assuming that the sum of both peak areas represents 100%

The product was isolated and analyzed using mass

spectr-ometry For D30A and WT Trx, no difference between the

mass of the intact protein and the oxidized one could be

detected (Table 2) In the case of W35A, a small increase of

the mass (< 40 Da) was evidenced

Enzymatic activity Enzymatic activity of the three Trx was measured before and after exposure to 100 Gy (Fig 4) Firstly, unirradiated D30A had the highest activity, compared to the WT and W35A forms Secondly, as expected, the activity of W35A compared to that of WT Trx was reduced by a factor of approximately two [25] Thirdly, activity of D30A was weakly modified only after irradiation with 100 Gy, while that of WT and of W35A increased by a factor of 1.5 and 1.2, respectively (Table 3)

Discussion

As already observed for several proteins or peptides [6,26– 29], in WT and D30A the N

3 radical first oxidizes a tryptophan residue that is subsequently reduced in the course of intramolecular charge transfer to a tyrosine residue (Reactions 3 and 4) The rate constants of Reaction 3 are in the same range as for tryptophan residues

in other proteins (Table 1) The reaction between N

3 radicals and the Trp residue is stoichiometric, as reported for other proteins [6] Mutating Asp30 by replacement with

an alanine did not affect this reaction Because the 510 nm band is missing in W35A, we suggest that in the case of WT and D30A, Trp35 is the residue oxidized by N

3 Evolution of the transient absorption spectra indicates that Trp35 is partly
repaired by a tyrosine residue acting as

a one-electron donor Trx contains two tyrosines From the known structure of WT and D30A Trx [30], the distances

•

with WT Trx, D30A and W35A These values are compared to those proposed for other peptides or proteins Data are an average of values given in [29,30,43] The distance is taken from Figs 4 and 5 of [43].

k (Reaction 3) (mol)1ÆLÆs)1)

k reaction

Trp-Tyr distance of the couple involved (A˚)

middle: W35A; right: D30A For each lane, 2 lg of protein was used The molecular mass standards used for W35A and D30A are shown on the gels For D30A, the arrows point out two new bands increasing with the dose.

between Trp35 and Tyr (53 and 85) were calculated at 29.8

and 18.4 A˚, respectively (Table 1) We therefore propose

that the tyrosine residue involved in the intramolecular

Trp•

fi Tyr transfer is Tyr85 These distances are much

larger than in the other proteins or peptides investigated to

date (Table 1), yet the rate constant of this step is also much

higher [5,6,31] It is currently agreed that the rate constant of

long-range intramolecular electron transfer decays

expo-nentially with the donor–acceptor distance [32,33] and that

this dependence would be the same for all proteins and

peptides [34] Our results clearly demonstrate that this

correlation does not apply for Trx For D30A, the

intramolecular rate constant is reduced by a factor of two

(Table 1) compared with that for the WT protein although

the distances between Trp35 and the tyrosine residues are

the same Sakata et al [35] suggested that a change in

orientation of the donor/acceptor residues could induce a

slower rate constant Whether this effect would be due to

electrostatic changes such as dipole moment different

orientation or to modifications of the structure of the

solvation layer is not known Here, no significant difference

between the Tyr and Trp residues orientation could be

demonstrated by superimposing their respective crystallo-graphic structures Asp30 has an important role in driving the hydrogen bond network linking its carboxylic group to the active site Changes in the kinetics of intramolecular electron transfer by mutation of Asp30 could thus be a consequence of hydrogen bond rearrangement at the active site

Weak bands at 390 and 480 nm, but no 510 nm band that could be assigned to Trp, was observed in the case of W35A (Fig 1C) Several explanations were proposed for the band at 480 nm Such a blue-shift was observed and assigned to Trp•

radical in casein and bovine serum albumin

as these proteins were transferred to a solvent of low polarity inducing changes in the protein environment and conformation [22,23] Under our conditions, a decrease in solvent polarity by addition of tert-butanol did not produce any shift of the Trp•

absorption band We thus propose that the bands at 390 and 480 nm could be related to oxidation

of methionine residues Indeed, Trx possesses two methio-nine residues at positions 41 and 79 Met79 is close to the carbonyl function of Phe31 (less than 4 A˚) Hence its reduction potential could be lower than that of Met41 which

is in a polar environment with solvent access [20] allowing Met79 oxidation by N

3 The end product would be a MetS+radical, which, in interaction with the oxygen atom

of the carbonyl function, would lead to Met S–O radical absorbing at 390 nm [36] In addition Met79 is at 5.2 A˚ from the sulfur atom of Cys39 and could form a Met S–S+ radical absorbing at 480 nm

Final products are different for the three proteins Aggregation was observed only for D30A, for doses above

100 Gy This aggregation was also seen with electrophoresis under reducing conditions, which excludes the formation of

a disulfide bond Surprisingly, aggregation did not correlate with the appearance of 315 nm absorption/420 nm fluores-cence bands, as could be expected for covalent tyrosine dimerization [37,38] Therefore, dityrosine is unlikely to be formed We propose that the polypeptide chain can also take part in the one-electron processes For example, in lysozymes oxidation of tryptophan residues leads to poly-peptide bond cleavage [6] Also, in hen egg white lysozyme, one-electron reduction of the 6-127 disulfide bond leads to peptide bond cleavage [39] We therefore propose hydrogen

Table 2 Mass analysis of intact and oxidized thioredoxin after

separ-ation using liquid chromatography.

Table 3 Activity of the different forms of Trx before and after

oxida-tion.

Thioredoxin

Non irradiated

Irradiated 100 Gy

Fig 3 Fluorescence spectrum of W35A and

pH 7) recorded at room temperature with

excitation at 315 nm Doses for W35A: 0 Gy;

403 Gy and 646 Gy Doses for D30A (inset):

0 Gy and 100 Gy.

loss from a Ca atom followed by aggregation through the

carbon-carbon bond This process occurs easily in polymers

[4] and glycine residues are good candidates [40] In

thioredoxin, oxidized forms other than aggregates are

formed Oxidation of tyrosine residues in aqueous solution

and in the presence of oxygen by OH radicals leads to

formation of 3,4-dihydroxyphenylalanine However, this

requires oxygen (which is absent in our system) and

therefore, we can exclude the formation of

3,4-dihydroxy-phenylalanine in the three forms of Trx after radiolytic

oxidation

No aggregate was formed from the W35A mutant but a

new fluorescent product appeared after oxidation As this

420 nm fluorescence band is not due to dimerization, it

could arise from a degradation product of the Trp13

residue Mass spectrometry indicates an increase of mass

lower than 40 Da This and fluorescence experiments

suggest that one-electron oxidation of W35A could produce

N-formylkynurenin at position 13 It would mean that

Trp13 is involved in the final step of the one-electron

oxidation of W35A by azide

Although many examples of enzyme inactivation have

already been reported (reviewed in [41,42]), no inactivation

of Trx was shown to result from oxidation This means that

no amino acid involved in insulin reduction activity is

irreversibly affected by oxidation and that the tertiary

structure is not altered to a large extent Moreover, an

opposite effect is observed for W35A, i.e this Trx is found

to be more active after a 100 Gy irradiation

Conclusion

The use of azide radicals generated by pulse radiolysis

allowed us to determine the reactivity of WT and two

mutant forms, D30A and W35A, of Trx toward

one-electron oxidants We were particularly interested in the reactivity of the Trp35 residue, as this is highly conserved in the active site and may be a part of the defence of living organisms toward reactive oxygen species We show here that oxidation of Trx (WT and D30A) with N

3 occurs first at the Trp35 residue The Trp•

radical subsequently undergoes intramolecular reduction

by a tyrosine residue The tyrosine residue involved in this transfer is probably Tyr85 Such intramolecular electron transfer thus protects Trp35, and hence the enzyme’s biological activity, in cases of oxidative stress When Trp35 is absent, the other Trp residue (Trp13) may be oxidized, however, indirectly through tyrosine and/or methionine oxidation followed by intramolecular electron transfer This suggests that protection against oxidation is not due to the accessibility of sensitive residues to free radicals, but rather to some kind of
repair through long range intramolecular electron transfer The main point is that no degradation of Trx was observed It means that in the case of oxidation stress, if thioredoxin reductase is active, oxidized thioredoxin can be recycled and the defenses of the cells are not affected

References

1 Holmgren, A (1985) Thioredoxin Annu Rev Biochem 54, 237– 271.

2 Watson, W.H & Jones, D.P (2003) Oxidation of nuclear thior-edoxin during oxidative stress FEBS Lett 543, 144–147.

3 Davies, M.J., Fu, S., Wang, H & Dean, R.T (1999) Stable markers of oxidant damage to proteins and their application in the study of human disease Free Rad Biol Med 27, 1151– 1163.

4 Spinks, J.W.T & Woods, R.J (1990) Introduction to Radiation Chemistry, 3rd edn Wiley Interscience, New York.

5 Weinstein, M., Alfassi, Z.B., De Felippis, M.R & Faraggi, M (1991) Long range electron transfer between tyrosine and trypto-phan in hen egg-white lysozyme Biochim Biophys Acta 1076, 173–178.

6 Stuart-Audette, M., Blouquit, Y., Faraggi, M., Sicard-Roselli, C.,

intramolecular long range electron transfer between tyrosine and tryptophan in lysozymes Evidence for the participation of other residues Eur J Biochem 270, 1–7.

7 Menchise, V., Corbier, C., Didierjean, C., Jacquot, J.-P., Bened-etti, E., Saviano, M & Aubry, A (2001) Crystal structure of the W35A mutant thioredoxin h from Chlamydomonas reinhardtii: the substitution of the conserved active site Trp leads to modifications

in the environment of the two catalytic cysteines Biopolymers 56, 1–7.

8 Krimm, I., Lemaire, S., Ruelland, E., Miginiac-Maslow, M., Jacquot, J.-P., Hirasawa, M., Knaff, D.B & Lancelin, J.-M.

Biochem 255, 185–195.

9 Eklund, H., Gleason, F.K & Holmgren, A (1991) Structural and functional relations among thioredoxins of different species Proteins 11, 13–28.

10 Chivers, P.T & Raines, R.T (1997) General acid/base catalysis in the active site of Escherichia coli thioredoxin Biochemistry 36, 15810–15816.

11 Dyson, H.J., Jeng, M.F., Tennant, L.L., Slaby, I., Lindell, M., Cui, D.S., Kuprin, S & Holmgren, A (1997) Effects of buried

Fig 4 Activity determination of the three forms of Trx using insulin

Non-irradiated W35A mutant, d; W35A after 100 Gy, s; non-Non-irradiated

WT Trx, j; WT Trx after 100 Gy, h; non-irradiated D30A mutant,

m; D30A after 100 Gy, n.

charged groups on cysteine thiol ionization and reactivity in

character-ization of mutants of Asp 26 and Lys 57 Biochemistry 36, 2622–

2636.

12 Stein, M., Jacquot, J.-P., Jeannette, E., Decottignies, P., Hodges,

M., Lancelin, J.M., Mittard, V., Schmitter, J.M &

Miginiac-Maslow, M (1995) Chlamydomonas reinhardtii thioredoxins:

structure of the genes coding for the chloroplastic m and cytosolic

h isoforms; expression in Escherichia coli of the recombinant

proteins, purification and biochemical properties Plant Mol Biol.

28, 487–503.

13 El Hanine Lmoume`ne, C., Conte, D., Jacquot, J.-P &

Houe´e-Levin, C (2000) Redox properties of protein disulfide bond in

oxidized thioredoxin and lysozyme: a pulse radiolysis study

Bio-chemistry 39, 9295–9301.

14 Favaudon, V., Tourbez, H., Houe´e-Levin, C & Lhoste, J.-M.

pro-teins A gamma-ray and pulse radiolysis mechanistic investigation.

Biochemistry 29, 10978–10989.

15 Buxton, G.V & Stuart, C.R (1995) Reevaluation of the

thio-cyanate dosimeter for pulse radiolysis J Chem Soc Faraday

Trans 91, 279–281.

16 Scha¨gger, H & von Jagow, G (1987) Tricine-sodium dodecyl

sulfate-polyacrylamide gel electrophoresis for the separation of

proteins in the range of 1–100 kDa Anal Biochem 166, 368–379.

17 Holmgren, A (1979) Thioredoxin catalyzes the reduction of

insulin disulfides by dithiothreitol and dihydrolipoamide J Biol.

Chem 254, 9627–9632.

18 Bensasson, R.V., Land, E.J & Truscott, T.G (1983) Flash

Pho-tolysis and Pulse Radiolysis Pergamon Press, Oxford.

19 Dudley, F.D., Santus, R & Grossweiner, L.I (1975) Laser flash

photolysis of aqueous tryptophan J Phys Chem 79, 2711–2716.

20 Pogocki, D & Schoneich, C (2002) Redox properties of Met (35)

in neurotoxic beta-amyloid peptide A molecular modeling study.

Chem Res Toxicol 15, 408–418.

21 Schoneich, C., Pogocki, D., Wisniowski, P., Hug, G.L &

Bobrowski, K (2000) Intramolecular sulfure-oxygen bond

for-mation in radical cations of N-acetylmethionine amide J Am.

Chem Soc 122, 10224–10225.

22 Joshi, R & Mukherjee, T (2003) Effect of solvent viscosity,

polarity and pH on the charge transfer between tryptophan radical

and tyrosine in bovine serum albumin: a pulse radiolysis study.

Biophys Chem 103, 89–98.

23 Joshi, R & Mukherjee, T (2002) Charge transfer between

tryp-tophan and tyrosine in casein: a pulse radiolysis study Biophys.

Chem 96, 15–19.

24 Packer, J.E., Mahood, J.S., Willson, R.L & Wolfenden, B.S.

(1981) Reactions of the trichloromethylperoxy free radical

(Cl 3 COO•) with tryptophan, tryptophanyl-tyrosine and

lysozyme Int J Radiat Biol 39, 135–141

25 Krause, G & Holmgren, A (1991) Substitution of the conserved

tryptophan 31 in Escherichia coli thioredoxin by site-directed

mutagenesis and structure-function analysis J Biol Chem 266,

4056–4066.

26 Pru¨tz, W.A & Land, E.J (1979) Charge transfer in peptides Pulse

radiolysis investigation of one-electron reactions in dipeptides of

tryptophan and tyrosine Int J Radiat Biol 36, 513–520.

27 Pru¨tz, W.A., Siebert, F., Butler, J., Land, E.J., Menez, A &

Montenay-Garestier, T (1982) Charge transfer in peptides.

Intramolecular radical transformations involving methionine, tryptophan and tyrosine Biochim Biophys Acta 705, 139–149.

28 Bobrowski, K., Poznanski, J., Holcman, J & Wierzchowski, K.L (1998) Long-range electron transfer between proline-bridged aromatic amino acids Adv Chem Series, Photochem Radiat Chem 254, 131–143.

29 DeFelippis, M.R., Faraggi, M & Klapper, M.H (1990) Evidence for through-bond long range electron transfer in peptides J Am Chem Soc 112, 5640–5642.

30 Menchise, V., Corbier, C., Didierjean, C., Saviano, M., Benedetti, E., Jacquot, J.-P & Aubry, A (2001) Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas

J 359, 65–75.

31 Faraggi, M., DeFelippis, M.R & Klapper, M.H (1989) Long-range electron transfer between tyrosine and tryptophan in pep-tides J Am Chem Soc 111, 5141–5145.

32 Marcus, R.A & Sutin, N (1985) Electron transfers in chemistry and biology Biochim Biophys Acta 811, 265–322.

33 Hush, N.S (1985) Distance dependence of electron transfer rates Coord Chem Rev 64, 135–157.

34 McLendon, G (1988) Long-distance electron transfer in proteins and model systems Acc Chem Res 21, 160–167.

35 Sakata, Y., Nakashima, S., Goto, Y., Tatemitsu, H., Misumi, S., Asahi, T., Hagihara, M., Nishikawa, S., Okada, T & Mataga N (1989) Synthesis of completely fixed porphyrin-quinone com-pounds and the mutual orientation effect on electron transfer.

J Am Chem Soc 111, 8979–8981.

36 Scho¨neich, C., Pogoski, D., Hug, G.L & Bobrowski, K (2003) Free radical reactions of methionine in peptides: Mechanisms relevant to b-amyloid oxidation and Alzheimer’s disease J Am Chem Soc 125, 13700–137013.

37 Malencik, Q.A., Sprouse, J.F., Swanson, C.A & Anderson, S.R (1996) Dityrosine: Preparation, isolation and analysis Anal Bio-chem 242, 202–213.

38 Audette, M., Blouquit, Y & Houe´e-Levin, C (2000) Oxidative dimerisation of proteins: role of tyrosine accessibility Arch Bio-chem Biophys 76, 673–681.

39 Berge`s, J., Kassab, E., Conte, D., Adjadj, E & Houe´e-Levin, C (1997) Ab initio calculations on arginine-disulfide complexes modelling the one-electron reduction of lysozyme Comparison to

an experimental reinvestigation J Phys Chem 101, 7809–7881.

40 Rauk, A., Yu, D., Taylor, J., Shustov, G.V., Block, D.A & Armstrong, D.A (1999) Effects of structure on alpha C-H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation Biochemistry 38, 9089–9096.

41 Houe´e-Levin, C & Sicard-Roselli, C (2001) In Radiation

eds), pp 553–584 Elsevier, Amsterdam.

42 Von Sonntag, C (1987) The Chemical Basis of Radiation Biology Taylor & Francis, London.

43 Bobrowski, K., Holcman, J., Poznanski, J., Ciurak, M & Wierzchowski, K.L (1992) Pulse radiolysis studies of

peptides J Phys Chem 96, 10036–10043.