Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3

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Electrode Potentials of L-Tryptophan, L-Tyrosine, 3-Nitro-Ltyrosine, 2,3-Difluoro-L-tyrosine and 2,3,5-Trifluoro-L-tyrosine Leila Mahmoudi, Reinhard Kissner, Thomas Nauser, and Willem Hendrik Koppenol Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00019 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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A. Title: Electrode Potentials of L-Tryptophan, L-Tyrosine, 3-Nitro-L-tyrosine, 2,3-Difluoro-Ltyrosine and 2,3,5-Trifluoro-L-tyrosine

B. Funding Source Statement: This research was supported by grant 200021_149430 of the Swiss National Science Foundation and by the Swiss Federal Institute of Technology. C. Byline: Leila Mahmoudi,† Reinhard Kissner, Thomas Nauser, and Willem H. Koppenol* Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich CH-8093, Switzerland * Corresponding author: Willem H. Koppenol, Institute of Inorganic Chemistry, Swiss Federal Institute of Technology (ETH) Zurich, Vladimir-Prelog-Weg 1, HCI H105, CH-8093 Zürich, Switzerland; Tel.: +41-44-632-2875; E-mail: [email protected]. †

Present Address:

Department of Internal Medicine, Zurich University Hospital, Zurich CH-8091, Switzerland

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Abstract Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were L-tryptophan, L-tyrosine, N-acetyl-L-tyrosine methyl ester, N-acetyl-3-nitro-L-tyrosine ethyl

ester, N-acetyl-2,3-difluoro-L-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-L-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for L-tryptophan, L-tyrosine and N-acetyl-3-nitro-L-tyrosine ethyl ester were obtained. Electron transfer, accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst’s law. E°’(Trp•,H+/TrpH) and E°’(TyrO•,H+/TyrOH) at pH 7, are +0.99±0.01 V, and +0.97±0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°’ of approximately 0.06 V. Thus, in small peptides, we recommend values of +1.00 V and +0.96 V for E°’(Trp•,H+/TrpH) and E°’(TyrO•,H+/TyrOH). The electrode potential of N-acetyl-3-nitro-L-tyrosine ethyl ester is higher, while, because of mesomeric stabilization of the radical, those of N-acetyl-2,3difluoro-L-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-L-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of (Trp•,H+/TrpH) and E°’(TyrO•,H+/TyrOH) are nearly equal, they would be, in principle, interchangeable. Protoncoupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.


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Introduction In 1965, Winfield1 proposed that aromatic residues could be reversibly oxidized and participate in electron transfer between cytochromes. However, such an oxidation requires strong oxidants with an electrode potential > 1 V which are generally not available. Exceptions are, for instance, the special pair of chlorophylls in Photosystem II and the di-iron center in ribonucleotide reductase, which do oxidize tyrosines.2-5 In the case of Class Ia ribonucleotide reductase, the electron “hole” is transferred over an astonishing 35 Å to oxidize a cysteine, which initiates the conversion of nucleotides to their 2'-deoxy form. The pathway most likely involves, within the β2-subunit, another tyrosine, and possibly a tryptophan, and two other tyrosines in the α2 subunit of ribonucleotide reductase. To delineate the path various modified tyrosines have been incorporated in the two subunits.5 Electron transfer from a tyrosine residue to a tryptophanyl radical in dipeptides, studied by pulse radiolysis, was first reported in 1979.6 As the neutral tryptophanyl radical absorbs near 510 nm, and the tyrosyl radical near 405 nm, this process is conveniently followed by time-resolved spectroscopy: tryptophan is rapidly (µs) oxidised by N3• generated by pulse radiolysis. In that6 and in later studies7-9 it was established that in peptides and proteins (i) formation of the tyrosyl radical is concomitant with the disappearance of the tryptophanyl radical; (ii) transfer is rapid, with rates that decrease from 105 s−1 to 104 s−1 with an increasing number − up to three − of glycine spacers between tryptophan and tyrosine; (iii) the activation energy is relatively small and varies from approximately 20 kJ/mol in peptides to 45 kJ/mol in β-lactoglobulin, and (iv) most importantly, there is an equilibration between the tyrosyl radical and tryptophan with K values near 10 in favour of the tyrosyl radical near neutral pH. These K-values limit the difference in electrode potential between E°'(Trp•,H+/TrpH) and E°'(TyrO•,Η+/TyrOH) to approximately 60 mV at that pH. The rate of electron transfer in dipeptides was confirmed in a laser flash photolysis study.10 The 3 ACS Paragon Plus Environment

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mechanism of electron transfer in model peptides with 3 to 5 prolines as spacers between tyrosine and tryptophan has been studied by Bobrowski and coworkers.11 They concluded that electron transfer takes place through bonds as opposed to through space. To find an absolute value, at least one potential must to be known by direct method. In 1987, Harriman12 determined a value of +1.015 V for the Trp•,H+/trpH couple, and +0.93 V for TyrO•,Η+/TyrOH by cyclic voltammetry. As the amino acid radicals dimerize rapidly, the cathodic peak is absent, but a determination is still possible based on an equation derived by Saveant and Vianello,13 and by Nicholson.14 Unfortunately, an error was made by Harriman12 in adapting this equation to the anodic oxidations of tyrosine and tryptophan, and this error was propagated by DeFelippis et al.15 A correct derivation is given in Supporting Information. Around 1990, Klapper and coworkers published a series of papers15-18 in which they re-examined electron transfer from tyrosine to tryptophan. Their pulse radiolysis experiments with tryptophan- and tyrosine-containing peptides yielded results that were very similar to those obtained by Prütz and coworkers,6-9 although the interpretation is different for at least one peptide: for Trp-Gly-Tyr, Prütz et al.8 reported absorbances of the tyrosyl and tryptophanyl radicals after 80 µs (6 half-lives) from which a K of 8 is calculated. However, Faraggi et al.17 list a difference of > 80 mV, which corresponds to K > 20. For TyrGluTrp, they17 report a difference of 65 mV, although an estimate based on the beginning and final absorbance at 510 nm (their Figure 1a17 suggest a K of 5 and thus a ∆E°' of 40 mV at pH 7.0). These authors also examined the peptides by differential pulse polarography, with results that are in approximate agreement with those derived from the pulse radiolysis experiments.15 In this study, the authors also established that proton transfer is not rate-limiting. From the kinetics and equilibria of reactions of well-known inorganic compounds with tryptophanyl and tyrosyl radicals, generated by pulse radiolysis, they established a range for the reduction 4 ACS Paragon Plus Environment

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potential of the tryptophanyl/tryptophan couple from +1.00 V to +1.09 V, and from +0.90 V to +0.97 V for that of the tyrosyl/tyrosine couple at 25°C and pH 7. They reported these standard electrode potentials as +1.05 ± 0.01 V and +0.94 ± 0.01 V, respectively.16 Later they extended these studies to di- and tri-peptides that contained tryptophan and tyrosine, and also determined electrode potentials by differential pulse polarography. The effect of a neighbouring amino acid was found to be small.15 The value for tyrosine, +0.94 V, contrasts with that obtained by differential pulse polarography, +0.82 V.19 In a recent review,20 values at pH 7 of +0.91 V and 1.03 V for E°'(TyrO•,Η+/TyrOH) and E°'(Trp•,H+/TrpH) were proposed. The electrochemically determined values by Harriman12 and DeFellipis et al.15 were not used for the reason given. Among other considerations, the value for E°'(TyrO•,Η+/TyrOH) was related to that of E°(PhO•,/PhO−), +0.803 V.21,22 We note that the difference between the reported12,16,20 electrode potentials for E°'(Trp•,H+/TrpH) and E°'(TyrO•,Η+/TyrOH) is approximately twice as large as that observed directly by following the oxidation of tyrosine by the tryptophanyl radical in oligopeptides and some proteins. Here, we present electrode potentials of tryptophan, tyrosine, and some of the modified tyrosines that were used in the studies of ribonuclease reductase.23-25 We show that the value we obtained for tyrosine in water is slightly higher than previously reported while that for tryptophan is somewhat lower, such that the difference between the two potentials is now in better agreement with the results from pulse radiolysis studies of tryptophan- and tyrosine-containing peptides and proteins.

Experimental Chemicals. All common reagents were of analytical grade or better and obtained from SigmaAldrich (Buchs, Switzerland), Merck Millipore (Zug, Switzerland) and ThermoFisher 5 ACS Paragon Plus Environment

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(Reinach, Switzerland). N-acetyl-2,3-difluoro-L-tyrosine methyl ester and N-acetyl-2,3,5trifluoro-L-tyrosine methyl ester were synthesized by Kanchana Ravichandran of the research group of professor J. Stubbe at the Massachusetts Institute of Technology as described in the literature.26 Ultrapure water was prepared with a Merck Millipore (Zug, Switzerland) Advantage device. Apparatus. We recorded cyclic voltammograms with an AMEL2049 potentiostat connected to an AMEL 568 function generator (AMEL SpA, Milano, Italy), or with a PGSTAT 128N (Metrohm Autolab, Utrecht, Netherlands) integrated potentiostat/function generator/digitizer. We used a 33 µm carbon micro electrode (ALS Co., Tokyo, Japan) as working electrode for cyclic voltammetry at scan rates up to 400 Vs–1, and pH values of 0.8, 3.8 and 7.4. The pH was set to about pH = 0.8 with 0.18 M H2SO4, with 0.5 mM H2SO4 / 0.18 M K2SO4 to pH = 3.8 and with 0.09 M KH2PO4 / 0.28 M K2HPO4 to pH = 7.4. The ionic strength in these solutions is around 0.54 M; with the carbon micro electrode a pair of broad anodic and cathodic waves is observed between 0 and 0.6 V vs. Ag/AgCl in all electrolytes. Addition of amino acids did not affect these waves. The counter electrode was a 2 mm glassy carbon rod, and the reference electrode was a Ag/AgCl (3 M KCl) electrode (Metrohm, Herisau, Switzerland). The reference potential was checked at regular intervals against that of the [Fe(CN)6]3-/[Fe(CN)6]4- couple +0.358 V, and the KCl solution was replaced when the potential deviated from this value. . Preparation of solutions. The amino acids were dissolved to concentrations between 50 µM and 1.0 mM in supporting electrolyte, with the exception of N-acetyl-L-2,3difluorotyrosine methyl ester and N-acetyl-L-2,3,5-trifluorotyrosine methyl ester. These hydrophobic compounds dissolve very slowly. Therefore, about 4 mg of each were treated with 0.4 ml 0.1 M NaOH under stirring and the solutions were warmed to 40 °C. After 30 minutes, most of the compounds had dissolved. 100-200 µl of this solution was added to 10 ml of the supporting electrolyte solutions. The pH values in the experimental solutions were 6 ACS Paragon Plus Environment

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determined after voltammetry by potentiometric measurement with a glass electrode. The degree of hydrolysis of the ester group of the fluorinated tyrosines after dissolution in 0.1 M NaOH was examined by potentiometric titration with hydrochloric acid. The fraction that shows a pKa of 3.5, and that therefore can be associated with a free carboxylate function, is less than 10%. Square wave voltammetry. Experiments were carried out with the same PGSTAT 128N, but a 2 mm glassy carbon electrode (Metrohm, Herisau, Switzerland) was used instead of the carbon microelectrode. Modulation frequency was 200 Hz, with 20 mV amplitude, and the scan rate was 1 Vs–1. Reference and counter electrodes were the same as used in the cyclic voltammetry experiments, and the same concentrations of amino acids were dissolved in the same supporting electrolytes. Pulse radiolysis. A Febetron field emission accelerator (L-3, Applied Technologies, San Leandro, CA) was used. Aqueous solutions of peptides containing 1 tyrosine and 1 tryptophan (cyclo-Tyr-Trp and H-Trp-Gly-Tyr-OH) and a large excess of NaN3 were saturated with N2O and then irradiated. This procedure ensures that all primary radicals, eaq−, HO•, and H• are converted to N3• within a few µs. 27 Under these conditions, N3• is produced with a yield of 0.63 µM/Gy. The oxidation of tryptophan by N3• 28 is kinetically favored over that of tyrosine, despite the slightly lower electrode potential of the TyrO•, H+/TyrOH couple. The equilibrium between tryptophanyl and tyrosyl radicals was determined in phosphate buffer, pH = 7.0, in a 6 cm optical cell with H-Trp-Gly-Tyr-OH and cyclo(Tyr-Trp). Trp• and Tyr• concentrations were monitored at 510 and 405 nm, respectively. Low doses of 2 Gy were applied. Errors are given as ± 2s.

Results L-Tyrosine and N-acetyl-3-nitro-L-tyrosine ethyl ester produce anodic waves during oxidative


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voltammetric scans on carbon fiber electrodes in all electrolytes used, but hardly any cathodic waves in the reductive scans (Figures 1A and 1B). Only at the highest scan rates, there is a slight increase in the cathodic current near 0.9 V, but without a distinct peak (e.g. trace g, Figure 1B).

Figure 1 here

As in previous studies where cyclic voltammetry was used,12,15 most traces show these characteristics, because the radicals produced by the oxidation disappear too fast by recombination to be detected in the cathodic scan. In the case of L-tyrosine and N-acetyl-3nitro-L-tyrosine ethyl ester, we find that the onset of the anodic wave does not shift more than 0.03 V with scan rates higher than 100 Vs–1, see Figures 1A and 1B, while the uncertainty of the potential measurement at the onset was ±0.005 V. This finding indicates that electrochemical electron transfer is highly reversible but limited by diffusion, and that an electrode potential can be derived. Matsuda29 has shown that, for reversible electron transfer, eq 1 holds:

Ep − Ep/2 = 2.2 RT/nF

(1)

which results at 25°C and for n = 1 in 0.056 V. This difference between Ep and Ep/2 is observed for L-tyrosine and N-acetyl-3-nitro-L-tyrosine ethyl ester at scan rates of 10 Vs–1 or less, and at all pH values used. The electrode potential follows from eq 2.29

Ep = E°’ + 1.109 RT/nF

(2)


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In eqs 1 and 2, Ep is the peak potential, Ep/2 the potential at the inflection point of the voltammogram, E°’ the electrode potential under non-standard conditions,30 R the gas constant, T the temperature in Kelvin, n the number of electrons, here 1, and F the Faraday constant. The cyclic voltammograms obtained with N-acetyl-L-2,3-difluorotyrosine methyl ester and N-acetyl-L-2,3,5-trifluorotyrosine methyl ester show quasi-reversible behavior because the anodic wave shifts to higher potential by 50 mV when the scan rate is changed from 1 Vs−1 to 10 Vs−1. Interestingly, a weak cathodic wave is observed with both compounds (Figure 2), and its position moves to lower potential, concomitant with the shift of the anodic wave, upon an increase in scan rate. We estimated E°’ values from the midpoint potentials between anodic and cathodic waves.

Figure 2 here

L-Tryptophan, like L-tyrosine and N-acetyl-3-nitro-L-tyrosine ethyl ester, shows only anodic

waves during oxidative voltammetric scans in all electrolyte solutions used (Figure 3). For the determination of E°’(Trp•, H+/ TrpH) we restricted the scan rates to less than 50 Vs–1, such that the wave maxima do not shift nor broaden with scan rate. For tryptophan, E p − E p / 2 is approximately 0.070 V at 10 Vs–1 and pH = 0.8, but 0.057 V at pH = 3.8 and 7.4.

Figure 3 here.

As with L-tyrosine and N-acetyl-3-nitro-L-tyrosine ethyl ester, E°’ values of L-tryptophan are obtained with Matsuda’s equation29 for reversible electron transfers. For L-tryptophan, we observe that only at concentrations below 0.1 mM the peak 9 ACS Paragon Plus Environment

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current at a given concentration was identical at all pH values. Therefore, E°’ values determined at c = 0.05 mM tryptophan only were used for the correlation of E°’ with pH. Figure 4 shows the pH-dependence of E°’ for L-tyrosine, N-acetyl-L-tyrosine methyl ester, N-acetyl-3-nitro-L-tyrosine ethyl ester and L-tryptophan.

Figure 4 here.

Table 1 here.

Square wave voltammetry was applied to solutions of L-tyrosine, N-acetyl-3-nitroL-tyrosine ethyl ester and L-tryptophan for comparison to the results obtained with cyclic

voltammetry (See Figures S2 and S3). Both positive and negative edge detections were carried out. This procedure yields pairs of peaks; one then estimates the equilibrium potentials by determining the midpoint between the two maxima. The results are shown in Table 2.

Table 2 here.

For N-acetyl-3-nitro-L-tyrosine ethyl ester, the same E°’ value as with cyclic voltammetry was found. However, for L-tyrosine and L-tryptophan, the pulsed method yields potentials that are 40 mV lower than those obtained with cyclic voltammetry. Equilibria between tyrosyl and tryptophanyl radicals in H-Trp-Gly-TyrOH and cyclo(Trp-TyrOH) were determined by pulse radiolysis. [Trp•] and [TyrO•] vs. time were monitored at 510 nm and 405nm, respectively. The dose was kept low (2 Gy), in order to minimize intermolecular reactions between radicals. Because the reaction of N3• is much faster with L-tryptophan than with L-tyrosine at 10 ACS Paragon Plus Environment

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pH 7.4, the kinetics trace at 510 nm goes through a maximum and settles at an absorbance level greater than 0, see Figure 5. With [Trp•]eq = [N3•]o − [Tyr•]eq ,we determine

K=

[Tyr • ]eq [Trp• ]eq

= (9 ± 3) for H-Trp-Gly-TyrOH, and K =

[Tyr • ]eq [Trp• ]eq

= (13 ± 3) for cyclo(Trp-Tyr).

Figure 5 here.

From these two K values, we calculate ∆E°’ = (0.06±0.01) V and (0.07±0.01) V.

Discussion We determined electrode potentials for the proton-coupled electron transfer of several aromatic amino acids with cyclic voltammetry at a carbon micro electrode.33 This is the most suitable electrochemical method for equilibrium potential determinations, because it does not only yield the potential, but also valuable information on the reversibility and kinetics of the electrode reaction, compared to most other electrochemical approaches. A major drawback is the modest sensitivity which requires concentrations that might be not accessible for reasons of solubility or availability of the compound. We therefore used microelectrodes, which increases sensitivity because of a better ratio of electrolytic vs. capacitive current. In addition, microelectrodes made from carbon fibers make better contact with aqueous solutions than conventional glassy carbon electrodes; these also do not favor adsorption of functionalized organics as many metals do. If the voltammetric current waves fulfill the reversibility conditions mentioned in the results section, we can derive equilibrium potentials, even if the wave in the return scan is hardly present or even absent. A correction which takes in account the decay of the products − in our case the bimolecular reaction of two radicals − produced during the forward scan needs 11 ACS Paragon Plus Environment

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to be applied, because the decay may affect the position of the maximum. A suitable equation that takes in account product dimerization was derived by Nicholson:14

E p = E° −

RT 4.78π 3DO RT n Fv ln − ln 3nF 2 DR 3nF kc0 RT

(3)

Eq 3 is a special form of a more general expression introduced by Savéant and Vianello.13 Here, k is the rate constant of the bimolecular recombination, DO and DR the diffusion coefficients of oxidized and reduced species, c0 is the bulk concentration of the amino acid, and v the scan rate. An adapted version of eq 3 was used to obtain E° from Ep of aromatic amino acids, the radicals of which are known to undergo dimerization.12 Unfortunately, an error was made in transforming eq 3, and the incorrect expression, eq. 4, was also used in a later report.15 Details concerning this error can be found in Supporting Information.

E p = E ° − 0.9

RT RT 2kc0 RT + ln nF 3nF 3nFv

(4)

RT RT kc0 RT − ln nF 3nF n Fv

(5)

The correct equation is

E p = E ° + 1.04

which, for nFv < kc0RT, predicts a shift of Ep to lower values. If the term that includes the rate constant becomes small or even zero, eq 5 reduces to an expression that is very similar to the peak potential relation derived for normal reversible electrode reactions (eq 6):29


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E p = E ° + 1.04

RT nF

(6)

If nFv >> kc0RT, the scan rate is faster than the decay of the radicals and Ep should have the value predicted by the conventional eq 2, given the electrode process is still reversible at these scan rates. In the case of N-acetyl-3-nitro-L-tyrosine ethyl ester, we observe at high scan rates that Ep hardly shifts and that a weak cathodic current becomes visible, which indicates that we closely approach the condition of nFv >> kc0RT. Quite unexpectedly, data obtained by cyclic voltammetry and interpreted with the incorrect eq 5 are in reasonable agreement with results by pulse radiolysis and differential pulse voltammetry.12,15 It is not possible to reconstruct how these electrode potentials were obtained because no raw data were presented, Instead of cyclic voltammetry, differential pulse voltammetry has been used extensively to determine E°’ of aromatic amino acid couples and some derivatives, including peptides.18 Advantages are high sensitivity, which helps to overcome problems of solubility or availability, and a signal shape which is easier to analyze. However, two conditions must be met for correct E°’ determinations from differential pulse voltammograms. Good reversibility of the electrode reaction is mandatory, as well as the absence of chemical reactions of the electrochemical products. L-tyrosine, for example, does not fulfill the second condition: as mentioned, upon reversible oxidation to the tyrosyl radical by proton-coupled electron transfer, the radicals undergo rapid dimerization. This is especially relevant for any technique where pulsing is applied: it periodically generates high concentrations of the tyrosyl radical on the surface of the electrode, which decays rapidly because the recombination of tyrosyl radicals is a second-order reaction. The electrode reaction thus does not reach equilibrium, and therefore the anodic current maximum is reached earlier than if it depended only on diffusion. This is a likely reason for why potentials obtained for L-tyrosine by differential 13 ACS Paragon Plus Environment

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pulse voltammetry are lower than those by cyclic voltammetry. Our square wave voltammetry results are in agreement with this interpretation, because square wave voltammetry is also a pulsed technique. Most interestingly, experiments with N-acetyl-3-nitro-L-tyrosine ethyl ester yield the same E°’ with both electrochemical methods. In cyclic voltammograms taken at 350 Vs–1, this compound shows a small cathodic current (Figure 1B), unlike tyrosine or tryptophan. This observation is an indication that the combination of the corresponding phenoxyl radical with the electron-withdrawing substituent leads to a slower dimerizes than just a phenoxyl radical; this must also be the case for the fluorinated tyrosines (Figure 2). Thus, the surface concentrations are closer to those where a true electrochemical equilibrium can be achieved, and the potentials estimated reflect the correct values much better. The value of E°’(TyrO•,H+/TyrOH) obtained by Tommos and coworkers34 with amino acid isolated inside a protein further supports the influence of the recombination reaction on E°’ determinations, because decay by dimerization is largely prevented for steric reasons. Another critical parameter and possible source of error in differential pulse experiments is the amplitude that was applied; for potential determinations this value must be specified because it affects the correction that must be applied to obtain the correct electrode potential. However, the most comprehensive report15 does not mention the pulse amplitude, and whether, or how, it was used to calculate electrode potentials. Because the electrode potentials determined for L-tyrosine by pulse radiolysis and differential pulse voltammetry are close to each other, the value of +0.94 V was accepted. Now that it is clear that the analysis of the data obtained by electrochemistry is flawed, we may expect that results from pulse radiolysis and electrochemistry do not agree. This is indeed the case: pulse radiolysis yields +0.94±0.01 V,16 as discussed in the Introduction, while our electrochemical determination results in +0.97±0.01. Similarly, the values for the tryptophanyl/tryptophane couple are +1.05±0.01 V16 by pulse radiolysis and +0.99±0.01 V by 14 ACS Paragon Plus Environment

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electrochemistry. However, there is a fundamental difference between the two techniques. A determination by pulse radiolysis is indirect: it relies on establishing equilibrium with a redox couple of which the electrode potential is well known. We note that no consistent value was obtained by equilibration with several well-defined inorganic couples.16 Furthermore, during pulse radiolysis experiments, there may be close contact between the reaction partners, or even formation of adducts. Additionally, side reactions may affect the equilibrium observed. In contrast, during electrochemical experiments, a single amino acid reacts with a more or less inert electrode, and the reference couple is in a separate compartment. Thus, an equilibrium constant calculated with electrode potentials obtained from voltammetric experiments should represent the energetics of the proton-coupled electron transfer process that are minimally disturbed. We note that the energetics of electron transfer from tyrosine to the tryptophanyl radical as predicted by their electrode potentials of the respective couples from electrochemistry are in quite good agreement with those determined by pulse radiolysis in oligopeptides, but do not agree with the individual determinations of the electrode potentials by pulse radiolysis. The overall pH dependences of the electrode potentials of Y•,H+/YH couples, Figure 4, follow the Nernst equation except for N-acetyl- L-tyrosine methyl ester. Given the pKa of 4.2 of the tryptophanyl radical,35,36 it is understandable that the point near pH 1 lies below the extrapolated line. There are not sufficient data points to establish an independent pKa for this radical. However, we have no explanation for the observation that the electrode potential of N-acetyl-L-tyrosine methyl ester is less than expected at that same pH. The presence of an electron-withdrawing −NO2 group increases the electrode potential by only 0.14 V (Table 1), which is less than that observed in an aprotic solvent.37 The superior solvation of H+ in water may be the reason for the small increase. The minor change in E°’ caused by the introduction of the nitro group explains why a mutant of the β2 subunit of Escherichia coli class Ia ribonucleotide reductase, one that contains 3-nitro-L-tyrosine at position 122 (3-NO2Y122) 15 ACS Paragon Plus Environment

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instead of L-tyrosine, can still be oxidized to the phenoxyl radical by dioxygen, as catalyzed by the di-iron center.23 The fluorotyrosines show reversible behavior. We ascribe this to mesomeric stabilization of the radical, which also reduces the electrode potential, although the fluorine substituent is electron-withdrawing. Ribonucleotide reductase with global substitution of tyrosine by 3-fluorotyrosine still has 65% of the activity of the wild type.26 The presence of 2,3,5-trifluoro-L-tyrosine in place of L-tyrosine at position 122 impedes the enzyme kinetics and partially changes the reaction path.25 During cyclic voltammetry, 2,3,5trifluoro-L-tyrosine shows slower, quasi-reversible electron transfer kinetics compared to Ltyrosine or N-acetyl-3-nitro-L-tyrosine ethyl ester. Given that, in solution, recombination is the most likely reaction, fluorination would make this pathway less likely, because it blocks access to the phenyl ring. We conclude that E°’ values for tryptophan, tyrosines and modified tyrosines can be determined reliably by cyclic voltammetry and other electrochemical techniques that provide information about the reversibility of the reaction under investigation. Interestingly, the electrode potential obtained for tyrosine is quite close to that obtained by Tommos and coworkers34 for tyrosine in a protein that is designed to prevent direct contact between tyrosine and the surface of the electrode − they do not differ by more than 20 mV.34,38 The combination of absolute electrode potentials determined by electrochemistry with differences from pulse radiolysis experiments provides access to values that cannot be determined by electrochemical means, such as the cysteinyl/cysteine electrode potential.39 The difference between the electrode potentials of the Trp•,H+/TrpH and TyrO•,H+/TyrOH couples measured electrochemically is only 20 mV. From pulse radiolysis results one calculates a difference of 40 mV to 60 mV. Values of +0.96 V and +1.00 V for E°’(TyrO•,H+/TyrOH) and E°’(Trp•,H+/TrpH), respectively, can be used in peptides at pH 7, see Figure 6. 16 ACS Paragon Plus Environment

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Biochemistry

Figure 6 here.

We conclude that, in water, trp is oxidized at a potential that is very close to that of tyrosine. Thus, these two amino acids are, in principle, interchangeable. The Pourbaix diagrams of tryptophan and tyrosine40 show pKa’s of 4.235,36 and 10.131 for TrpH•+ and TyrOH, respectively. The protein environment may influence these pKa’s and allows thereby finetuning of the electrode potentials: a change of 1 pKa unit changes the electrode potential by 59 mV near neutral pH as the curve in the Pourbaix diagram moves to the left or to the right by one pH-unit. In addition, the proton released upon oxidation must be accommodated, which will influence the kinetics and thermodynamics. The rate of electron transfer over a distance of 20 Å can be accelerated at least 20-fold by a “stepping stone”, an aromatic residue41 that allows the electron hole to reside temporarily42 on that residue. Such electron transfer is efficient between redox couples with similar electrode potentials.43 Based on results presented here, the electrode potentials should be approximately +0.98 V. Cysteine, although having a suitable electrode potential, E°’(Cys•,H+/CysH) = +0.93 V39 was found to be less efficient.44 A proton-coupled electron transfer chain has been observed in cryptochromes. These proteins play a role in the animal circadian clocks and, in migratory birds, in sensing the earth’s magnetic field. Cryptochromes are structurally related to photolyases. In Arabidopsis thaliana, FAD oxidizes upon photoactivation TrpH 400. Trp• 400 is reduced by TrpH 377, and Trp• 377 oxidizes TrpH 324. These three tryptophans are conserved. The total distances is approximately 18 Å.45 Trp• 324 may be reduced by a tyrosine in some proteins.46 Cryptochromes also contain several tyrosines. Given the similarity in electrode potentials, it is quite possible that tyrosines also are involved in proton-coupled electron transfer. Their 17 ACS Paragon Plus Environment

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involvement may have gone unnoticed as the absorption of the tyrosyl radical at 406 nm can be obscured by absorption changes of the flavin in that region of the spectrum. Given the agreement between electrode potentials obtained for tyrosine in water, reported here, and in an artificial protein,34 these potentials can be used to estimate the energetics of electron transfer in proteins. In ribonucleotide reductase, the transfer of the electron deficiency takes place from the two-iron center to Tyr 122, to Tyr 356 (possibly via trp 48) in the β2 subunit, to tyr 731, Tyr 730 and finally to Cys 439 in the α-subunit. The total distance is over 35 Å.4 The exact location of Tyr 356 is not known. If the distance between Tyr 122 and Tyr 356 is more than 10-15 Å, Trp 48 is likely involved as a stepping stone. The oxidation of Cys 439 by the nearby47 TyrO• 730 would be thermoneutral, given the reported electrode potentials at pH 7: +0.93±0.02 V and 0.94±0.03 V for the TyrO•,H+/TyrOH and Cys•,H+/CysH couples, respectively, and a K near 1 has been reported.48 Given that we report an electrode potential for TyrO•,H+/TyrOH couple that is slightly higher, +0.96 V, we assume that E°’(Cys•,H+/CysH) is also slightly higher than +0.94 V, although it is difficult to define that potential, as Cys• undergoes rapid intramolecular H-abstraction.49 Experimentally, glutathione reduces tyrosyl radicals, although not very fast,50 and intramolecular oxidation of cysteine by a tyrosyl radical on the ms time scale has been observed in alcohol dehydrogenase.28 In ribonucleotide reductase, the Cys• 439 abstracts a hydrogen from C’3 of a ribonucleotide, the first step of the modification of RNA to DNA. The electrode potential at pH 7 of the (C’•3, H+/C’3H) couple can be estimated. We use 2-propanol as a model compound; the bond dissociation enthalpy of the C-H bond at C2 is 381 kJ/mol.51 The solution energies of the radical and the parent compound are assumed to be the same; one then calculates52 a standard electrode potential of +1.55 V, or +1.13 V at pH 7, with an estimated error of 0.10 V. The abstraction of the C’3 hydrogen is thus slightly uphill, in agreement with an ab initio calculation.47 Experimentally, a rate of at least 1.4 • 104 s−1 has 18 ACS Paragon Plus Environment

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been determined by time-resolved spectroscopy after the photochemically-induced oxidation of Tyr 356.53 The irreversible step is loss of water at C’2 and formation of a double bond between C’3 and C’2. Overall, the Gibbs energy change of the oxidation of C’3 by TyrO• 122 is thus close to zero.


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Acknowledgement The fluorinated tyrosines and N-acetyl-3-nitro-L-tyrosine ethyl ester were a gift of Prof. J. Stubbe of the Massachusetts Institute of Technology. We thank Dr. Walter Prütz, Professor JoAnne Stubbe and Professor Cecilia Tommos for discussion.

The Supporting Information (Figures S1 – S3 and a derivation of the peak potential correction for coupled chemical reaction) to this publication can be found at (http://pubs.acs.org).


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References

1. Winfield, M. E. Electron transfer within and between haemoprotein molecules. J. Mol. Biol. 1965, 12, 600-611. 2. Babcock, G. T.; Barry, B. A.; Debus, R. J.; Hoganson, C. W.; Atamian, M.; McIntosh, L.; Sithole, I.; Yocum, C. F. Water oxidation in photosystem II: From radical chemistry to multielectron chemistry. Biochemistry 1989, 28, 9557-9565. 3. Umena, Y.; Kawakami, K.; Shen, J. R.; Kamiya, N. Crystal structure of oxygenevolving photosystem II at a resolution of 1.9 Å. Nature 2011, 473 (7345), 5560. 4. Nordlund, P.; Reichard, P. Ribonucleotide reductases. Annu. Rev. Biochem. 2006, 75, 681-707. 5. Minnihan, E. C.; Nocera, D. G.; Stubbe, J. Reversible, long-range radical transfer in E. coli class Ia ribonucleotide reductase. Acc. Chem. Res. 2013, 46 (11), 25242535. 6. Prütz, W. A.; Land, E. J. Charge transfer in peptides. Pulse radiolysis investigation of one-electron reactions in dipeptides of tryptophan and tyrosine. Int. J. Radiat. Biol. 1979, 36, 513-520. 7. Prütz, W. A.; Butler, J.; Land, E. J.; Swallow, A. J. Direct demonstration of electron transfer between tryptophan and tyrosine in proteins. Biochem. Biophys. Res. Commun. 1980, 96, 408-414. 8. Prütz, W. A.; Land, E. J.; Sloper, R. W. Charge transfer in peptides. J. Chem. Soc. , Faraday Trans. 1 1981, 77, 281-292. 9. Butler, J.; Land, E. J.; Prütz, W. A.; Swallow, A. J. Charge transfer between tryptophan and tyrosine in proteins. Biochim. Biophys. Acta 1982, 705, 150162. 21 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10. Reece, S. Y.; Stubbe, J.; Nocera, D. G. pH dependence of charge transfer between tryptophan and tyrosine in dipeptides. Biochim. Biophys. Acta 2005, 1706 (3), 232-238. 11. Bobrowski, K.; Holcman, J.; Poznanski, J.; Ciurak, M.; Wierzchowski, K. L. Pulse radiolysis studies of intramolecular electron transfer in model peptides and proteins. 5. Trp.-Tyr. radical transformation in H-Trp-(Pro)n-Tyr-OH series of peptides. J. Phys. Chem. 1992, 96, 10036-10043. 12. Harriman, A. Further comments on the redox potentials of tryptophan and tyrosine. J. Phys. Chem. 1987, 91, 6102-6104. 13. Savéant, J.-M.; Vianello, E. Étude de la polarisation chimique en régime de variation linéaire du potential. Cas d'une désactivation spontanée, rapide et irréversible du produit de la réduction. C. R. Hebd. Séances Acad. Sci. 1963, 256, 25972600. 14. Nicholson, R. S. Some examples of the numerical solution of nonlinear integral equations. Anal. Chem. 1965, 37 (6), 667-671. 15. DeFelippis, M. R.; Murthy, C. P.; Broitman, F.; Weinraub, D.; Faraggi, M.; Klapper, M. H. Electrochemical properties of tyrosine phenoxy and tryptophan indolyl radicals in peptides and amino acid analogs. J. Phys. Chem. 1991, 95 (8), 3416-3419. 16. DeFelippis, M. R.; Murthy, C. P.; Faraggi, M.; Klapper, M. H. Pulse radiolytic measurement of redox potentials: The tyrosine and tryptophan radicals. Biochemistry 1989, 28, 4847-4853. 17. Faraggi, M.; DeFelippis, M. R.; Klapper, M. H. Long-range electron transfer between tyrosine and tryptophan in peptides. J. Am. Chem. Soc. 1989, 111, 5141-5145.


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Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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18. Weinstein, M.; Alfassi, Z. B.; DeFelippis, M. R.; Klapper, M. H.; Faraggi, M. Long range electron transfer between tyrosine and tryptophan in hen egg-white lysozyme. Biochim. Biophys. Acta 1991, 1076, 173-178. 19. Yee, C. S.; Seyedsayamdost, M. R.; Chang, M. C. Y.; Nocera, D. G.; Stubbe, J. Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: Insertion of 3-nitrotyrosine at residue 356 as a probe of the radical initiation process. Biochemistry 2003, 42 (49), 14541-14552. 20. Armstrong, D. A.; Huie, R. E.; Koppenol, W. H.; Lymar, S. V.; Merényi, G.; Neta, P.; Ruscic, B.; Stanbury, D. M.; Steenken, S. Standard electrode potentials involving radicals in aqueous solution: Inorganic radicals. Pure and Applied Chemistry 2015, 87 (11-12), 1139-1150. 21. Costentin, C.; Louault, C.; Robert, M.; Savéant, J.-M. The electrochemical approach to concerted proton—electron transfers in the oxidation of phenols in water. Proc. Natl. Acad. Sci. USA 2009, 106 (43), 18143-18148. 22. Savéant, J.-M. Electrochemical approach to proton-coupled electron transfers: recent advances. Energy Environ. Sci. 2012, 5 (7), 7718-7731. 23. Yokoyama, K.; Uhlin, U.; Stubbe, J. A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase. J. Am. Chem. Soc. 2010, 132 (43), 15369-15379. 24. Yokoyama, K.; Smith, A. A.; Corzilius, B.; Griffin, R. G.; Stubbe, J. Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase. J. Am. Chem. Soc. 2011, 133 (45), 18420-18432. 25. Minnihan, E. C.; Young, D. D.; Schultz, P. G.; Stubbe, J. Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase. J. Am. Chem. Soc. 2011, 133 (40), 15942-15945. 23 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26. Seyedsayamdost, M. R.; Reece, S. Y.; Nocera, D. G.; Stubbe, J. Mono-, di-, tri-, and tetra-substituted fluorotyrosines: New probes for enzymes that use tyrosyl radicals in catalysis. J. Am. Chem. Soc. 2006, 128 (5), 1569-1579. 27. Hayon, E.; Simic, M. Absorption spectra and kinetics of the intermediate produced from the decay of azide radicals. J. Am. Chem. Soc. 1970, 92, 7486-7487. 28. Land, E. J.; Prütz, W. A. Reaction of azide radicals with amino acids and proteins. Int. J. Radiat. Biol. 1979, 36 (1), 75-83. 29. Matsuda, H.; Ayabe, Y. Zur Theorie der Randles-Sevcikschen KathodenstrahlPolarographie. Z. Elektrochem. 1955, 59, 494-503. 30. Cohen, E. R.; Cvitaš, T.; Frey, J. G.; Holmström, B.; Kuchitsu, K.; Marquardt, R.; Mills, I.; Pavese, F.; Quack, M.; Stohner, J.; Strauss, H. L.; Takami, M.; Thor, A. J. Quantities, Units and Symbols in Physical Chemistry. IUPAC Recommendations 2007; 3 ed.; IUPAC & RSC Publishing: Cambridge, UK, 2008. 31. Martin, R. B.; Edsall, J. T.; Wetlaufer, D. B.; Hollingworth, B. R. A complete ionization scheme for tyrosine, and the ionization constants of some tyrosine derivatives. J. Biol. Chem. 1958, 233 (6), 1429-1435. 32. Sokolovsky, M.; Riordan, J. F.; Vallee, B. L. Conversion of 3-nitrotyrosine to 3aminotyrosine in peptides and proteins. Biochem. Biophys. Res. Commun.

1967, 27 (1), 20-25. 33. McCreery, R. L. Advanced carbon electrode materials for molecular electrochemistry. Chem. Rev. 2008, 108 (7), 2646-2687. 34. Berry, B. W.; Martínez-Rivera, M. C.; Tommos, C. Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical. Proc. Natl. Acad. Sci. USA

2012, 109 (25), 9739-9743.


Page 24 of 38

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35. Posener, M. L.; Adams, G. E.; Wardman, P.; Cundall, R. B. Mechanisms of tryptophan oxidation by some inorganic radical-anions: A pulse radiolysis study. J. Chem. Soc. ,Faraday Trans. I 1976, 72, 2231-2239. 36. Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Singh, A. Oxidation of tryptophan and N-methylindole by N3., Br2.-, and (SCN)2.- radicals in light- and heavy-water solutions: A pulse radiolysis study. J. Phys. Chem. 1991, 95, 3639-3643. 37. Hapiot, P.; Pinson, J.; Yousfi, N. Substituent effects on the redox properties of phenolates in acetonitrile. One-electron redox potentials. New J. Chem. 1992, 16, 877-881. 38. Ravichandran, K. R.; Liang, L.; Stubbe, J.; Tommos, C. Formal reduction potential of 3,5-difluorotyrosine in a structured protein: Insight into multistep radical transfer. Biochemistry 2013, 52 (49), 8907-8915. 39. Madej, E.; Wardman, P. The oxidizing power of the glutathione thiyl radical as measured by its electrode potential at physiological pH. Arch. Biochem. Biophys. 2007, 462, 94-102. 40. Prütz, W. A.; Butler, J.; Land, E. J.; Swallow, A. J. The role of sulphur peptide functions in free radical transfer: A pulse radiolysis study. Int. J. Radiat. Biol.

1989, 55 (4), 539-556. 41. Cordes, M.; Giese, B. Electron transfer in peptides and proteins. Chem. Soc. Rev.

2009, 38 (4), 892-901. 42. Cordes, M.; Köttgen, A.; Jasper, C.; Jacques, O.; Boudebous, H.; Giese, B. Influence of amino acid side chains on long-distance electron transfer in peptides: Electron hopping via "stepping stones". Angew. Chem. Int. Ed. 2008, 47 (18), 3461-3463.


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43. Cordes, M.; Jacques, O.; Köttgen, A.; Jasper, C.; Boudebous, H.; Giese, B. Development of a model system for the study of long distance electron transfer in peptides. Adv. Synth. Catal. 2008, 350 (7-8), 1053-1062. 44. Wang, M.; Gao, J.; Müller, P.; Giese, B. Electron transfer in peptides with cysteine and methionine as relay amino acids. Angew. Chem. Int. Ed. 2009, 48, 42324234. 45. Solov'yov, I. A.; Domratcheva, T.; Schulten, K. Separation of photo-induced radical pair in cryptochrome to a functionally critical distance. Scientific Reports

2014, 4, 3845. 46. Chaves, I.; Pokorny, R.; Byrdin, M.; Hoang, N.; Ritz, T.; Brettel, K.; Essen, L. O.; van der Horst, G. T. J.; Batschauer, A.; Ahmad, M. The cryptochromes: Blue light photoreceptors in plants and animals. Annu. Rev. Plant Biol. 2011, 62 (1), 335364. 47. Argirevic, T.; Riplinger, C.; Stubbe, J.; Neese, F.; Bennati, M. ENDOR spectroscopy and DFT calculations: Evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase. J. Am. Chem. Soc. 2012, 134 (42), 17661-17670. 48. Folkes, L. K.; Trujillo, M.; Bartesaghi, S.; Radi, R.; Wardman, P. Kinetics of reduction of tyrosine phenoxyl radicals by glutathione. Arch. Biochem. Biophys. 2011, 506 (2), 242-249. 49. Schöneich, C.; Mozziconacci, O.; Koppenol, W. H.; Nauser, T. Intramolecular 1,2and 1,3-hydrogen transfer reactions of thiyl radicals. Isr. J. Chem. 2014, 54 (3), 265-271. 50. Domazou, A. S.; Gebicki, J. M.; Nauser, T.; Koppenol, W. H. Repair of protein radicals by antioxidants. Isr. J. Chem. 2014, 54 (3), 254-264.


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51. Luo, Y.-R. Tabulated BDEs of C-H bonds. In Handbook of Bond Dissociation Energies in Organic Compounds, CRC Press: 2002. 52. Koppenol, W. H. Oxyradical reactions: From bond-dissociation energies to reduction potentials. FEBS Lett. 1990, 264, 165-167. 53. Olshansky, L.; Pizano, A. A.; Wei, Y.; Stubbe, J.; Nocera, D. G. Kinetics of hydrogen atom abstraction from substrate by an active site thiyl radical in ribonucleotide reductase. J. Am. Chem. Soc. 2014, 136 (46), 16210-16216.


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Table 1. Nernst equation parameters for five different tyrosines and for L-tryptophan at 298 K

a

Compound

Phenolic pKa

Slope (mV/pH)

E° (V)

E°’ (pH=7, V)

L-tyrosine

10.131

-58.5±1.0

1.39±0.01

0.97±0.01

N-acetyl-L-tyrosine methyl ester

10.1a

-58.0±1.0b

1.38±0.01c

0.97±0.01

N-acetyl-2,3-difluoro-L -tyrosine methyl ester

7.826

-58.0±1.0

1.36±0.01

0.94±0.01

N-acetyl-2,3,5-trifluoroL-tyrosine methyl ester

6.426

-55.0±1.0

1.33±0.01

0.91±0.01

N-acetyl-3-nitroL-tyrosine ethyl ester

7.232

-59.7±1.0

1.53±0.01

1.11±0.01

L-tryptophan

---

-56.2±1.0

1.41±0.01

0.99±0.01

Assumed to be not significantly different from the value for L-tyrosine;

b

Between pH=3.8 and pH=7.4; c

Extrapolated, while omitting the value at pH=0.8


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Table 2. Comparison of E°’ (pH = 7, V) values obtained by cyclic voltammetry with and square wave voltammetry.

Compound

Cyclic Voltammetry

Square Wave Voltammetry

L-tyrosine

0.97±0.01

0.93±0.01

L-tryptophan

0.99±0.01

0.95±0.01

1.11±0.01

1.11±0.01

N-acetyl-3-nitroL-tyrosine ethyl ester


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Figure Captions

Figure 1. Cyclic voltammograms. A: 1.0 mM L-tyrosine. Conditions: 0.18 M H2SO4, pH=0.8, carbon micro electrode. Scan rates: a, 100 Vs–1; b, 150 Vs–1; c, 200 Vs–1. B: 1.0 mM N-acetyl3-nitro-L-tyrosine ethyl ester. Conditions: 0.18 M K2SO4/0.005 M H2SO4, pH=3.8, carbon micro electrode. Scan rates: d, 100 Vs–1; e, 200 Vs–1; f, 300 Vs–1; g, 350 Vs–1.

Figure 2. Cyclic voltammograms of 0.5 mM N-acetyl-L-2,3-difluorotyrosine methyl ester (grey line) and N-acetyl-L-2,3,5-trifluorotyrosine methyl ester (black line). Conditions: 0.18 M H2SO4, pH = 0.8, carbon micro electrode, scan rate 1 Vs–1.

Figure 3. Cyclic voltammograms of 0.05 mM L-tryptophan. Conditions: 0.18 M H2SO4, pH=0.8, carbon micro electrode. Scan rates: a, 10 Vs–1; b, 20 Vs–1; c, 50 Vs–1.

Figure 4. pH dependence of E°’ for N-acetyl-L-tyrosine methyl ester (triangles), L-tyrosine (squares), L-tryptophan (diamonds) and N-acetyl-3-nitro-L-tyrosine ethyl ester (circles). Dashed lines represent ideal Nernst plots (298 K) fitted to the E°’ values at pH = 3.8 and 7.4. Every point is the average of multiple determinations. The errors are smaller than the symbols used.

Figure 5. A: Kinetics trace for the equilibration of Trp• and Tyr• radicals in H-Trp-Gly-TyrOH; average of 5 measurements. B: Same for cyclo(Trp-Tyr); average of 5 measurements. The concentration of Trp• was monitored at 510 nm. Conditions: peptide concentration, 0.030 mM; NaN3, 10 mM; phosphate buffer, 1 mM; pH = 7; N2O saturated; dose, 2 Gy.


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Biochemistry

Figure 6. Overview of the old and new electrode potentials of the Trp and Tyr couples. The values are estimated to have errors of 10 mV. The red arrows are the differences in electrode potential determined by the techniques indicated. The red arrow below the horizontal line shows the compromise with the electrode potentials in bold.


Biochemistry

Figures

0.8

A

1.0

c b a

B g f e d

0.4

i / μA

0.6

i / μA

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0.2

0.0 e d

-0.2

f

g

-0.4

-0.6 -0.8

-0.3

0.2

0.7

-0.4

1.2

0.1

0.6

E / V vs. Ag/AgCl

E / V vs. Ag/AgCl

Figure 1


1.1

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20

10

i / μA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0

-10 0.0

0.5

1.0

E / V vs. Ag/AgCl

Figure 2


1.5

Biochemistry

120 c 60

i / nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

b a

0

-60

-120 -0.2

0.3

0.8

1.3

E / V vs. Ag/AgCl

Figure 3


Page 35 of 38

1.5

E°' / V

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1.3

1.1

0.9 0

2

4

6

pH Figure 4


8

Biochemistry

6

6

B

A

5

5

4

4

Abs•1000

Abs • 1000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

3

3

2

2

1

1

0

0

0

50

100

150

200

0

50

t / µs

100

t / μs

Figure 5


150

200

Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Pulse radiolysis Electrochemistry

Tyr, old 0.93

Tyr 0.96

Trp, old

Trp

0.97 V(vs NHE)

0.99

1.00

∆ E°' = 0.04 V

Figure 6


1.02

Biochemistry

TOC Graphics

- e–

- H+ R

OH

+ H+

–

R

O

+ e–

+ e–

R

+•

OH

+ H+

R

O

•

OH

O

- H+

- e– R

R

•

8.9 cm x 2.7 cm

- H+ R

OH

+H

- e–

+

–

R

O

+e

+e

–

O

+•

R

OH

Scalable 16 cm x 4.9 cm


+H

+

R

•

OH

R

- H+

- e– R

–

•

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 38

O

Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3

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