Spectroscopic Analysis of Tyrosine Derivatives: On the Role of the

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Spectroscopic Analysis of Tyrosine Derivatives: On the Role of the Tyrosine-Histidine Covalent Linkage in Cytochrome c Oxidase Mariana Voicescu,†,‡ Youssef El Khoury,† David Martel,§ Martine Heinrich,† and Petra Hellwig*,† Laboratoire de Spectroscopie Vibrationnelle et Electrochimie des Biomole´cules, UMR 7177, Institut de Chimie, CNRS-UniVersite´ de Strasbourg, 1 rue Blaise Pascal, 67070 Strasbourg, France, and Laboratoire d’Electrochimie et de Chimie Physique du Corps Solide, Institut de Chimie, UMR 7177 CNRS, UniVersite´ de Strasbourg, 4 rue Blaise Pascal, 67070 Strasbourg, France ReceiVed: May 25, 2009; ReVised Manuscript ReceiVed: August 2, 2009

2′-(1-Imidazolyl)-4-methylphenol (C-N bonding in the ortho′ position at the phenyl group), a model compound for a tyrosine-histidine covalent linkage, was studied with a combined electrochemical and UV-vis/IR spectroscopic approach. Electrochemical analysis of the 2′-(1-imidazolyl)-4-methylphenol model compound by the means of cyclic voltammetry yielded a potential of 0.48 vs ferrocene (1.15 V vs NHE) for the oxidation of the deprotonated form, the reaction being kinetically irreversible. A tentative assignment of the electrochemically induced Fourier transform infrared (FTIR) difference infrared spectra is presented that indicates the deprotonation of the tyrosine before oxidation and importantly the strong influence of the solvent on the spectral properties and on the mechanism of radical formation. Fluorescence lifetimes and pre-exponential factors of the tyrosine-histidine model compounds are presented and discussed in comparison to tyrosine. The tyrosine-histidine fluorescence lifetime is found to be solvent dependent. The influence of the solvent on the reaction mechanism is proposed with regard to the mechanism of electron coupled proton transfer in proteins that include covalently linked amino acid side chains, like the cytochrome c oxidase. 1. Introduction The aromatic side chain of tyrosine (Tyr) is involved in longdistance electron-transfer reactions in enzymes, reactions that are essential steps in the enzyme’s catalytic mechanism. Oneelectron oxidation/reduction of a Tyr side chain occurs in prostaglandin H synthase, galactose oxidase, ribonucleotide reductase, and photosystem II.1-5 It was found that the protein environment of the Tyr can control the function of the redox active amino acid,6-8 among the free amino acid radicals identified in proteins, the redox couple tyrosyl radical/Tyr being essential in many biological processes. Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and catalyzes the reduction of dioxygen to water in energy transduction membrane.9,10 The structural analysis of bovine CcO showed that Tyr244 is covalently linked to His240 between the ortho carbon of the hydroxyl group of phenol and the ε-nitrogen of the imidazole ring. While His240 is one of the ligands of CuB, Tyr244 seems to be a possible redox center in which it holds an extra oxidation equivalent in the P intermediate of its enzymatic reaction.11-14 The proton transfer from Tyr244 to a ferric peroxide generates a hydroperoxo adduct and subsequently CuB was suggested to provide an electron via His240-Tyr244cross-link to cleave the OsO bond of the ferric hydroperoxide; the cross-linked Tyr could serve as a proton donor during the O2 reduction.15-19 The metals in the active site provide three of the four electrons required for OsO bond * Corresponding author. E-mail: [email protected]. † Laboratoire de Spectroscopie Vibrationnelle et Electrochimie des Biomole´cules. ‡ Permanent Address: Romanian Academy, Institute of Physical Chemistry “Ilie Murgulescu”, Splaiul Independentei 202, 060021, Bucharest, Romania. § Laboratoire d’Electrochimie et de Chimie Physique du Corps Solide.

cleavage by oxidation of the Fe2+/CuB+ binuclear center to oxoferryl (Fe4+dO) and HOsCuB2+. In this regard, it is discussed that in the cross-linked Tyr-His, the Tyr residue might supply both the proton and the forth electron required for the transformation via hydrogen atom transfer from the neutral Tyr. Thus, a tyrosyl radical would be generated, placing CcO in the company of a growing number of proteins that utilize redox active amino acids during catalysis.20 It was shown that these aspects are dependent on the physicochemical properties of the Tyr that are changed as a result of the covalent bond to His.11 A deprotonation of the Tyr residue before electron transfer seems likely, and it was discussed at which part of the cycle the tyrosine deprotonates. The protonation states of the Tyr residue have been analyzed for different reaction intermediates and thus for different pH values. The direct observation of protonation reactions during the catalytic cycle of CcO is reported by Nyquist et al.,21 and the deprotonation of a redoxactive site Tyr, plausibly Tyr288, in the F intermediate, is suggested and confirmed by Gorbikova et al.24 Structural and chemical changes of the PM from Paracoccus denitrificans CcO were investigated by Iwaki et al.22,23 In these experiments, transitions from the oxidized to the PM state were initiated by perfusion with CO/oxygen buffer. The investigation of Tyrring-d4 labeled protein had large effects on PM minus oxidized difference spectra, leading to the attribution of a protonated Tyr hydroxyl group present for the oxidized state of the binuclear center. In addition, the deprotonation of a Tyr residue with the formation of the PM intermediate was presented.22 With both Tyr- and His-labeling, and comparing the isotope effects on the amide I regions in PM minus O spectra, it was evidenced that amide carbonyl bonds of Tyr and His are major contributors, and subsequently, a structural alteration in PM is suggested. In addition, it is concluded that the Tyr in the oxidized state of

10.1021/jp9048742 CCC: $40.75  2009 American Chemical Society Published on Web 09/15/2009

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SCHEME 1: Molecular Structure of the Investigated Model Compounds

the enzyme is most likely in the neutral state.23 Using the CO photolysis approach for reaction intermediate formation, the protonation state of Tyr in all the main intermediates throughout the catalytic cycle is reported by Gorbikova et al.24,25 It was found that the deprotonated Tyr280 must be reprotonated later on in the catalytic cycle to serve as a proton donor for the next oxygen reduction event. Moreover, the complete reprotonation of the Tyr is linked to the formation of the one-electron reduced state coupled to the reduction of the CuB site.24 The pH effect was studied by Gorbikova et al.,25 and it was presented that in the PR intermediate, at pH 6, and after O-O bond breaking, the anonic tyrosinate is formed.25 Also, the development of electric potential during formation of the PR species suggests transfer of a proton oriented perpendicularly to the membrane plane and over a distance of 4 Å, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr280 and the bound oxygen.25 Recently, insights into the functional role of the Tyr-His linkage in CcO were reported.26-32 2-Imidazolyl-1-yl-4-methylphenol was synthesized, and it was found that the covalently linked imidazole ring perturbs the acidic dissociation constant of the phenol by more than 1.5 orders of magnitude. This modulation is due to stabilization of the phenolate through an inductive effect that could be even more pronounced in the protein by coordination of the imidazole to CuB. The crosslinked Tyr-His facilitates proton delivery to the binuclear center in the CcO.28,33 Recently, 1-ortho-phenol(acetyl)histidine methyl ester, a cross-linked Tyr-His compound, was synthesized as a chemical analogue of the active site of CcO.34 The structure of the cross-linked compound was investigated by IR, 1H, and 13C NMR, mass spectrometry, and single-crystal X-ray analysis. It was found that the difference in the Fourier transform infrared (FTIR) spectrum, associated with the oxidation of the crosslinked compound, enables the detection of significant perturbation of the phenoxyl radical vibrational bands.34 A model compound of Tyr244-His240 of bovine CcO was synthesized and analyzed by UV resonance Raman, UV absorption, and pH titration.35 Some imidazole vibrations were resonance enhanced upon ππ* transition of phenol, although they were not observed for the corresponding equimolar mixture of imidazole and paracresol, indicating delocalization of π electrons between the imidazole and phenol rings via the covalent linkage.35 In this report, additional studies and further insights regarding covalently bonded Tyr-His are obtained using a spectroscopic characterization by UV-vis absorption, time-resolved fluorescence spectroscopy, and FTIR/electrochemical analysis. In these lines, we focus on 2′-(1-imidazolyl)-4-methylphenol (C-N bonding in ortho′ position at the phenyl group), a model compound for a Tyr-His covalent linkage, the linkage that is most similar to that found in the CcO. The major interest refers to the tyrosyl radical formation in electron transfer and hydrogen abstraction, which can be evidenced from the specific environ-

ment or the specific interactions of Tyr with the specific protein environment. A tentative assignment of the vibrational modes is presented. Fluorescence lifetimes and pre-exponential factors of Tyr and 2′-(1-imidazolyl)-4-methylphenol model compounds are presented and discussed. Electrochemical analysis of the 2′-(1-imidazolyl)-4-methylphenol model compound was made by means of cyclic voltammetry. The solvent effects are discussed. Finally the reaction of the Tyr-His is discussed in the light of the current debate on the CcO mechanism. 2. Experimental Section 2.1. Materials. The structure of the studied compounds is shown in Scheme 1. Tyr-His model compounds (YH and C I) were synthesized by the research team of Prof. van der Donk, Illinois, USA.36 L-Tyrosine was purchased from Sigma. The solutions were prepared in ethanol (EtOH), in EtOH-H2O, EtOH-HCl, and EtOH-NaOH mixtures as well as in acetonitrile (ACN). The EtOH, of spectroscopic grade, was purchased from Merck. The puriss ACN, from Sigma-Aldrich, was used without any purification. Tetrabutylammonium hexafluorophosphate (NBu4PF6) and lithium perchlorate (LiClO4) were purchased from Sigma and Fluka, respectively. The Ludox, AS40 colloidal silica, 40 wt % suspension in water, was purchased from Sigma-Aldrich. 2.2. Methods. The absorption measurements were recorded at room temperature using a CARY 300 SCAN, UV-vis spectrometer (Varian). The spectrometer parameters were the following: average time, 0.1 s; date interval, 1 nm; scan rate, 600 nm/min; SBW, 2 nm; beam mode, double. In the used solvent mixtures, the final concentration of the compounds was 5 × 10-5 M in EtOH, 0.16 mM NaOH, 0.16 mM HCl, and 16% water, respectively. Fluorescence Decays. The fluorescence decays for Tyr-His model compounds were collected using a Fluorolog FL 3-22, Horiba Jobin Yvon, by a time-correlated single-photon counting (TCSPC) technique. The excitation source was an electroluminescent diode (NanoLED-295, Horiba Jobin Yvon) emitting at 295 nm with a bandwidth of about 12 nm and giving pulses of about 730 ps full width half-maximum. The NanoLED diode operated at a 1 MHz repetition rate, coaxial delay of 70 ns, time-to-amplitude converter (TAC) range of 50 ns, and 14.5 nm band-pass. The decay curves were stored in 1024 channels of 0.056 ns/channel. The NanoLED pulse was recorded using a diluted Ludox solution (0.01%). The concentration of the compounds were 1.5 × 10-5 M (in EtOH) and 3 × 10-5 M (in ACN); all measurements were performed at 20 °C. To determine the fluorescence lifetime of the compounds, the fluorescence decay data were fitted by a single- or a triple-exponential function

F(t) ) R1 exp(-t/τ1) + R2 exp(-t/τ2) + R3 exp(-t/τ3)

Spectroscopic Analysis of Tyrosine Derivatives using Decay Analysis Software with reconvolution (DAS6), from Horiba Jovin Yvon. The data were fitted using nonlinear least-squares methods.37 The quality of the data fit was judged using statistical parameters and graphical tests. The reduced chi-squared values were close to 1. The weighed residuals were low and uniformly distributed around zero. The average fluorescence lifetimes, 〈τ〉, were calculated from the following relation: 〈τ〉 ) ∑Riτi2/∑Riτi.38 In all fluorescence measurements, solutions were placed into a 10 mm precision cell made of special optical glass for fluorescence, the final working volume being of 1000 µL. MID-IR Analysis. The solutions of Tyr-His model compounds were prepared in EtOH and ACN, respectively, at 11 mM in the absence and in the presence of 0.1 M NaOH and 0.1 HCl, respectively. The measurements were recorded at room temperature for the spectral regions from 1800 to 1000 cm-1 using a FTIR - Spectrometer Vertex 70 (Bruker). The working parameters were: Resolution, 4 cm-1; Sample Scan Time 128; Background Scan Time 128; Source Setting, MIR; Beam Splitter, KBr; Scanner Velocity, 20 kHz; Detector Setting, LNMLT Photovoltaic. Spectra were recorded with the help of the ATR reflection unit the so-called Harrick MV2 (diamond prism). Cyclic Voltammetry. Measurements were carried out using an AutoLab PGSTAT 302. A homemade three-electrode electrochemical cell has been used. Two platinum (Pt) electrodes were used as a counterelectrode and pseudoreference electrode, respectively. The cell was initially purged with Argon, and this also provided a well-mixed bulk solution. A gold electrode (2 mm in diameter) was used as a working electrode, and prior to the measurements, it was polished with 0.05 µm alumina powder (micropolish Buehler) on a microcloth polishing cloth (Buehler), further sonicated in ultrapure water, washed by ethanol and dried. The working electrode was chemically modified with an aqueous solution of 2 mM Cysteamine39 for 20 min and then immersed into pure water (for 10 min) and in ACN (for 5 min), respectively. As the supporting electrolyte 0.1 M LiClO4 was used, and the concentration of the sample was 1 mM, in ACN. Electrochemical determinations of Tyr and Tyr-His were carried out in the electrochemical cell previously mentioned, with 3 mL of supporting electrolyte solution. The Tyr and Tyr-His samples were then added to the required concentration. The cyclic voltammetric sweeps were recorded in the potential range of 0-1.7 V for Tyr at 50 mV/s (after 1.7 V, solvent oxidation was observed) and of 0-1.55 V, for Tyr-His at 10 mV/s. The obtained values have been scaled with the help of control experiments with ferrocene for each experimental condition and a value of 0.67 V then added in order to obtain the NHE values. Both values are presented throughout the text. FTIR/Electrochemical Analysis. The spectroelectrochemical cell40 was used for FTIR measurements in the 1900-1000 cm-1 spectral region equipped with calcium fluoride windows. A gold grid was used as the working electrode; it was chemically modified with a 2 mM cysteamine solution for 1 h and then washed with distilled water.39 The counter and the pseudoreference electrodes were Pt wires. The working electrode was then set in the cell and pushed against the window to form a thin layer of solution (about 10 µm). A potential step of -1.3-1 V vs ferrocene (-0.63-1.67 V vs NHE) was applied. The sample solutions were prepared as 30 mM in ACN, and NBu4PF6 was added to a final concentration of 0.1 M. The FTIR spectrometer (Bruker IFS 28) was equipped with a deuterated triglycine sultate (DTGS) detector. The scanner velocity was 10 kHz, and 6 × 256 scans were averaged at a resolution of 4 cm-1. The measurements were performed at 5 °C, using a water

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Figure 1. Absorption spectra of 5 × 10-5 M YH model compound in different mixtures of EtOH (with 16% water, 0.16 mM HCl and 0.16 mM NaOH) (A) and in ACN (B).

thermostat. An equilibration time of 3-7 min, the exact time defined for each compound, solvent, and pH, was used, in order to allow the slow electrochemical reaction to take place completely. Electrochemically induced difference spectra were recorded and processed as previously described by Hellwig et al.39 3. Results and Discussion 3.1. UV-vis Absorption Measurements. Figure 1A shows the electronic absorption spectra of (5 × 10-5 M) YH solutions in EtOH, EtOH-water, EtOH-HCl, and EtOH-NaOH. The absorption spectrum has mainly two electronic bands at about 239 nm, which are characteristic to the anion state and assigned to the La (1B1u) transition of para-substituted phenol, and at 289 nm, specified for Tyr absorption. A bathochromic shift for the low energy absorption band (S0-S1* transition) from 289 to 312 nm was evidenced in the case of EtOH-NaOH. This is due to deprotonation of the phenol hydroxyl group in alkaline solution and at the same time the π electrons delocalization in phenol-imidazole that takes place. No significant shifts were found in the case of EtOH-water and EtOH-HCl solutions; no specific solvent interactions take place. However, a decrease in intensity could be observed, Figure 1A. The same behavior (results not shown) was found for YH in ACN and protonated in a solution of 0.1 M HCl (absorption band at 286 nm) as well as deprotonated in a solution of 0.1 M NaOH (absorption band at 313 nm). In Figure 1B, the absorption spectra of YH and C I Tyr-His model compounds, in ACN, are presented in direct comparison. For YH, an absorption band at 286 nm and two shoulders at about 240 and 329 nm, were found. A hypsochromic shift due to the nature of the solvent could be observed. Previously, for

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Figure 2. (A) MID-IR absorption spectra of the YH in EtOH (a), protonated in a solution of 0.1 M HCl (b), and deprotonated in a solution of 0.1 NaOH (c). The YH concentration is 11 mM. (B) MID-IR absorption spectra of the YH in ACN (a), protonated in a solution of 0.1 M HCl (b), and deprotonated in a solution of 0.1 NaOH (c). The YH concentration is 11 mM.

YH in 20 mM Tris-HCl buffer pH 7.4 an absorption band at 283 nm was found.41 The appearance of the shoulders into the absorption spectrum is due to the overlapping of the vibrational bands. In the case of the C I compound, a broad absorption band at about 306 nm could be seen due to Tyr absorption. The displacement is due to C-C bonding, in phenol-imidazole linkage by comparison to C-N bonding and in phenol-imidazole linkage as in the case of YH compound. The absorption bands at about 256 and 265 nm correspond to the La (1B1u) transition of para-substituted phenol as well as to the overlapping of the vibrational bands due to the specific solvent interactions. Only small changes in the absorption spectrum of the YH model compound were observed if the solvent type was changed (i.e., EtOH and ACN). 3.2. IR Measurement. Figure 2A shows the MID-IR absorbance spectra of YH model compound in EtOH (curve a), in the presence of 0.1 M HCl (curve b) as well as in the presence of 0.1 M NaOH (curve c). According to Wolpert et al.42 and Hellwig et al.,33 it was observed that both the imidazole as well as the phenol are protonated in the presence of HCl and deprotonated in the presence of NaOH. In pure solvent (Figure 2Aa), the tyrosine ring is protonated whereas the imidazole ligand remains neutral. We note that the assignments presented are tentative and made by comparing spectra of the isolated compounds. Shifts and splittings are expected due to the reduced symmetry of each ring after covalent bonding. In the deprotonated form, in Figure 2Ac, signals at 1664 and 1510-1487 cm-1 are attributed to the ν(CC) vibrations of the

Voicescu et al. Tyr ring. Signals from the ν(CN) vibrational mode of the imidazole may also be involved here. The modes at 1448 cm-1 arise from ν(CC) and δdef(CH) of the Tyr, the mode at 1294 cm-1 from the ν(Cring-O) vibrational mode. The contributions at 1059/1074 cm-1, Figure 2Ac, may arise from ν(CN) vibrations of the imidazole ring. Upon protonation of the tyrosine, as seen in the data in pure solvent (Figure 2Aa), shifts of all signals can be reported. Obviously, the vibrations are highly coupled. The ν(CC) vibrations of the Tyr ring and the ν(CN) vibration of the imidazole ring are suggested to be included in the signals structure at 1523 and 1495 cm-1, and the ν(Cring-O) vibrational mode is involved in the 1285 cm-1 signal. Signals below 1140 cm-1 arise from ν(CN) and δ NH (ring) modes. Addition of HCl will protonate the imidazole ring, but not change the tyrosine ring features. In Figure 2Ab, however, a full shift of the spectrum is observed, confirming the strong coupling of the molecules vibrations. The essential features are expected in the same spectral area, and in addition, ring modes of the protonated modes may be involved in the signals at 1542 cm-1. A final assignment is difficult to make. Due to the strong coupling of the vibrations, isotope labeling at most positions of the molecule would shift the overall ensemble. The information that can nevertheless be obtained from the infrared data is the signals structure as a function of the protonation state. For a clearer analysis of the potential signals in a proteins environment, we may also investigate the influence of different solvents. Figure 2B shows the data as obtained in acetonitrile. Interestingly, the spectra of the partially protonated form as obtained in the pure acetonitrile and fully protonated, obtained after addition of HCl (curves a and b, respectively), correspond to the data in EtOH. The fully deprotonated form of the compound, however, seems to be highly sensitive toward the solvent, and major changes in signals position and intensity occur. The spectral feature at the position typical for the ring vibrations between 1508 and 1445 cm-1 is very broad and significantly more intense. The ν(Cring-O) vibrational mode area, at about 1317-1248 cm-1, shows the same behavior. Additionally upshifts of the signals indicate the weaker hydrogen bonding, which seems to be possible for the molecule in EtOH. The data obtained in acetonitrile is certainly closer to the proteins environment, since the Tyr-His in cytochrome c oxidase is found in the membrane part close to the active site. 3.3. Time-Resolved Fluorescence Measurements. The intrinsic fluorescence from proteins is regarded as an effective method to study protein conformation and dynamics. The structural information is associated with the sensitiveness of the emission spectrum of tryptophan (Trp) and Tyr residues to the nature of their environments. Fluorescence lifetime measurements have been used to evidence the existence of different and unique protein conformations.43 Conventionally, the decay is resolved in terms of exponential components, and the values of the decay rates and pre-exponential factors of each component are associated with a particular conformation and with the relative population of each conformation.43,44 The fluorescence decay kinetic of 1.5 × 10-5 M YH in EtOH (at λem ) 320 nm) gave a monoexponential fit. The random distributions of weighted residuals (standard deviations) are presented in Figure 3. The fluorescence lifetime value is 2.09 ns (χR2 ) 1.21). That corresponds to a single excited state lifetime. The average fluorescence lifetime value of the YH in 20 mM Tris-HCl is 2.03 ns (χR2 ) 0.99).41 According to literature,45 specific conformations stabilized by H-bonds are

Spectroscopic Analysis of Tyrosine Derivatives

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Figure 3. Fluorescence decay curve of 1.5 × 10-5 M YH model compound in EtOH; λem ) 320 nm; NanoLED pulse, λex ) 295 nm and random distribution of weighted residuals; χR2 ) 1.21; resolution 0.056 ns/channel.

TABLE 1: Fluorescence lifetime and pre-exponential factors of YH model compounds in ACNa compound Y YH C I

τ1, ns

R1

τ2, ns

R2

τ3, ns

R3

〈τ〉, ns

χR2

0.75 0.2256 0.20 0.6326 4.37 0.1418 3.12 1.31 2.22 1 2.22 1.04 2.81 1 2.81 1.30

a The compound concentration is 3 × 10-5 M; λem ) 320 nm, λex) 295 nm; χR2 presents the quality of the fit; the residual goodness of fit values were determined for one- and three-component fits.

responsible for the heterogeneous fluorescence intensity decay of Tyr derivatives in aqueous solution. For 3 × 10-5 M YH in ACN, using the same excitation wavelength, the fluorescence lifetime value is 2.22 ns (χR2 ) 1.04), Table 1. Comparatively, free Tyr in ACN has been investigated. It can be observed (see Table 1) that the fluorescence decay kinetic is a combination of triple-exponentials and three decay components τ1 (0.75 ns) with R1 ) 0.2256, τ2 (0.20 ns) with R2 ) 0.6326, and τ3 (4.37 ns) with R3 ) 0.1418. These indicate the presence of more than one conformer in the excited state, differentially emitting the energy. This reflects the heterogeneity of the Tyr environment and the change in its conformation. The lifetime value of C I model compound (3 × 10-5 M) in ACN is 2.81 ns (χR2 ) 1.30), Table 1. The difference in fluorescence lifetime observed (0.59 ns) is due to the C-C covalent linkage Tyr-His, in the ortho-position of the phenol group. The average lifetimes values found for the fluorescence decay of YH and C I model compounds in ACN are higher than the lifetime values of this model compound in aqueous solution.41 The average fluorescence lifetime values for YH (2.22 ns) and C I (2.81 ns) model compounds in ACN are smaller than the average fluorescence lifetime value of free Tyr (3.12 ns). It should be pointed out that for free Tyr in ACN, the relative contribution of the shortest lifetime, 0.20 ns with a preexponential factor of 0.6326, represents the majority. Such a shift in the relative amplitude reflects an increase in the population associated with the fast-decaying conformer. Therefore, the fraction of Tyr residue in a given conformation is higher when the lifetime is shorter. Moreover, this could be due to the formation of conformers that produces effective quenching of Tyr fluorescence; the fluorescence emitting conformer being sensitive to events in its immediate vicinity.

The solvent effect is partially due to the solvophobic interaction as well as to charge transfer interactions. It is known that water has a high dielectric constant (ε ) 80), and ionic charge interactions in water are relatively weak in comparison to electrostatic interactions in the interior of a protein, where the dielectric constant (ε ) 2-40) has approximately a factor of 1:40 to that of water, 1:2. The ε value of ACN is 36. Consequently, the strength of an electrostatic interaction in the interior of a protein, where the dielectric constant is low, may be significant. The model compound studied here corresponds to a tyrosine residue located in a buried and hydrophobic part of a membrane protein. Measurements performed in water do not correspond to the internal environment of a membrane protein. We note that electrostatic interactions in water are lower than those in other solvents because of the water’s high dielectric constant, which results from the tendency of the large dipoles of water molecules to align with any electric field.46 In addition, the polarity of the solvent plays an important role in the YH fluorescence quenching mechanism in the Tyr emission for the molecule where the His covalently linked to Tyr. The covalent linkage is significant for the differences observed in the fluorescence lifetime in EtOH and ACN. This is important for the intrinsic fluorescence of Tyr residue in the proteins like CcO, where the covalent linkage Tyr-His is the present. It is well-known that there is a variety of redox states of CcO where the spectroscopically observed signals appear not to account for all the electrons that have been donated to the binuclear Haem-Cooper active site.13 3.4. Cyclic Voltammetry Analysis. Tyr is found to be electroactive at most metal electrodes, and its characterization is of interest for the elucidation of the interfacial behavior of proteins.47,48 Recently, studies on tyrosine side chains as an electrochemical probe of stacked β-sheet protein structures were reported.49 Electrochemical Tyr oxidation is well-known to be a quite complex process, and experimental conditions such as electrode material and the pH value affect its properties. The oxidation peak is found to be kinetically irreversible in all cases studied, connected with radical formation. Its exact position is pH dependent.50-53 The possibility of polymerization is also discussed.54 For all these studies, carbon electrodes were used, mostly GCE, the measurements being done in a similar pH range. Figure 4A shows the cyclic voltammogram obtained at a gold electrode for 1 mM YH in ACN, in the presence of 0.1 M NaOH, at a potential sweep rate of 10 mV/s. Using ferrocene as a reference (E ) 0.46 V), a well-defined oxidation peak was found at +0.48 V, attributed to Tyr oxidation. Adding 0.67 V for NHE values, we obtain 1.15 V for the oxidation of the molecule. The value is in line with earlier observations on similar compounds.36 It may be pointed out that the peak intensity decreases with the number of scans, and a slightly more positive oxidation peak, at about 0.70 V, is found. This shift is associated with the strong adsorption of the species on the electrode surface; a small dependence of the surface structure of the electrode of the electro-oxidation products is taken into consideration. It can be also seen in Figure 4A, that no peak was observed in the reverse scan, confirming, as mentioned above, that the oxidation reaction is kinetically irreversible and that the kinetics of oxidation is more rapid than that of the reduction. The cyclic voltammogram of 1 mM Tyr in ACN in the presence of 0.1 M NaOH and for a potential range of 0 to 1.7 V at 50 mV/s scan rate is shown in Figure 4B, for direct comparison. We could observe an oxidation peak at +1.50 V,

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Figure 5. FTIR difference spectra of the YH model compound in ACN for a potential step from -1.3 to +1 V (-0.63-1.67 V vs NHE) in the spectral range from 1800 to 1060 cm-1: 0.1 M NBu4PF6 as supporting electrolyte. The signal region of the solvent was omitted for clarity.

Figure 4. Cyclic voltammetric profile of adsorbed 1 mM YH in ACN, in the presence of 0.1 M NaOH, at 10 mV/s (A) and 1 mM Y in ACN, in the presence of 0.1 M NaOH, at 50 mV/s (B): 0.1 M LiClO4 as the supporting electrolyte. Note: The values of E ) 0.94 V, for YH, and E )1.50 V, for Y, respectively. The correction vs the ferrocene redox couple led to values at 0.48 and 1.04 V, respectively. (1.15 and 1.71 V vs NHE)

and in comparison to ferrocene, we determine the oxidation peak of Tyr at +1.04 V. Adding 0.67 V for NHE values, we obtain 1.71 V for the oxidation of the molecule. No significant reduction peak is evident, and again, the oxidation peak intensity decreases with the increase of the number of cycles (decrease not shown), indicating that an adsorption was induced on the electrode surface. Also, Tyr polymerization is taken in consideration. Therefore, the imidazole may change the redox potential and increase the irreversibility. Directly comparing free Tyr (Epeak_oxidation ) 1.04 V) and YH (Epeak_oxidation ) 0.48 V) vs ferrocene and Tyr (Epeak_oxidation ) 1.15 V) and YH (Epeak_oxidation ) 1.71 V) vs NHE, both in ACN, a significant lowering of the oxidation potential is found, which is in line with the previous suggestion that imidazole withdraws electron density from the phenolate.28,55 We note that both values describe the oxidation of the deprotonated form. 3.5. FTIR Coupled Electrochemical Analysis of Tyrosyl Radical in the YH Model Compound. To complete the investigation of the YH model compound, electrochemically induced FTIR difference spectroscopy was performed. The electrochemically induced FTIR difference spectra are used to identify the interactions and reorganizations of Tyr residues upon electron transfer and coupled proton transfer reactions.33 Figure 5 presents the reduced minus oxidized (bold line) FTIR difference spectrum of the YH model compound for a potential step from -1.3 to 1 V, and inverse (dotted line) in ACN and in the presence of NaOH. The potentials are given relative to the ferrocence redox couple. The positive signals of the bold line correspond to the reduced and the negative to the oxidized form. The positive signals would thus correlate to the data obtained

by absorbance measurements in ACN, and several signals correlate with the spectrum shown in Figure 2Bc, indeed. This includes the vibrational ring mode at 1664 cm-1 and the δ(C-O)/ν(C-C) vibrational modes around 1321-1294 cm-1. Signals corroborating with the oxidized form are seen with a broad feature at 1524 cm-1. This position is close to the vibrational mode of the radical proposed for isolated tyrosines by Berthomieu et al.56 It is most likely including coordinates from the ν(CsO)/ν(CdC) vibration. A very small signal at 1755 cm-1 is seen. It may be due to a fraction of the radical that is present in the carbonyl conformer, as discussed with Figure 6. Further signals in the lower frequency range can be detected at 1277 and 1215 cm-1. We note that between 1479 and 1360 cm-1 the C≡N vibrations from ACN overlap the data, and this part of the spectrum was thus omitted. The spectra presented here seem to be different to the ones presented by Cappuccio et al.34 on the first look; however, this can be easily explained by the ligands added to the later model, since they change the symmetry and thus the spectroscopic properties. When the YH model compound was measured in aqueous solution, in 20 mM Tris-HCl pH 7.4, for a potential step from 0 to 0.7 V, a very strong stretching mode was found at 1717 cm-1 and attributed to oxidized ν(CHO) Tyr, and the contributions appearing at 1489/1519 cm-1 were assigned to the tyrosyl radical formation.41 The solvent seems to strongly change the preferred structure and increase the CdO conformer. A model for the possible electron and the coupled proton transfer paths via tyrosyl radical formation in the YH model compound is shown in Figure 6. The presence of the carbonyl function is essentially excluded in the ACN experiments; however, it was found in aqueous solution. These data give an insight into the IR spectra of both conformers. 4. Conclusions Tyrosine is one of the most important amino acid residues in protein structures. In this work we present some insights on the role of the histidine covalently attached to tyrosine, the linkage that is present and highly conserved in proteins like cytochrome c oxidase. This linkage was previously studied by several groups, and it is widely accepted that imidazole

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Figure 6. Proposed mechanism of tyrosyl radical formation.

substitution lowers the pKa of phenol and decreases the redox potential of the phenol derivative due to its σ-electron withdrawing ability.28,55 The electrochemical studies made here reveal a kinetically irreversible mechanism showing that the imidazole substitution does not only lower the redox potential for the oxidation but decreases even more the efficiency of the back reaction. This is confirmed by the strong overpotential of more than -1.2 V that needs to be applied for obtaining a reversible FTIR spectrum. The slow kinetics of the back reaction might explain why tyrosine, after being oxidized, is able to be stabilized so efficiently as a radical in proteins. We focused on the solvent dependence of the mechanism of electron coupled proton transfer in the Tyr-His model compound. The presence of the conformer with a carbonyl function in the oxidized form seems unlikely on the basis of the electrochemically induced FTIR difference spectra (cf. Figure 6), and the phenoxyradical seems stabilized. The data obtained in acetonitrile is certainly closer to the proteins environment, since the Tyr-His in cytochrome c oxidase is found in the membrane part close to the active site. The solvent effect is partially due to the solvophobic interaction as well as charge transfer interactions. In addition, the polarity of the solvent plays an important role in the YH fluorescence quenching mechanism in the Tyr emission for the molecule where the His is covalently linked to Tyr. The covalent linkage is significant for the differences observed in the Tyr fluorescence lifetime in ethanol (2.09 ns) and acetonitrile (2.22 ns). This is important for the intrinsic fluorescence of Tyr residue in the proteins like cytochrome c oxidase, where the covalent linkage Tyr-His is present. As mentioned in the introduction, the protonation state of the covalently linked Tyr280 (Paracoccus denitrificans numbering) is a crucial question, that was addressed by a number of FTIR spectroscopic studies on cytochrome c oxidase.22-25,33 The model compound used there shows for the deprotonated tyrosine a signal structure at 1321 and 1294 cm-1 and would confirm the attribution of this spectral area to be critical for this covalently bound tyrosine. We note the protonation will change in function of reaction intermediate formation and details of data acquisition. In addition,

within the protein, the local pK values may significantly change during real time electron and proton transfer for each intermediate, an analysis of each step is necessary. In conclusion, the ring oxygen of tyrosine tends to lose its proton when the residue is oxidized to its radical form. This reaction is strongly supported by the histidine linkage, and its kinetic reversibility is low. As a result, the proton-coupled electron transfer is more favorable, a characteristic that may allow the tyrosine relay or acceptor to be switched on and off by protein environments or conformation changes that favor or disfavor proton transfer. Acknowledgment. This work was supported by CNRS (Centre National de la Recherche Scientifique), Universite´ de Strasbourg, ANR (Agence National de Recherche). M.V. is grateful for financial support from the CNRS. The authors are indebted to Prof. van der Donk’s team, UIUC, Illinois, USA, for Tyr-His model compounds synthesis. References and Notes (1) Tsai, A.; Kulmacz, R. J.; Palmer, G. Spectroscopic evidence for reaction of prostaglandin H-synthase-1 Tyrosyl radical with arachidonic acid. J. Biol. Chem. 1995, 270, 10503–10508. (2) Whittaker, M. M.; DeVito, V. L.; Asher, S. A.; Whittaker, L. W. Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase. J. Biol. Chem. 1989, 264, 7104–7106. (3) Sjoberg, B. M.; Reichard, P.; Graslund, A.; Ehrenberg, A. The tyrosine free radical in ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 1978, 253, 6863–6865. (4) Barry, B. A.; Babcock, G. T. Tyrosine radicals are involved in the photosynthetic oxygen -evolving system. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7099–7103. (5) Boerner, R. J.; Barry, B. A. Isotopic labeling and EPR spectroscopy show that tyrosine residue is the terminal electron donor, Z, in manganesedepleted photosystem II preparations. J. Biol. Chem. 1993, 268, 17151– 17154. (6) Hoganson, C. W.; Babcock, G. T. Protein - tyrosyl radical interactions in photosystem II studied ESR and ENDOR Spectroscopy: Comparison with ribonucleotide reductase and in Vitro tyrosine. Biochemistry 1992, 31, 11874–11880. (7) Barry, B. A.; Einarsdottir, O. J. Phys. Chem. B 2005, 109, 6972– 6981.

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