6102
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J . Phys. Chem. 1987, 91, 6102-6104
unchanged. The intensity of the 'Ag 'Bu* emission band also increases relative to those from 'Ag 'Ag* with increasing solvent polarizability and with increasing temperature. We have observed similar behavior upon changing the solvent from hexane (a,= 0.31) to hexadecane (a,= 0.35). Table I1 lists the shifts in the absorption and fluorescence maxima as well as those of the 'Bu* emission band for the arene probes. As expected for excitation to a 'Bu* state, the absorption maximum of each probe is most sensitive to the solvent change. The emission maxima of the DPH probes do not shift upon changing the solvent. This can be attributed to the fact that this emission is predominantly from an 'Ag* state. The emission band on the blue edge of the fluorescence envelope, however, does show sensitivity to the solvent. Not only are substantial shifts of the peak at -385 nm observed (210 and 270 f 30 cm-' for DPH and 4H4A, respectively) but the intensity of this peak relative to that of the maximum also increases with increasing a,. There is no corresponding emission band observed in the spectra of the DPB compounds. However, the fluorescence maximum is sensitive to the solvent for these chromophores. Interestingly, the 4B4A emission appears to be much less sensitive to a,than is DPB. It thus appears that, although the DPB fluorescence occurs from the 'Bu* state, that of 4B4A may contain a high degree of IAg* character. This is supported by the lifetime data shown in Table 111. 4B4A exhibits a long lifetime component (1.1-1.25 ns) that is not observed for DPB. Also, the kf value calculated for each of these compounds, except DPB, increases with increasing as,suggesting that the energy separation between the first two excited-state surfaces decreases (Eb > E,) in all cases except that of DPB. Whether the substituents added to DPB in creating 4B4A causes a reversal in the level ordering of the chromophore or not, the interaction between the two states is clearly greater."
As we progress from trans-stilbene to the longer arenes, DPB and DPH, the photophysics governing fluorescence and photoisomerization certainly become complex. However, Arrhenius studies of the diphenylpolyenes have shown that in each case a small, but significant, barrier to nonradiative decay exists. The large decreases observed in the activation energies calculated for 4B4A and 4H4A demonstrate that seemingly small perturbations to these systems can have pronounced effects on the nature of the excited state. The comparison between the behavior of these molecules and that of their surfactant derivatives in different media may provide us with a more complete picture of the photophysical properties of such systems and how they can be influenced by such factors as solvent viscosity and polarizability. Ultimately, we should be able to apply this enhanced understanding of the behavior of potential probe molecules to the study of more complex systems. More detailed investigations of the effects of solvent and temperature on the fluorescence and isomerization behavior of these four molecules are currently under way in our laboratories. Acknowledgment. We thank the National Science Foundation (grant No. CHE-8616361) for supportpf this research. L.M. is grateful to the FundaqZo de Amparo A Pesquisa do Estado de SBo Paulo for a fellowship. (19) The addition of alkyl substituents to DPH causes a red shift in the absorption spectrum while the same substitution of DPB results in a blue shift of the 4B4A absorption. Taken with the lifetime data in which T~~~ > T~~~~ while that of 4B4A shows a significant com nent of its lifetime which is greater than that of DPB, it appears that the state may be stabilized by substitution in the case of DPH (increasing the coupling between 'Ag' and 'Bu*) while it appears to be destabilized in the case of DPB. Molecular orbital calculations of the effect of alkyl substitution on the ground- and excited-state energy levels of linear diphenylpolyenes may reveal the cause of such apparently opposing effects.
'%*
Further Comments on the Redox Potentials of Tryptophan and Tyrosine Anthony Harriman Davy Faraday Research Laboratory, The Royal Institution, London W l X 4BS, England (Received: August 5, 1987)
Redox potentials for one-electron oxidation of tryptophan and tyrosine, as well as for a few simple indoles and phenols, have been determined by cyclic voltammetry. Mostly, the values observed are in reasonable agreement with those determined earlier by pulse radiolysis but the value observed for tryptophan (Eo = 1.015 V vs NHE at pH 7) is much higher than that derived from pulse radiolysis. The relative magnitude of the redox potentials for tryptophan and tyrosine shows a marked pH dependence.
Introduction Radiation damage to proteins is a subject of enormous significance, especially since it is recognized now that charge migration can facilitate transport of the radical center away from the initial site. Thus, harmful cross-links can be formed at amino acids far removed from the molecule where the primary radiolytic reaction occurs. To assess the importance of such processes, several attempts have been made to employ pulse radiolysis to determine the one-electron redox potentials of common amino acids. Using the method of competitive kinetics, there is general acceptance',* that the redox potentials of tryptophan and tyrosine are around 0.5-0.6 and 0.6-0.7 V vs NHE, respectively, at pH 13. Acid/base equilibria, associated with ground-state and radical species, serve to raise these redox potentials at lower pH but the relationship is nonlinear. Furthermore, whilst there is agreement that the redox potential of tyrosine is more oxidizing than that of tryptophan at pH < 3 there is some controversy3 about the relative values
in neutral solution. This is a particular problem for tryptophan; redox potentials of 0.60, 0.64, 0.87, and 0.98 V have been re~ 0 r t e d . l Because ~~ of the uncertainty about the redox potential for the tryptophan couple tryp
+ H+ + e = trypH
it is necessary to obtain an independent and reliable value for this important amino acid. (1) Jovanovic, S. V.; Harriman, A,; Simic, M. G. J . Phys. Chem. 1986, 90, 1935. ( 2 ) Butler, J.; Land, E. J.; Prutz, W. A,, Swallow, A. J. J . Chem. SOC., Chem. Commun. 1986, 348. (3) Butler, J.; Land, E. J.; Swallow, A. J.; Prutz, W. A. J . Phys. Chem. 1987, 91, 3113. (4) Grossweiner, L. I . Curr. Top. Radiat. Res. Q. 1976, 11, 141. ( 5 ) Butler, J.; Land, E. J.; Prutz, W. A,; Swallow, A. J. Eiochim. Eiophys. Acta 1982, 705, 150.
0022-365418712091-6102$01.50/0 0 1987 American Chemical Society
Letters
08
06
The Journal of Physical Chemistry, Vol. 91, No. 24, 1987 6103 EO
(V) 1.2
1
d
1 1
L
1.2
1.4
v
1.0
0.61
0.8'0.'6
I
0
v s NHE
!
I
1
4
2
I
I
6
I
I
8
l
l
b
Figure 1. Typical cyclic voltammograms recorded for (a) tryptophan and (b) tyrosine in neutral v a t e r . For all the compounds studied, the peak half-widths were 43 5 mV, the peak currents were proportional to initial concentration (C) and square root of the scan rate (u), and plots of peak potential vs log u or log C were linear.
*
TABLE I: Redox Potentials Measured for Selected Phenols and Indoles in Aqueous Solution EO,
'
I
l
PH
b
1.0
1
V vs N H E
compound
pH2
pH7
pH13
tryptophan tyrosine p-methoxyphenol phenol p-cresol indole indole-3-acetic acid
1.15 1.22 0.945
1.015 0.93 0.655 0.86 0.77 0.97 0.84
0.65 0.72 0.44
c
Results and Discussion It has been shown recently that cyclic voltammetry can be used to derive redox potentials for unstable radicals in aqueous solution.6 The agreement between redox potentials for the N,-/N,- and S 0 2 - / S 0 2 couples measured by pulse radiolysis and cyclic voltammetry is excellent7~* and it is clear that this multidisciplinary approach will increase in popularity. We have used cyclic voltammetry to augment our earlier pulse radiolysis studies and obtained redox potentials for one-electron oxidation of tryptophan, tyrosine, and several simple indoles and phenols in aqueous solution. In each case, the compound (1 mM) was dissolved in water containing KC1 (0.2 M) and buffer ( 5 mM) and purged thoroughly with N2. A highly polished, glassy carbon working electrode was used in conjunction with an SCE reference and a Pt counter electrode. Typical cyclic voltammograms are displayed in Figure 1; it was found that all the compounds exhibited diffusion controlled electrode behavior but the oxidation product was unstable with respect to chemical reaction (e.g., dimerization) and no peaks were observed on the reverse scan.9 Under such conditions,10 the observed peak potential (E,) is related to the redox potential ( E O ) by E , = Eo - 0.9(RT/nFj + ( R T / 3 n F ) In (2kCRT/3nFv) where C i s the initial concentration, u is the potential scan rate, and k is the rate constant for the bimolecular chemical reaction. Redox potentials were determined for each compound, as a function of pH by measuring peak potentials at different scan rates and initial concentrations. The results are displayed in Figure 2 and selected values are collected in Table I. A few indoles and (6) Cyclic voltammetry cannot be used to determine redox potentials for all unstable radicals in aqueous solution. However, it has been used successfully in cases where the radical undergoes rapid dimerization to form an electrode-inactive product. See for example: Andrieux, C. P.; Nadjo, L.; Saveant, J. M. J . Electroanal. Chem. 1970, 26, 147. (7) Alfassi, 2. B.; Harriman, A,; Huie, R. E.; Mosseri, S.; Neta, P. J. Phys. Chem. 1987, 91, 2120. (8) Neta, P.; Huie, R. E.; Harriman, A. J . Phys. Chem. 1987, 91, 1606. (9) The cyclic voltammograms recorded for p-methoxyphenol exhibited a peak on reverse scans due to formation of benzoquinone; see: Hawley, D.; Adams, R. N. J . Electroanal. Chem. 1964, 8, 163. (IO) Nicholson, R. S. Anal. Chem. 1965, 37, 667.
,
0.6
0
, 2
,
I
4
I
I
I
6 PH .
I
8
,
,
10
,
I
12
'
Figure 2. Plots of measured E o vs p H for (a) tryptophan (b) tyrosine, and (c) p-methoxyphenol. Trace (d) compares the data obtained for tryptophan and tyrosine.
phenols" were studied at pH 7 to provide some data for comparison. The observed electrochemical data show qualitative agreement with known properties of the compounds. Thus, both p-methoxyphenol and tyrosine gave Eo values that were independent of pH above the ground-state pK and Eo values that increased by ca. 60 mV/pH below the pK. From Figure 2, approximate pK values of 10.6 and 10.3 respectively were obtained for p-methoxyphenol and tyrosine compared to respective literature values'si2 (1 1) Pulse radiolytic studies (ref 14) have estimated Eo for phenol to be >0.80 V at pH 7 whilst direct electrochemical methods have also found Eo = 0.86 V (Pungor, E.; Szepesvary, E. Anal Chim. Acta 1968,43, 289). Some phenols, e.g., p-nitrophenol, adsorbed very strongly to the electrode surface and could not be studied under these conditions. (12) Ionisation Constants of Organic Acids in Aqueous Solution; Pergamon: Oxford, U.K., 1979.
J. Phys. Chem. 1987, 91, 6104-6106
6104
of 10.2 and 9.9. The Eo values found for tryptophan were pHindependent below the pK of the radical (pK = 4.7) and decreased by 60 mV/pH at higher pH. For p-methoxyphenol, pulse radiolysis s t ~ d i e s ' ~have J ~ derived redox potentials of 0.32, 0.40, and 0.46 V at pH 13.5 and 0.6 V at pH 7. The cyclic voltammetry studies gave E o values within the error limits of the pulse radiolysis results. For tyrosine, cyclic voltammetry gave Eo values of 0.93 and 0.72 V, respectively, at pH 7 and 13 compared to values of 0.85 and 0.64 V, respectively, obtained by pulse radio1ysis.I Considering some of the assumptions used to derive the latter values, the agreement is acceptable. For tryptophan, cyclic voltammetry gave higher Eo values than obtained from any of the pulse radiolysis studies and in neutral solution the discrepancy is pronounced. In particular, the Eo measured by cyclic voltammetry at pH 7 is 1.015 V compared to the pulse radiolysis values of 0.64 V obtained by Jovanovic et al.' and 0.87 V reported by Butler et al.* In neutral solution, redox potentials measured for substituted indoles and phenols (Table I) are clearly related to Hammett coefficients for the substituent. However, the amino acid side chain present in both tryptophan and tyrosine raises the Eo of the molecule above that expected for a simple alkyl side chain.15 This is an important finding that merits further study; it appears to confirm the hypothesis' that the amino acid side chain can exert a strong influence on the redox properties of indole. Also, it indicates the dangers of estimating redox potentials for amino acids from simple model compounds and Hammett coefficients.* Comparison of the data obtained for tryptophan and simple indoles (Table 11) infers that the redox potential for tryptophan cannot (13) Steenken, S.; Neta, P. J . Phys. Chem. 1979, 83, 1134. (14) Steenken, S.; Neta, P. J . Phys. Chem. 1982, 86, 3661. (1 5 ) Comparison of the measured Eo values for tryptophan and tyrosine with the alkyl-substitutedmodel compounds given in Table I infers that the amino acid side chain raises Eo by about 160 mV.
be 0.64 V, as obtained recently by pulse radiolysis,' and the cyclic voltammetric value of 1.015 V seems to be the correct figure. It is clear from Figure 2 that the efficiency and direction of electron transfer between tryptophan and tyrosine are related to pH. That tyrosine radicals oxidize tryptophan at pH C 3 and pH > 12 has been demonstrated by pulse radiolysis studies.',* It has also been demonstrated that tryptophan radicals will oxidize tyrosine in neutral solution, although the bimolecular rate constants are low.' According to Figure 2, there is a small thermodynamic driving force for electron transfer in neutral solution. tryp
+ tyrH = trypH + tyr.
AGO = -8 kJ mol-'
Such a modest driving force would explain the relative slowness of electron transfer at pH 7 . However, it has been shown that phenols are easily oxidised only when present in the deprotonated form.' Oxidation of a phenol in neutral solution requires subsequent loss of a proton. tyr.
+ H + + e = tyrH
Thus, oxidation of tyrosine by tryptophan radicals involves an overall hydrogen atom transfer and this could impose a kinetic barrier due to intermediate formation of an inner-sphere complex. Indeed, tryptophan radicals oxidize p-methoxyphenol in neutral solution at a rate well below the diffusion-controlled limit, although there is a substantial thermodynamic driving force. tryp
+ pmpH = trypH + p m p
AGO = -35 kJ mol-'
Because of such slow reactions, which may not involve simple outer-sphere electron-transfer steps, the use of phenols as redox indicators in neutral solution should be avoided; this is almost certainly responsible for the earlier erroneous Eo value obtained for tryptophan.' Acknowledgment. This work was supported by the S.E.R.C.
Excited-State Absorption Spectroscopy of Ortho-Metalated I r ( III)Complexes K. Ichimura, T. Kobayashi, Department of Physics, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
K. A. King, and R. J. Watts* Department of Chemistry, University of California, Santa Barbara, California 931 06 (Received: September 17, 1987)
Excited-state absorption spectra have been determined for the lowest excited state of three complexes of Ir(II1) which contain combinations of 2,2'-bipyridine (bpy) and the ortho-metalating ligands 2-phenylpyridine (ppy) and benzo[h]quinoline. The two complexes containing bpy, Ir(ppy),(bpy)+ and Ir(bzq),(bpy)+, have similar excited-state absorption spectra which consist of three bands in the ultraviolet, visible, and near-infrared regions. These three bands are characteristic of bipyridine radical anions and indicate a MLCT excited state in which a metal electron has been promoted to a A* orbital of bpy. The excited-state absorption of Ir(ppy), also displays three bands in the ultraviolet, visible, and near-infrared regions. However, these bands are less clearly resolved and have somewhat lower extinction coefficients than do the excited-state absorption bands of the complexes which contain bpy. The position of the ultraviolet excited-state absorption band of Ir(ppy), is at 370 nm whereas this band appears at 390 nm in Ir(ppy)2(bpy)+and in Ir(bzq),(bpy)+. This band position is useful in distinguishing charge transfer to the bpy ligand from charge transfer to ppy.
Excited-state absorption spectroscopy has proven to be instrumental in characterizations of metal-to-ligand charge-transfer (MLCT) excited states of transition-metal complexes of 2,2'bipyridine (bpy) and related ligands. Formation of this type of excited state typically results in electronic absorption features in the ultraviolet and visible spectral regions (340-390 and 450-550 nm, respectively) which are characteristic of bipyridine radical 0022-3654/87/2091-6104$01.50/0
anions. A third broad, weak absorption feature in the near-infrared region (600-900 nm) completes the pattern of absorption bands observed for bipyridine radical anions,Is2but this weak band has not yet been reported for a MLCT excited state in metal-bpy ( 1 ) Creutz, C. Comments Inorg. Chem. 1982, I , 293. (2) Konig, E.; Kremer, S . Chem. Phys. Lett. 1970. 5 , 8 7 .
0 1987 American Chemical Society
