Highly Efficient Charge Transfer in Peptide Cations in the Gas

J. Phys. Chem. 1996, 100, 18567-18585

18567

Highly Efficient Charge Transfer in Peptide Cations in the Gas Phase: Threshold Effects and Mechanism R. Weinkauf,* P. Schanen, A. Metsala,† and E. W. Schlag Institut fu¨ r Physikalische und Theoretische Chemie, TU-Mu¨ nchen, 85747 Garching, Lichtenbergstrasse 4, Germany

M. Bu1 rgle and H. Kessler Institut fu¨ r Organische Chemie und Biochemie, TU-Mu¨ nchen, 85747 Garching, Lichtenbergstrasse 4, Germany ReceiVed: March 27, 1996; In Final Form: August 26, 1996X

We present new experimental data demonstrating specific, photoactivated positive charge migration in isolated peptide radical cations. The effect exhibits a threshold behavior, which we can directly correlate with energetics of local electronic states. A new very efficient mechanism for charge transfer in cations is proposed that involves an extended coulomb state (EC) of shakeup character. Our investigations are performed on laserdesorbed, cooled, neutral peptides in the gas phase. Charge localization in the peptide is achieved by resonant UV two-photon ionization at an aromatic chromophore. Charge flow in the cations can be activated by absorption of a first visible (VIS) photon. Presence of charge in the aromatic chromophore is probed by resonant absorption of a second VIS photon and monitored by dissociation. While this charge detection is found to work in isolated, positively charged chromophores or amino acids, it is efficiently quenched in some peptides. We explain this by photoactivated charge transfer and charge storage in nonaromatic groups of the peptides. At threshold this process is found to be strongly dependent on amino acid substitution even far away from the site of photoactivation. For analysis we first set up a local molecular orbital model for peptide cations and subsequently obtain a landscape of local electronic cation states formed by local hole and low-lying extended coulomb states. Charge transfer is found to be a through-bond mechanism involving energetically accessible electronic states along the path of charge flow. Charge transfer between hole states is mediated with very high efficiency through saturated carbon bridges by extended coulomb states. This new mechanism seems to be generally applicable to large extended molecular radical cations. Only barriers of the size of a full length of a certain defined amino acid are found to block charge transfer. The model qualitatively accounts for the order of the rates of the processes involved.

I. Introduction The dynamics of proteins in its many facets is patently important and its nature is without doubt peculiar to the properties of the constituent amino acids. One of the most essential aspects of this dynamics involves the extremely rapid transfer of charges and excitation energy often serving as an energy pump for biological systems. These processes can be successfully seen and studied already in medium-sized polypeptides. We introduce a new model for the understanding of charge-transfer processes in cationic peptides and demonstrate its applicability to a series of tailor-made polypeptides. Charge transfer is a fundamental process in bioenergetics such as in photosynthesis.1-3 In native charge-transfer systems an electron is transported between donor and acceptor through large peptides and proteins.3-6 Here “through-bond” and “throughspace” mechanisms can contribute to charge transfer. Because of the complexity of the peptides, the importance of individual amino acids in controlling electron transport is not yet understood in detail. We will demonstrate here that it can be highly specific and strongly dependent from amino acid composition. In typical charge-transfer model systems special donoracceptor constituents are used to enable local photoexcitation * To whom correspondence should be addressed. † Present address: Institute of Chemistry, Akadeemia tee 15, EE 0026 Tallinn, Estonia. X Abstract published in AdVance ACS Abstracts, November 1, 1996.

S0022-3654(96)00926-4 CCC: $12.00

which triggers the charge-transfer process. We are investigating charge transfer and charge migration in native peptide cations without additional donor and acceptor units attached, in order to understand the detailed contribution of the individual amino acids to the charge migration process not mitigated by bridgehead structures at both ends. Bridges spacing donor and acceptor complexes are usually consisting of rigid saturated carbon chains, a molecular system far away in its properties from peptide chains. Only few experiments on model charge-transfer systems with donor and acceptor spaced by peptide oligomer bridges have been carried out.7-9 Oligopeptides, due to their chainlike valence structure and their functional groups, spaced by short saturated carbon bridges (Figure 1a), provide a special one-dimensional model systems. It is this linear, repetitive structure which is causing special effects in transport of positive charge through peptides as we will demonstrate. The positive charge conductivity of peptides can be regarded as being analogous to a molecular wire, similar to concepts presented by Ratner and co-workers.10 We here show that a very efficient charge-transfer mechanism is active in radical cations of peptides. For treating charge transfer in the gas phase, the relevant potentials of course do not include solvent effects (see ref 11) but rather refer to normal coordinates of the molecule. In this case charge transfer becomes a pure intramolecular relaxation © 1996 American Chemical Society

18568 J. Phys. Chem., Vol. 100, No. 47, 1996

Figure 1. (a) Structure of the peptide Ala-Ala-Ala-Tyr. We typically use peptides with a single aromatic chromophore in the C-terminal position and nonaromatic amino acids in the chain and at the N-terminus. (b) In a local picture of the peptide by resonant twophoton excitation a local ionization at the aromatic side chain is possible (see text). Without further photon activation the positive charge stays there because of the low ionization potential of the chromophore. (c) Laser mass spectrum of Ala-Ala-Ala-Tyr: UV photon excitation of the radical cation results in an internal energy of 4-5 eV. The observed fragment ion of mass 44 Da can be identified as a positive charged N-terminal fragment (see a). Its occurrence can be exclusively explained by charge migration.

process12-15 similar to internal conversion14,15 taking place between two electronic states located at different sites of the molecule. The theoretical description leads to an energy gap law for the charge-transfer rate15 similar to that found for internal conversion.16,17 For neutral molecules in solvents intramolecular energy is not conserved, and the charge-separated state is strongly stabilized by solvent shielding. In the gas-phase molecular18-20 and cluster charge-transfer systems21-23 mostly implode and form exciplexes. This is due to the large coulomb force resulting from charge separation. Bixon, Jortner, and others12,13 have analyzed charge transfer in neutral isolated supermolecules and suggested that charge separation should have a limited range of 7 Å. So far there is only one case where evidence for longrange electron transfer in the gas phase has been observed.20 There is no distance restriction expected for anions and cations.12 As this indicates the situation in cations for charge transfer is quite different than that for neutrals: (i) By local ionization of peptides a positive hole is created in the HOMO orbital of an aromatic chromophore. Due to this surplus charge, transfer becomes a “charge shift” process. (ii) The motion of the charge does not substantially affect the global geometry because of the nonbonding character of

Weinkauf et al. the electronic states involved and the lack of electron-hole coulomb attraction. This is clearly in contrast to neutral chargetransfer systems where due to the large coulombic forces (gas phase) or the strong shielding of the solvent (liquid) the geometry of the charge-transfer state is typically strongly distorted. (iii) The electronic states involved are essentially localized. They correspond either to localization of the positive charge at different functional sites in the peptide cation or to shakeup states. (iv) In cations quartet states are situated at high energies. Relaxation processes of excited states therefore are exclusively intra- or interchromophore processes of “internal conversion” type. (v) In linear extended cations shakeup states can be extremely low in energy due to coulomb forces (extended coulomb states). They can efficiently mediate charge transfer through short saturated carbon bridges. The low energy of such states appears as a general characteristic of linear extended radical cations. We believe that these states are the essential element facilitating charge migration in most large molecular radical cations. (vi) Because of the small energetic spacings of electronic states an energetic order of local electronic states can be tailored in a staircase fashion: Charge transfer to the other side of the peptide then becomes a multistep process probably without any barrier. To our knowledge such a barrierless “through-bond” multistep mechanism has so far not been investigated. The arguments given in i-vi show that investigation of the mechanism of charge flow in peptide cations in the gas phase represents a new approach to the study of charge transfer in pure proteins. New insights in the mechanism of charge migration in radical cations are provided. Although the experimental investigation of peptides in the gas phase is in some ways much more complicated, the above-mentioned advantages greatly simplify the interpretation of data for these molecular systems and permit us to understand the mechanism by a molecular description in local molecular orbitals together with their couplings. It should be noted that gas-phase investigations of ionization, charge shift and dissociation processes have essential advantages: (i) Ionization has large cross section because no charge recombination after ionization as in solvents can occur. (ii) No reorganization of the solvent has to be considered. (iii) The detectability of isolated ions and electrons in the gas phase is high. Investigations of electron transfer in peptide cations without cofactors are known only for few solution phase experiments.24-28 In those experiments, however, protonation and deprotonation was involved. Little has been done so far on peptide radical cations under isolated conditions.29-31 Special tailor-made peptides have been synthesized for these investigations which can be photoexcited locally. Subsequent charge migration is steered by choosing a suitable amino acid sequence. Thus control of the position of the charge will be possible. Usually we have a single aromatic chromophore in the peptide to ensure resonant photoexcitation and hence local ionization of the chromophore site. As an example of a typical sample the peptide alanyl-alanyl-alanyl-tyrosine (Ala-Ala-AlaTyr) is shown with its structure in Figure 1a. As discussed previously29 resonant (1+1)-UV photon absorption at the aromatic side chain of tryptophan (Trp), tyrosine (Tyr), or phenylalanine (Phe) is site selective. Local ionization is easily achieved due to electronic selection rules and energetic conditions (see Figure 1b). This initial charge localization is the inherent precondition for the observation of charge flow in the

Charge Transfer in Peptide Cations cation. In our previous paper29 and in Figure 1 we show that after UV excitation of peptide cations charge migration can occur. This is derived by the type of fragment ions found in the mass spectra. For example in the UV laser dissociation mass spectrum of the Ala-Ala-Ala-Tyr cation (Figure 1c) a fragment ion of mass 44 Da is observed, which is undoubtedly due to the positively charged immonium fragment ion shown on the left hand in Figure 1a. Despite that the positive charge was initially located at the hydroxyphenyl chromophore ((1+1)ionization) the charge is finally found at the N-terminal fragment ion. Such charge transfer at high internal energies has been observed for Leu-Leu-Tyr,29 Leu-Leu-Leu-Tyr, Ala-Ala-Tyr, Ala-Ala-Tyr-Ala-Ala, and other peptides.32 The process of charge migration exists in peptide cations and must have a threshold. Our goal here is to investigate this threshold and to study how charge transfer can be correlated to the properties of the peptides and its amino acid subunits. Charge transfer directly after ionization has been shown to occur by Levy and co-workers for some bichromophoric molecules33 and by us for N,N-dimethylphenylethylamine.34 For peptides containing only one aromatic amino acid, it is obvious that electron transfer directly after ionization is impossible for energetic reasons (see local ionization energies in Figure 1b and Table 2). Charge migration in these peptide cations requires photoactivation and takes place in excited electronic states. The absorption shift to the green, which can be found for aromatic molecules upon electron removal and the fact that charged nonaromatic amino acids as glycine and leucine do not absorb in this wavelength range allows site- and charge-sensitive photoexcitation (see section V.II). Detection of a doubly resonant two-photon excitation of the peptide cations is then realized via dissociation. Note that the kind of fragment ion in such an experiment is not of interest but only the total fragment ion current. If charge is stored outside the aromatic chromophore throughout the duration of the laser pulse, no fragmentation will be detected. In this work we describe our pump-probe technique in detail and present new results for peptides differing in size, in chromophores, and in nonaromatic constituents. For some peptides we observe two-photon absorption in the cation, for some not. Investigations show that the effect of quenching of the two-photon excitation is not simply correlated to the molecular size or to absorption changes. Several alternative explanations are discussed in detail, but charge transfer is argued to be the only reasonable mechanism. Furthermore, we present a model of charge transfer in peptide cations based on a local molecular orbital picture and a landscape of local electronic states. This picture allows us to estimate the energetics of the process. We find that energetically accessible electronic states of functional groups are efficiently coupled by low energetic extended coulomb states. In case of charge transfer we find a staircase-like situation without any barrier for charge flow. Time scales of rates are qualitatively in agreement with this model. II. Experimental Setup Large biological molecules are thermally unstable. Thus, for observation in the gas phase, they have to be evaporated by special laser desorption techniques.35 Ionization of neutrals is performed by multiphoton ionization by UV laser pulses, a technique that has been proven to be a suitable tool for mass spectrometrical analysis.30,31 The experimental setup used here is similar to that of our previous experiments.29 In the first vacuum chamber the nonvolatile molecules and peptides were laser desorbed by Nd:

J. Phys. Chem., Vol. 100, No. 47, 1996 18569 YAG laser pulses (1064 nm, 1 mJ/pulse, 200 ns pulse duration). The sample holder was a stainless steel rod homogeneously covered by the peptide sample by means of electrospray. The sample rod was turned by a stepping motor to ensure that for each cycle a new surface was desorbed. The desorption process took place in a closed channel (diameter 1 mm, 6 mm long) which is filled by Ar gas through a pulsed nozzle (3 bar backpressure, nozzle diameter 400 µm). In the Ar beam the molecules are cooled and transported through a skimmer into the ionization chamber of a reflectron time-of-flight instrument. The pressure there was typically 10-6 mbar. Only neutral molecules can reach the ion source due to the permanently applied voltages of +1600 V (Repeller) and +1000 V (second ion source electrode). The local ionization of the intact and cold neutral molecules is performed by resonant two-photon UV ionization with the frequency doubled output of an excimer laser pumped dye laser (UV pulse energy 100 µJ, focus diameter 500 µm, pulse width 5-7 ns, wavelength range 260-290 nm). By carefully adjusting the UV laser intensity, it is possible to achieve “soft” ionization.29-31 Thus, neither neutral fragmentation nor subsequent ionization of fragments contribute to our mass spectra, nor is a further UV photon absorbed by the cation. The latter statement can be derived from the low dissociation threshold for formation of the immonium ion as discussed previously.29 Photodissociation is exclusively performed by a second dye laser in the visible (VIS) wavelength region, which was pumped by the same excimer laser. We employed a time delay of 0 and 10 ns between ionization laser (laser 1, UV) and photofragmentation laser (laser 2, VIS) and found that the results have been independent of this delay and fixed it to 10 ns. Typical pulse energies of the visible laser radiation have been 1.5 mJ at a focus diameter of 1 mm. The pulse width was 7-10 ns at wavelengths of 480-550 nm. To ensure complete overlap between the UV and VIS lasers, resonant multiphoton dissociation of benzene was monitored for the adjustment before and after each peptide experiment. Mass separation was performed in a reflectron time-of-flight mass spectrometer. The ion signal was digitized by a transient recorder and stored in a computer at a repetition rate of 6 Hz. Mass spectra have been averaged over 100 shots. Calculation of electronic state energies and molecular structures have been performed on a silicon graphics work station and a CONVAX by MOPAC at a PM336 level taking into account configuration interaction. Due to the time-consuming calculations for peptides, we confined our investigations at this level of theory to the tripeptide Gly-Gly-Tyr. Structures of other peptides have been calculated at a MNDO level. The energetic positions of extended coulomb states in peptides have been estimated by calculation of such states for small molecules at a PM3 level including configuration interaction. We are aware that due to the three singly occupied orbitals these energetic values only qualitatively describe the situation (see section V.II.I). III. Concept of the Site-Sensitive Charge Probe In the tailor-made peptides we employ here, charge transfer can occur only in electronic excited states of the cation (see section V.II). For site-selective activation and site-selective charge probing we make use of the fact that the absorption of aromatic chromophores changes strongly upon electron removal. This dependence on the charge state of the chromophore produces a switching effect since the VIS absorption is switched on or off. For the understanding of the relevant processes in peptides we first need a short review of the photoinduced processes in isolated aromatic chromophores.

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Figure 2. Photoabsorption of aromatic chromophores turns from ultraviolet to green by electron removal. (a) UV-VIS ionization and ion excitation scheme in aromatic molecules. Multiresonant multiphoton excitation either direct or after internal conversion can be detected by dissociation. (b) Molecular orbital scheme and photoexcitation steps: Excitation of the cation is explained as electron hole shifting downward in the manifold of bonding electronic states. Due to the small spacing of the bound states excitation by several green photons is always multiresonant. (c) VIS photon dissociation spectrum of the benzene cation, X-B transition: resonant absorption of several VIS photons (500-550 nm) is detected by dissociation. The dissociation can take place at the three photon energy level. In peptides because of the high density of vibronic transitions broad absorption bands can be expected.

III.I. Photoexcitation in Isolated Aromatic Cations. The absorption of aromatic molecules strongly changes upon electron removal. Whereas neutral aromatic molecules absorb UV (first band at 240-290 nm), their corresponding radical cations absorb resonantly both UV and visible light. This is well-known from fluorescence spectroscopy,37 photodissociation spectroscopy,33,38-43 HeI photoelectron spectra,44,45 and absorption spectra of amino acid ions in glasses.46 The reason for the absorption change upon electron removal is easily understood by the molecular orbital scheme (Figure 2) of the aromatic chromophores, here benzene. Each electronic state (Figure 2a) represents an electron configuration in the molecular orbital scheme (Figure 2b). Positions of the molecular orbitals can be taken from band positions in HeI photoelectron spectra as described in section V.II. Resonant (1+1) UV excitation via a π* orbital results in ionization (UV1, UV2 in Figure 2a,b). By ionization a hole in the HOMO orbital is created. Note that now in all photoexcitation processes the positive hole is moved downward to lower molecular chromophore orbitals (see Figure 2b VIS1,2 and UV3). Absorption of UV photons in aromatic cations is resonant as proven in various experiments29-31 (UV3 in Figure 2b). In small peptide cations such UV photon absorption can

Weinkauf et al. be detected by dissociation. At low UV laser intensities photoabsorption in the cation can be avoided and exclusively parent cations are produced. In charged aromatic chromophores also resonant photoexcitation processes can be performed by transitions in the manifold of bonding orbitals (VIS1,2 in Figure 2a,b). Due to their small spacing the resulting phototransitions are in the infrared (X-A transition) and green wavelength range (X-B and B-E transition, in Figure 2a). In peptides we use the B-X transition for activation of charge transfer. In the B-state VIS photon excitation is also resonant with highly excited electronic states (E state in Figure 2a). The existence of such resonant electronic states can be taken from HeI PE spectra44,45 and the fact that UV photons (hνUV ) 2hνVIS) are resonantly absorbed (see above). Thus, in aromatic cations typically a multiphoton excitation with wavelengths of 480530 nm is multiresonant. Lifetimes of Excited Electronic States. Excited electronic states in aromatic cations typically have short lifetimes.37,47,48 This lifetime shortening is attributed to fast and ultrafast47,48 internal conversion (IC). In cations quartet states are higher in energy and internal conversion is the only relaxation mechanism of the first excited states.49 In the MO picture, after photoexcitation of the cation to the B states, the internal conversion process fills the deeper hole with a HOMO electron (see Figure 2b). A large IC rate in aromatic cations can be understood by the relatively small energy gap of 1.5-2 eV between electronic states and the energy gap law.17 Ultrafast processes are due to potential intersections in one or several reaction coordinates and mostly appear at higher energies (for benzene for example at the C state level47,48). In our chromophores the internal conversion rate acts like an internal clock which is measuring other competing processes in comparison to its own period. Therefore in the B state, which we excite in the VIS photoactivation step, further photoabsorption has to compete with fast internal conversion. In nanosecond laser pulse excitation usually optical pumping rates are small (1/100 ps). Therefore they cannot compete with internal conversion processes, and further photon absorption occurs indirectly. After fast internal conversion to the electronic cation ground state, the B state can be reexcited by a second VIS photon but from a higher vibrational level. Explicitly for C2H2+ we could show that resonant vibrational states with lifetimes of 2 ps can be detected by dissociation after a multiphoton excitation.39 The two-photon resonance has been proven as well to hold for the fluorobenzene cation38 where excitation to the excited state (first green photon) and photodissociation (several green photons) was separated in time by some microseconds. This agrees well with Franck-Condon considerations which show that such a transition would be favored by small differences in vibrational quantum numbers. Hence we generalize this observation to all our aromatic chromophore cations and rule out that internal conversion processes are blocking the second VIS photoabsorption. Either direct or via internal conversion (see Figure 2a) multiresonant multiphoton absorption is a signature of charged aromatic chromophores and hence inherently a result of the presence of the positive charge. Annihilation of the positive charge would turn back the absorption to the UV wavelength range and interrupt photoabsorption of VIS photons. Dissociation occurs after a resonant two-photon or multiphoton excitation. Thus, the yield of dissociation is an appropriate signal for detection of the efficiency of the resonant VIS two-photon absorption processes in the cation. This concept was previously applied by us and others for spectroscopy of short lived electronic excited states in cations.39-43,50,51 In analogy to

Charge Transfer in Peptide Cations

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Figure 3. (a) Detection scheme for charge transfer in peptide cations, example Leu-Leu-Tyr: UV1,2: Site selective ionization at Tyr. VIS1: photoactivation. CT: charge transfer to the nonaromatic chain. VIS2: detection of the positive charge in the chromophore. IC: internal conversion and subsequent VIS2 excitation. Diss: dissociation after two VIS photoabsorption. (b-d) Two-color UV-vis mass spectra. Note the large differences in the two-photon absorption behavior despite the same chromophore. We here clearly observe an effect in which the whole peptide is involved. Selective local and fragment free two-photon UV ionization at 287 nm is not shown. VIS1 and VIS2 photon excitation of the cations is performed in the wavelength range 490-550 nm. (b) In Gly-Gly-Trp two photon VIS absorption is detected by dissociation. (c) In Leu-Leu-Trp resonant two-photon VIS absorption is efficiently quenched (no fragmentation). We take this as indication of charge flow out of the aromatic chromophore at the one photon VIS energy. (d) In Leu-Gly-Leu-Trp fragmentation by absorption of two VIS photons is observed. We interpret this by glycyl acting as barrier (see text).

multiphoton ionization, by tuning laser wavelength and detection of all fragment ions, spectroscopy of the resonant intermediate states of cations is possible. This technique can be applied to the cations of the amino acids tryptophan, tyrosine,29 and phenylalanine. As an example for the multiresonant VIS multiphoton absorption in aromatic cations, the spectrum of the X-B transition of the benzene cation is shown in Figure 2c. The spectrum was obtained by monitoring the signal of the C6H5+ fragment ion as a function of the VIS laser wavelength. The first fragmentation channel produces C6H5+ and is reached by a three-VIS-photon excitation. The observed structure corresponds to vibrational features of the B state. These structures cover the wavelength range 500-550 nm and continue below 500 nm (C state). The assignment of the vibrational features is described elsewhere.40 We know that the peak width of the spectrum is mostly determined by saturation and power broadening because of the high intensity needed for the three-photon step to achieve dissociation.38 A lifetime of some picoseconds or shorter can be assumed for the B state.38 The multiple resonant VIS multiphoton excitation is a general feature of all aromatic chromophores. It is worth noting that the photodissociation spectrum agrees well with the first electronic band in the HeI PE spectrum.44 Differences in vibrational structures are due to different selection rules caused by the high symmetry in benzene. Such symmetry selection rules are weakened in tyrosine and tryptophan, and thus absorption bands of aromatic cations can be predicted as energy differences of bands in HeI PE spectra.44,45 In section V.II we will make use of this concept for amino acids. III.II. Site-Selective Activation and Site-Selective Charge Probe in Peptide Cations. For site-selective activation and site-selective charge probing in the peptide cations we make use of the optical properties of the charged aromatic chro-

mophores described above. As for photoabsorption of aromatic chromophores embedded in amino acids, it is known from HeI photoelectron spectra52-55 that the energies of the local electronic chromophore states are not or only slightly shifted. In our tailormade peptides the chromophore is always substituted at the C-terminus and hence spaced from the side chains of the next amino acid by two saturated carbon atoms and the peptide bridge (five σ bonds). Therefore, we assume that their electronic energies and absorption cross sections stay the same in peptides. The optical spectra in peptide cations are, however, expected to be broad and diffuse due to the large density of vibrational states and a continuous tuning of the excitation wavelength is not required. In Figure 3a the scheme of the charge-sensitive detection in peptide cations is shown for Leu-Leu-Trp. The excitation scheme of the chromophore is the same as in Figure 2a. After site-selective ionization the first resonant VIS absorption (VIS1 in Figure 3a) photoactivates the peptide cation. This process is resonant for all the tailor-made peptides we investigate. As mentioned above photoactivation performs a transport of the positive hole to a lower molecular orbital of the chromophore. In contrast to the case of the isolated chromophore at this energy level, charge transfer from the nearest chain unit can occur and annihilate the positive hole in the aromatic chromophore (see Figure 3a: CT). This charge transfer process is in direct competition to the intrachromophore internal conversion rate, and we only observe can two limiting cases in which either charge transfer is very fast or slow in comparison with the intrachromophore internal conversion rate, which, in this function, acts as a clock. The charge annihilation in the chromophore returns back to its neutral ground-state absorbing UV. No VIS photoabsorption is observed for the charged s-bonded amino acid cations such as Gly, Ala, and Leu, either as N-terminal or chain amino acid in the peptide in the

18572 J. Phys. Chem., Vol. 100, No. 47, 1996 wavelength range 480-530 nm (see Figure 3a and section V.II). Therefore as long as the positive charge is stored in the nonaromatic part of the peptide during the laser pulse no second photon absorption is observed. No dissociation occurs in peptides at the one-VIS-photon energy level due to energetic reasons (section V.II). Photoabsorption of two-VIS-photons leads to fragmentation. At 4.4-5 eV internal energy dissociation takes place. Neither the kind of fragment ions nor charge transfer at the two-photon level is part of our immediate interest: Simply the yield of fragment ions of any kind is used for detection of the presence of the charge in the chromophore site after photoactivation. In short, “dissociation” of our tailor-made peptides with VIS light means no charge transfer after activation; “no dissociation” means that after photoactivation charge transfer and storage of the charge outside the chromophore occures. In our first paper on this subject we could show that the VIS two-photon excitation in the cation works for the amino acids tyrosine and tryptophane and for the tripeptide Gly-Gly-Trp but interestingly not for LeuLeu-Tyr. We explained this by charge transfer in the Leu-LeuTyr peptide cation at the one-photon energy level.29 IV. Results In this part we present new results of UV-VIS two-color experiments at tailor-made small peptides and give an overview of all peptides investigated up to now. Initial charge localization in peptides is performed by resonant (1+1) UV two-photon ionization of aromatic chromophores. The UV wavelengths for ionization have been chosen in accord with the neutral resonances in the amino acids.29,56,57 For each peptide it was possible to obtain a completely fragment-free UV ionization mass spectrum, which indicates that peptide absorption stops after resonant two-photon UV ionization (see section V). In our first publication on this subject we always displayed the UV ionization mass spectra which have the parent cation as a single peak. In this work, for simplicity, we display only the two laser UV-VIS mass spectra. Photoexcitation of the peptide cations was performed 10 ns after ionization by a second laser. Photoactivation and charge probe is performed by the same visible laser (pulse width 5 ns). Charge probing in the aromatic chromophore has been detected by cation fragmentation in a reflectron time-of-flight mass spectrometer. We showed previously that two VIS photons can be absorbed in the wavelength range 480-530 nm in isolated amino acids tryptophan and tyrosine. In the following we show how photoabsorption in these chromophores depends on composition of the peptide. Tryptophan-Containing Peptides. In Figure 3b-d the UV-VIS two-color mass spectra of some tryptophan-containing peptides are shown. All peptides possess the aromatic amino acid tryptophan as single aromatic chromophore and only the nonaromatic parts are exchanged. Because the VIS photoabsorption takes place in the identical chromophore the properties of the three peptides should be very similar. As Figure 3b-d shows, the differences in the two-color UVVIS mass spectra are substantial: Fragmentation is observed for Gly-Gly-Tyr and Leu-Gly-Leu-Tyr, whereas no fragmentation is observed for Leu-Leu-Trp. The comparison of the peptide sequences and the corresponding UV-VIS mass spectra in Figure 3b-d clearly shows that the effect of quenching of the VIS two-photon absorption is neither a simple internal chromophore effect nor a simple effect caused by one of the nonaromatic amino acids. If only one amino acid is added (as in Figure 3c-d) the two-photon absorption behavior of the

Weinkauf et al. peptide is entirely changed. For example because the substitution of glycyl versus leucyl has been performed at the nonneighboring amino acid of tryptophan (see Figure 3d) a simple change of the absorption cross section of the charged aromatic chromophore or a shift of its absorption wavelength can be ruled out as very unlikely. According to our charge detection scheme described in section III, this bifurcational behavior is directly correlated with an absorption of a second VIS photon (charge in the chromophore, see Gly-Gly-Trp) or quenching of the VIS absorption by charge transfer (charge moves to the chain, see Leu-Leu-Trp). We exclude experimental reasons for the differences in our UV-VIS mass spectra of Figure 3b-d. To ensure that all parameters were optimized, we had to perform several tests in each experiment: (i) Laser overlap between the UV and the VIS laser was tested before and after each peptide mass spectrum, by switching to a benzene resonance. (ii) In addition the VIS laser wavelength was tuned stepwise for each peptide between 480 and 530 nm. (iii) The laser pulse energy of the VIS laser was varied between 50 and 2 mJ without substantially changing the bifurcational character of the spectra. In the latter experiment (iii) the differences of the two-photon cross section can be estimated: Whereas at laser pulse energies of 2 mJ VIS light (5 ns pulse width) more than 90% of isolated tryptophan and the tripeptide Gly-Gly-Trp can be dissociated, vanishing amount of dissociation is found for the tripeptide LeuLeu-Trp. Hence we do not find effects of a few percent but more of several orders of magnitude. To explain our result for Leu-Leu-Trp, the lifetime of the excited isolated chromophore state B has to be shortened severely by charge annihilation. By our rate model a charge transfer rate faster than 1/(50 ps) was estimated. The complex behavior of the peptides in Figure 3b-d shows that some matching of electronic states in the amino acids is necessary for charge transfer at such a low energy level to understand the mechanism. The results have to be correlated to the energetic positions of molecular orbitals of tryptophan, leucyl and glycyl as will be discussed in section V.II. A Barrier for Charge Transfer. The motivation for the investigation of the peptides Leu-Gly-Leu-Trp (see Figure 3d) and Leu-Gly-Gly-Leu-Trp (not shown here) was the search for a barrier for charge flow. For both molecules two-photon VIS chromophore excitation and fragmentation is observed in contrast to Leu-Leu-Gly. Obviously glycyl in combination with tryptophan blocks charge migration and acts as a barrier. This must have energetic reasons as we show below. The results of Leu-Gly-Leu-Trp and Leu-Gly-Gly-Leu-Trp are especially interesting because they demonstrate several effects: (i) An absorption shift of the excited state in the indole chromophore is not responsible for the quenching of the twophoton absorption. For example, in Leu-Gly-Leu-Trp in comparison to the case Leu-Leu-Tyr the neighboring amino acid of tryptophan was not changed: Absorption shifts due to various substitutions performed seven σ bonds away from the chromophore can be neglected. (ii) Charge transfer proceeds through the peptide chain bonds. Otherwise a head-to-tail (i.e., “through space”) transfer would have been able to occur even in the presence of the glycyl barrier (see section V.II). (iii) We assume that charge is transferred to the first leucyl (see above) but then cannot overcome the barrier of glycyl and charge is then transferred back into the aromatic chromophore within our laser pulse duration of 5 ns. Backflow of the charge into the chromophore leads to the ground state X (probably via

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J. Phys. Chem., Vol. 100, No. 47, 1996 18573

Figure 4. Charge transfer to the other side of the peptide: (a, b) MPI mass spectra of a sample mixture of Phe-Leu (a) and Leu-Tyr (b) at the ionization wavelengths of 266.25 and 281.75 nm. (b) Selective ionization of tyrosine can be achieved at 281.75 nm. We use this wavelength for site-selective ionization of the peptide composed of both parts. (c, d) UV-VIS mass spectra of Leu-Leu-Leu-Tyr (c) and Phe-Leu-Leu-Tyr (d). (c) “No dissociation” indicates that after photoactivation charge is transferred out of the chromophore and stored in nonaromatic sites of the peptide. (d) After local ionization and VIS photon activation in Tyr we explain dissociation now by absorption of a second VIS photon in the charged aromatic ring of Phe: for this, charge has been transferred to the other side of the peptide.

the A state). As explained previously the second VIS photon excitation then is again resonant with the X-B chromophore transition, but in a higher vibrational manifold. Charge Transfer in Biaromatic Peptides. To demonstrate that we really can move charges in peptide cations, we investigated a biaromatic peptide linked by nonaromatic amino acids: The concept is detection of charge arrival at the other side of the peptide. We gain this information by comparison of two UV-VIS mass spectra of Leu-Leu-Leu-Tyr and PheLeu-Leu-Tyr. A precondition for this experiment is the initial charge localization in Tyr. As is well-known by liquid- and gas-phase spectra of the amino acids Phe and Tyr,56,57 the UV absorption of Tyr is red-shifted against those of Phe. We first test the ionization to be localized at tyrosine by ionizing a mixture of the dipeptides Phe-Leu and Leu-Tyr at different wavelengths. In Figure 4a the mass spectrum of the mixture is shown at an ionization wavelength of 266.25 nm (resonant for Phe and Tyr). Both peptides appear in the mass spectrum. At a wavelength of 281.75 nm (resonant for Tyr only) the dipeptide Leu-Tyr is ionized selectively (see Figure 4b). Thus, at a wavelength of 281.75 nm in the peptide Phe-Leu-Leu-Tyr composed of both parts we locally ionize in the aromatic side chain of tyrosine. Charge transfer directly after ionization is impossible due to

energetic reasons. The ionization potential of tyrosine is by 0,4 eV lower than that of phenylalanine (see Table 2). This large difference in ionization potentials and the energetic order makes “through-space resonant charge exchange” directly after ionization impossible. In both peptides Leu-Leu-Leu-Tyr and Phe-Leu-Leu-Tyr cation photoactivation and charge probing by visible light (shown in Figure 4c,d) has been performed under absolutely equal conditions. The VIS photon excitation does not lead to dissociation for Leu-Leu-Leu-Tyr but does for Phe-Leu-LeuTyr. Apparently the second aromatic chromophore plays an active role in the photoabsorption of the cation. In accordance with our results given above the lack of fragmentation for Leu-Leu-Leu-Tyr indicates charge transfer to the peptide chain. This implies that in Phe-Leu-Leu-Tyr after VIS photoactivation the charge transfer at least to the leucyl can occur. The ionization potential at the N-terminus is 8.5 eV. If the charge would be once in the chain, in Phe-Leu-LeuTyr charge can be transferred to the second aromatic chromophore Phe because of its lower ionization potential of 8.4 eV. The side chain of phenylalanine then could act as a efficient trap for the positive charge. The second VIS photon then can be absorbed in the charged aromatic benzyl ring of the N-terminal Phe leading to fragmentation. Such a process would

18574 J. Phys. Chem., Vol. 100, No. 47, 1996 TABLE 1: UV-Vis Two-Color Results of Several Small Peptides: Fragmentation, Charge-Transfer (CT) Behavior (+, -), and Energy (∆E) Balance for the Charge-Transfer Steps (+: Exoenergetic) peptides

fragmentation

CT

Gly-Gly-Trp Leu-Leu-Trp Leu-(Leu)3-Trp Leu-Gly-Leu-Trp

+ +

5 Leu-(Gly)2-Leu-Trp

+

6 Gly-Gly-Tyr

-

+ + CT and back CT CT and back CT +

7 Leu-Leu-Tyr

-

+

1 2 3 4

8 Leu-(Leu)2-Tyr 9 Phe-Leu-Leu-Tyr 10 Ala-Ala-Tyr

+

+ +

-

+

∆E balance for CT steps 1, 2, 3, ... in eV +0.2, -0.1, +0.4 +0.2, +0.2, +0.7 +0.2, +0.2, +0.2, +0.2, +0.7 +0.2, +0.2, -0.1, +0.7 +0.2, +0.2, -0.1, -0.1, +0.7 +0.2 to nO C-term. +1.6 to nN (Tyr) +1.3 to nN in chain +1.8 to nN N-term. +0.2 to nO C-term. +1.6 to nN (Tyr) +1.6 nN in chain +2.1 to N-term. see above see above and text +2.2 to highest π (Phe) +0.2 to n0 C-term. +1.6 to nN (Tyr) +1.4 nN in chain +2.0 to N-term.

explain the occurence of a second VIS photoabsorption in accordance with the observation of dissociation. Overview over the Peptides Investigated. We investigated a variety of peptides and found either quenching or absorption of VIS light. An overview is given in Table 1. In most of the peptides charge transfer is observed. Two-photon VIS absorption is found only in cases where the amino acids glycyl and tryptophan were combined or in some biaromatic peptides. Obviously an energetic threshold for charge transfer in peptide cations exists. For peptides containing the chromophore tyrosine no two-photon absorption is observed despite the fact that the isolated amino acid tyrosine absorbs two photons of VIS light efficiently.29 All molecules listed in Table 1 have been investigated with VIS wavelengths in the range 480-550 nm. No substantial change in the yield of fragment ion production as a function of wavelength was observed. Especially for peptides where no quenching of the two-photon excitation was observed we tried without success to provoke charge transfer at higher photon energies. In Table 1 we include the energetics of the charge-transfer process along the chain: We only observe charge transfer in cases where the process is exoenergetic (∆E positive) along the path to the N-terminus. For a detailed explanation see section V.II. The collection of different peptides investigated delivers here more information than each individual result. In the following, by referring to Table 1, we explain some of these conclusions. For example, experiments 1 and 6 show that photodissociation is not a pure side-chain effect. Experiments 1, 2, and 4 show that photoabsorption is not a pure chromophore effect. Hence the full molecule is participating at the process taking place. Experiments 3 and 4 as well as 8 and 9 show that two-photon absorption is not due to a simple level shift of the electronic state in the chromophore. Through-bond charge transfer can be ruled out by experiments 2, 3, 4, and 5. Complete charge transfer to the other side of the peptide has to be assumed by comparing experiments 8 and 9. No length and size dependence of the effect is observed for peptides larger than two amino acids which indicates that two-photon dissociation still works and is not suppressed by a slow statistical decay (see 2, 3, and 7, 8). Back charge transfer even after the one helical can be ruled out by experiments 3 and 8. “Permanent” charge storing

Weinkauf et al. TABLE 2: Energetic Positions of the Lowest Electronic States in Some Amino Acids As Taken from HeI Photoelectron Spectraa molecule

EI band pos

energy

ref

leucine leucyl alanine alanyl glycine glycyl glycine NH-CH2-CH2-NH

N-terminal nN nN in chain N-terminal nN nN in chain N-terminal nN nN in chain C-terminal nO CH2 π like σCC π band π band nN in chain nO C-terminal π band π band π band nN in chain π band π band nO C-terminal π band N-terminal nN π band π band

8.5 eV 9.0 eV 8.6 eV 9.2 eV 8.8 eV 9.3 eV 10.5 eV 11.3 eV 11.8 eV 8.0 eV 8.7 eV 9.0 eV 10.4 eV 10.6 eV 7,4 eV 8.0 eV 9.0 eV 9.2 eV 10.5 eV 10.5 eV 8.4 eV 8.5 eV 8.8 eV 10.9 eV

estimated, 55, 71, 72 estimated, 55, 71, 72 53, 55 53, 55, 67 53, 55 53, 55, 70 53, 55b 44 44 53, 58 58 estimated, 53 52, 53b 58 52, 54, 58 52, 54, 58 estimated, 52, 67 52, 54, 58 52 58b 53, 58 58 44, 58, estimated 58

tyrosine

tryptophan

phenylalanine

b

a All values are taken from the onset of HeI photoelectron spectra. Same value taken as for glycine.

outside the aromatic chromophore is essential for our 5 ns detection scheme. We therefore do not consider of dipeptides. Short pulse excitation is required to obtain results here. Our experiments for various peptides show (see Table 2) that the process is inherently coupled with properties of the constituent individual amino acids. For the occurence of charge transfer in polypeptides apparently a certain energy balancing between local electronic states of the amino acids has to be fulfilled. V. Discussion We observe large differences in two-photon absorption yields in peptide cations which we explain by photoactivated charge transfer out of the aromatic chromophore and storage in a nonaromatic part of the molecule. As we will show below, we can readily explain all our observations for peptide cations with a unified model. Charge transfer has been demonstrated to occur in peptide cations at an internal energy larger than 4 eV. (section I29). This points to the fact that charge transfer exists in peptide cations and must have a threshold. In this work we concentrate on threshold effects at various tailor-made peptides in order to find the direct correlation of our results with peptide properties and properties of individual amino acids. We believe that in peptide cations the threshold for charge transfer lies in the energy range 2-2.5 eV. This corresponds to photoactivation by a one photon process in the wavelength range 480-530 nm. V.I. Alternative Possible Mechanisms for the Quenching of the Second VIS Photon Excitation Step in Peptide Cations. For completeness we discuss various alternate possibilities to explain our findings. We feel that we have strong evidence for a threshold behavior of the charge-transfer process occurring in peptides at an energy of 2-2.5 eV. Local ionization at the aromatic chromophore is performed by resonant (1+1) UV Ionization for all peptides investigated here. We here discuss only processes taking place in cations. Therefore, as mentioned above our experiment consists of three steps: activation (first VIS photon), charge probe (second VIS photon), and monitoring of two-photon excitation by dissociation. Therefore, to inves-

Charge Transfer in Peptide Cations tigate alternative mechanisms for the quenching of the second VIS photon excitation, photoabsorption and dissociation at the one- and the two-VIS-photon level and intrachromophore relaxation processes have to be investigated in detail. V.I.I. Effects of Variation in VIS Photoabsorption. For peptides containing the same aromatic chromophore but differing in length and amino acid composition, differences in optical absorption could occur due to either (i) different initial internal energies (ii) or shifts of electronic states at the one or two VIS photon level. (i) As for differences in internal energies: The laser desorbed molecules are cooled in a supersonic beam and ionized by resonant (1+1) UV ionization. Both processes do not contribute substantial internal energy to the peptides and in addition the relevant parameters are very similar for all peptides. Nevertheless, even an excess energy of 0,5 eV would result in temperatures far below room temperature because of the large numbers of internal degrees of freedom. Large internal energies in cations could be generated by further UV photon absorption in the cation previous to VIS excitation. Such UV absorption would be resonant as explained in section III.I. The fact that we can ionize without any fragmentation, however, demonstrates, that peptide ions are formed in their electronic ground state. (ii) As for possible shifting of electronic states in energy as a result of the peptide environment: Large shifts of electronic states either at the one or the two photon energy level can be excluded. The aromatic amino acids were always attached at the C-terminal side of the peptide and hence in all peptides, if the neighboring amino acid was changed, the site of substitution is spaced by five σ bonds. Small or vanishing shifts are also confirmed by the comparison of HeI photoelectron spectra of amino acids52-55 and analogous molecules58 to the HeI PE spectra of the isolated chromophores.44,45 The most convincing argument, however, is that the quenching of fragmentation or even the occurrence of fragmentation is correlated with substitution of nonneighboring amino acids (see Figure 3c,d) where the distance between site of substitution and chromophore is enormous. In addition the absorption band can be assumed to be broad in peptides due to the high density of vibrational states, thus making small shifts irrelevant for our experiment. Despite this, for resonant VIS excitation, wavelength have been varied between 480 and 530 nm. As for resonant excitation at the two-photon VIS level of the chromophore, we could show that for all peptides UV (hνUV ) 2hνVIS) absorption of the chromophore is possible. V.I.II. Effects of Rate Variation of Intrachromophore Relaxation Processes. Similar argumentation as above holds for influences of the environment on intrachromophore relaxation processes as intrachromophore internal conversion and isomerization. As discussed in section III.I, relaxation of the excited chromophore state by internal conversion is the predominant pathway and would not stop resonant VIS multiphoton excitation. Isomerization processes can be ruled out due to the low internal energy at the one photon VIS level. This is especially valid for all nonaromatic amino acids. In aromatic side chains the expected isomerization is the formation of a ring structure consisting of seven carbon atoms similar to the cycloheptatriene cation: One carbon atom of the alkyl chain would enter the aromatic ring forming a positively charged seven (Tyr and Phe) or six ring (Trp). For the phenylethyl cation it is known that its isomer, the methylcycloheptatriene, is 0.4 eV higher in energy and that the barrier between both structures is 1.1 eV.59 Other isomers are higher in energy or directly correlate to dissociation as for example the formation of the

J. Phys. Chem., Vol. 100, No. 47, 1996 18575 tropylium cation. We are well aware that after one-photon VIS excitation we could be above the isomerization threshold. However, even if at the one-photon VIS level this isomerization channel would be accessible on the time scale of 5 ns (which is improbable due to the high barrier), we expect that most of the molecules keep the six-ring structure because of the higher density of vibrational states there. Hence this would not completely switch on and off the photoabsorption process. In addition, this process should not depend on substitution far away from the chromophore as observed. V.I.III. Time Scale of Dissociation in Small Peptides. A possible explanation for not seeing the same VIS photodissociation for all peptides could perhaps be sought in molecular size effects and the resulting various dissociation thresholds: The peptides investigated differ in number of atoms and molecular composition. Both parameters can influence the energetics and dynamics of dissociation, our detection step here. This can be excluded in as discussed below. For dissociation at the one-photon VIS level, it is well-known that dissociation of large molecules place on a long time scale even for high internal energies.60,61 We know the local dissociation thresholds either N-terminal (IPN + 0.5 eV29) or C-terminal (IPC + 1.5 eV29). As shown previously29 the kind of dissociation depends from the relative position of the absolute dissociation energies (which depends from the local IP) and the internal energy. However for our peptides we find no correlation between the yield of fragment ions and our data. For example in Leu-Gly-Leu-Trp, due to the low ionization energy of Trp, at the one photon VIS energy level (2,5 eV) we would be only 1.0 eV above dissociation threshold. In such a large molecule no dissociation would be expected at such low excess energies.61 In contrast to this we observe fragmentation. This suggests a two-photon process. Other examples can be found which show that dissociation at the one-photon VIS energy does not occur. After absorption of two VIS photons, molecules are excited to an energy which is up to several electron volts (3.5-4.0 eV) above dissociation threshold. Due to the low dissociation threshold at this high internal energy fast dissociation is expected for all peptides of size up to at least four amino acids.61 In addition our results show that neither peptide length nor the number of atoms shows correlation with the dissociation observed. V.I.IV. Photo-Charge Transfer. For completeness we have to discuss the possibility that the first charge-transfer step in peptide cations could also be due to a direct photoexcitation. This would be an optical transition which excites an electron from the neighboring chain state into the HOMO orbital of the chromophore. Such a process would be possible in peptides containing tyrosine as a chromophore. We here observe absorption of visible light below the estimated chromophore absorption. No data, however, are available concerning direct optical excitation of charge-transfer states in large molecules. Optical excitation of charge transfer states has been observed to have a large transition moment in the weakly bound cluster cation N2O+‚Ar.62 We cannot completely rule out a similar mechanism in our peptides. We have good arguments to explain a fast charge transfer and do not favor such a direct phototransition as the relevant mechanism. Especially the results of Gly-Gly-Trp and LeuGly-Leu-Trp sustain this concept: Optical transition to the charge-transfer state by VIS light would be energetically possible but is not observed. Direct optical charge transfer would have to compete with chromophore excitation. Transition moments in aromatic chromophores, especially if they are charged, are

18576 J. Phys. Chem., Vol. 100, No. 47, 1996 large. To explain our results of complete highly efficient quenching of the two-photon VIS excitation, the optical transition moment for the charge-transfer excitation has to be at least a factor of 100 higher than that of the chromophore excitation, a value that is highly improbable. We therefore propose that a direct photo-charge transfer is not the main effect we observe here. Another argument against a photo-charge-transfer process is the exclusively observed local chromophore dissociation in 500 fs laser pulse excitation of the polypeptides gramicidin D and gramicidin S.63 Direct photo-charge transfer would have distributed charge and photoexcitation over the molecule even in femtosecond laser excitation, an effect that would lead to statistical dissociation in contrast to our observation. We therefore assume photo-charge transfer to play an minor role in the charge transfer we observe. Our two-color experiments show that photoabsorption of two photons of visible light is switched on or off in several peptides containing the same chromophore. The observed behavior is multifarious but as such is highly informative: It is different for different chromophoric amino acids, different nonaromatic chain amino acids, different peptide length, barriers, and biaromatic molecules. However, we can explain all these effects by a charge-transfer mechanism and hence feel that this is the most plausible explanation. As our results indicate, some matching between electronic states of the chromophores and peptide chain amino acids has to be fulfilled in order to allow charge transfer to occur. To verify this, we set up a zero-order local molecular orbital model to obtain a landscape of local ionization potentials for peptides. We discuss charge transfer as a hole transfer involving charge positions in local orbitals of functional groups. Extended coulomb states are found to mediate efficient charge transfer in peptide cations. V.II. Energetics of Charge Transfer in Peptide Cations. Our main goal here is the determination of the energetics of the charge-transfer process in peptide cations and the elucidation of the contribution of individual electronic states. So far there is no experimental data of electronic states available for peptides. It is, however, well-known that theoretical calculations at a MNDO or PM3 level can well describe the conformation of peptides. Calculations of the ground state, excited electronic states, shakeup states, and extended coulomb states of peptide ions at a high level of accuracy is very time consuming. This is the reason we look for support mostly from experimental data. For this we have to develop a concept for determining energetic positions of relevant electronic states of the peptides. Here we have to distinguish two kinds of electronic states: (i) Local hole states which correspond to electron removal from well-localized orbitals either “lone pair” orbitals or orbitals of the aromatic chromophores (one molecular orbital is half occupied). (ii) Electronic states which correspond to electron removal and simultaneous electron excitation into antibonding molecular orbitals. These states are called shakeup states64 and correspond to an electron configuration of three half-occupied orbitals. In linear chainlike molecules they can be extended and modified by coulomb forces and are called extended coulomb states. Whereas local hole states are observed in HeI photoelectron spectra of amino acids and related molecules, valence shakeup states are either not at all accessible by photoelectron spectroscopy (two electron excitation)49 or in some cases appear as small satellite structures.49 We there calculated their energetic position by theory. V.II.I. Electronic States That Correspond to Electron Removal from Well-Localized Orbitals (Hole States). In

Weinkauf et al. contrast to neutrals, in radical cations because of small reorganization energies and the lack of large coulomb energy terms the energetic positions of the positive charge in various sites of the peptide can be determined with high accuracy by HeI photoelectron spectra of amino acids and analogous molecules. This is a zero-order description of localized electronic states in peptides, but to our knowledge this is the first attempt to determine energetic positions of electronic states in peptides by experimental data. We therefore explain our concept in detail. A special property of peptides is the localization of lonepair and chromophore electronic states and their spacing by short saturated carbon bridges. As Figure 1a shows, the peptides, for our concern, can be analyzed in terms of several, wellspaced, functional parts: The amine group at the N-terminus on the left hand, the acid group at the C-terminus on the right hand, the nitrogen-oxygen site in the peptide chain skeleton, and the aromatic chromophore. In the following we derive the properties of the functional groups in peptides from their properties in isolated well understood small molecular subsystems. To simplify this task, we have to make some assumptions as follows: (i) For determination of energetic positions of electronic states in peptides, electronic states which are either lone pair or aromatic side chain states and which are spaced by saturated carbon links can be treated as independent. (ii) Couplings and energetic degeneracy of neighboring electronic states have to be treated separately. (iii) The energetic positions of the positive hole at various locations in the peptide chains are given by electronic band positions (ionization potentials) in HeI photoelectron spectra of amino acids and small analogous model molecules. (iv) By using Koopmans theorem,65 electronic band positions in HeI photoelectron spectra apart from the energetic positions of electron holes describe the energetic positions of molecular orbitals. The concept that electronic states which are spaced by saturated carbon links can be treated as independent is a “molecules in a molecule” concept, similar to the “composite molecule approach”.66 Due to the saturated carbon spacers we here assume no or weak direct electronic coupling. In general the assumptions i-iv have been found to be useful for amino acids52-55 and related molecules.58,67 For lone-pair orbitals in nitrogen and oxygen a local approximation is widely used in HeI photoelectron spectroscopy and referred to as “lone-pair” spectroscopy.58,66-72 By substitution of methyl groups it has been shown for amino acids and related molecules that lonepair orbitals only slightly shift their energetic positions if the site of substitution is spaced by two saturated carbon atoms. Similar considerations are valid also for interaction of lonepair orbitals with aromatic ring systems isolated by two saturated carbon bridges.58 Shifts and intensities of the electronic bands can be predicted by substituent additivity effects66 with an accuracy of (0.1 eV, in a similar way as the classical Woodward rules73,74 are used for the absorption shifts in neutral molecules. Electronic States in Amino Acids and Related Molecules. HeI photoelectron spectra of amino acids52-55 and related molecules58,67,72 have been measured previously by others. We make use of these results in order to determine the energetic position of the energetic position of molecular orbitals and the positive hole at various locations in peptides. Figure 5 shows reproductions of HeI photoelectron spectra of glycine (a),53 N-methylacetamide (b),67 and the aromatic amino acid tyrosine (c).53 The HeI photoelectron spectrum of

Charge Transfer in Peptide Cations

Figure 5. Determination of energetic hole positions in peptides by aid of “lone-pair” HeI photoelectron spectroscopy of amino acids and related molecules: spectra taken from refs 53 (a, c) and 67 (b). (a) Glycine: Local orbitals exists in the cation due to electron removal from lone pair orbitals of either nitrogen (nN) or oxygen (nO). (b) N-methylacetamide contains the peptide bond: the nN orbital is shifted to higher energies in comparison to (a). Note the wide energy gap between the first two and higher bands, which results in low cross section for VIS laser excitation. (c) Tyrosine: the HeI photoelectron spectrum consists mostly of structures due to the aromatic chromophore. Note the low ionization potential and the energetic position of excited electronic states which allow 2-fold resonance for VIS photoexcitation.

the simplest amino acid glycine (see Figure 5a53) contains two well-localized electronic states: The nN and the nO state which are due to a removal of a lone-pair electron of either nitrogen (nN) or of the oxygen in the CdO group (nO). These two orbitals are assumed to be independent and completely localized.53 The position of the electronic state corresponding to the ionization in the amine group is situated at 8.8 eV (onset). Its smooth onset can be understood by ab initio calculations: 75,76 The pyramidal amine group of the neutral amino acid becomes planar in the cation. We therefore take the very beginning of the onset as the position of the electronic origin. The shift of this state on a substitution is about 0.2 eV for CH3 (alanine) and 0.3 eV for CH2-CH2-(CH3)2 substitution (estimated from increments given for R, β, and γ substitution in refs 66 and 68-72). We take this amine lone-pair state and its energy value as characteristic of the highest occupied molecular orbital at the N-terminus. Strong localization of the nN orbital has been confirmed by electron momentum spectroscopy77 as well by own MP336 and own “ab initio” calculations.75,76 Photoelectron spectra of the isolated amine group as for example in ethylamine show a large gap between the ground and the first excited electronic state of the cation.44 Hence visible (480530 nm) photon excitation is nonresonant. In glycine the electronic state which is due to ionization of the C-terminal nO state is situated at an energy of 10.5 eV

J. Phys. Chem., Vol. 100, No. 47, 1996 18577 (onset). The πOO state ionization potential is found at 11.6 eV (onset53,55). Both energies are out of the range of the relevant processes for tryptophan-containing peptides, which makes them in our opinions into spectator orbitals. As for tyrosine containing peptides, however, the C-terminal nO state plays an important role for charge transfer. The electronic states of amino acids in the peptide chain (neither N nor C terminal) are well typified by the molecule N-methylacetamide. The HeI PE spectrum and its structure are shown in Figure 5b. The peptide bond is “simulated” in this molecule, resulting in a somewhat different spectrum in comparison to Figure 5a. The onset of the nN lone-pair state in the amide group is shifted to higher energies (9.3 eV) in comparison to the amine group (8.8 eV). Despite the nomenclature of others (πON53 and π267), we prefer the nomenclature nN because of the close energetic position to the lone-pair nN band in the PE spectrum of the amine group. In N-methylacetamide the oxygen lone-pair nO state is situated much lower in energy in comparison to glycine resulting in an overlapping with the nN state. This near-degenerancy of both states can be shown by substitution experiments as done in ref 67. Due to the small energetic gap we are aware that both the nN orbital and the nO orbital are strongly coupled. Both states together as a unit can be seen as localized around the amide group in the peptide chain. In the following, for simplification, we mostly refer to this states as a single “chain” state. The most interesting result from the HeI PE spectrum of Figure 5b is the large gap to the higher electronic bands which are due to ionization of the saturated carbon bridge. This energy gap is broad enough to explain the lack of absorption of photons of an energy below 2.5 eV in cations of non-aromatic amino acids (see arrow in Figure 5b). The same lack of VIS light absorption (2-2.5 eV) is valid for the amino acids alanine and leucine. In Figure 5c the HeI photoelectron spectrum of tyrosine measured by Cannington et al.53 is shown. By comparison to the HeI photoelectron spectrum of phenol44 it becomes evident that the observed bands in Figure 5c are due to the aromatic chromophore. For tyrosine subsequent photoexcitation two photons of energies between 2.2 and 2.4 eV is 2-fold resonant as shown by the two arrows in Figure 5c. This holds also for tryptophan and phenylalanine. In Table 2 the energetic positions of the relevant electronic states of the amino acids leucine, alanine, glycine, tyrosine, tryptophan, and phenylalanine either as isolated amino acids or as chain are given. Errors are mostly due to the broad structures of the electronic bands in the HeI photoelectron spectra which are due to the high temperatures (500 K) at which the photoelectron spectra have been taken. To identify the excited-state bands in HeI photoelectron spectra values have been taken from molecules analogous to amino acids.58,67 The energetic position of the C-terminal nO band in Tyr, Trp, and Phe has been estimated from Gly, because no identification of this band was possible in the corresponding HeI spectra. Similarly the “in-chain” nN band of Tyr and Trp was estimated to be the same as in Leu. Assembling a Local Molecular Orbital Model for Peptides. We assemble a local molecular orbital model for peptides by making use of the energetic positions of the highest molecular orbitals of the functional peptide sites as determined by photoelectron spectra (see Table 2). The energies of these orbitals as shown in Table 2 are taken to correspond to the positive hole in this localized orbital where the rest of the molecule is neutral. Reorganization effects due to charge removal are completely included. It is this energetic information of hole positions which is of direct interest to us.

18578 J. Phys. Chem., Vol. 100, No. 47, 1996

Weinkauf et al.

TABLE 3: Calculated and Experimental Data of the Tripeptide Gly-Gly-Tyr electronic states of the cation ground state + at chromophore excited state + at NH2 group

IP(PM3+CI)

IP(exp)

pos charge localization

R(N-C) (Å)

8.52 eV

8.0 eV (onset) 8.8 eV (onset)

95% + at chromophore 80% + at NH2 group

5.56

9.07 eV

5.87

We first constitute a molecular orbital picture for our peptides. We assume additivity of the local states as a zero-order model. This is a reasonable starting point as explained above and is supported by our own theoretical calculations level, taking configuration interaction into account. As an example, in Table 3 for the tripeptide Gly-Gly-Tyr experimental ionization potentials derived by our above-described technique are compared to theoretical values. The energies of the two electronic states of the peptide where the charge is on the chromophore or where the charge is in the amino group have been calculated by PM336 taking configuration interaction into account. The energetic positions of the amino group to the chain states are reproduced. Note especially the high charge localization in either the chromophore site (state prepared by ionization) or the amine site (positive charge site after charge transfer). The chromophore-amine distances are discussed in section V.IV. Calculations have been quite time consuming, and therefore we confined our investigations at the highest level (PM3, configuration interaction included) on Gly-Gly-Tyr. For other peptides MNDO calculations have been performed which also agree with our model. In Figure 6a-c a molecular orbital schemes for the peptides Gly-Gly-Trp (a), Leu-Leu-Trp (b), and Tyr-LeuLeu-Trp (c) are displayed. To show the local character of the molecular orbitals, we order them along the peptide chain. The CH2 pseudo-π orbitals at 11.3 eV and the σC-C carbon orbitals of the CH2-CH2 bridge at 11.8 eV are low in energy and not included. By ionization the positive hole is created in the HOMO orbital of the aromatic chromophore. Charge transfer needs further photoactivation. Excitation with VIS light (480530 nm) transports an electron from a lower π-orbital to the HOMO orbital (VIS1, Figure 6). By this the positive hole is transferred to a lower orbital. Charge transfer is a “throughbond” transfer involving HOMO orbitals. In Figure 6a the energetic situation of molecular orbital states is shown for the peptide Gly-Gly-Trp for which experimentally no quenching of two-photon absorption in the cation was observed. The position of the excited state hole of the indole chromophore is determined to be below the first chain orbital nn, which still belongs to the tryptophan amino acid. According to the energetic order of the bridge levels the first glycyl peptide bridge states nN and nO, however, are lower in energy than the hole position after chromophore excitation. Due to energetics, electron flow through this state is endoenergetic. We believe that transfer of the charge is energetically blocked at the glycyl in agreement with our observation that charge probe in the aromatic chromophore is positive. The relaxation pathway given in Figure 6a is the most natural if we consider the energy gap law for internal conversion16,17 to hold here. Exchanging glycyl by leucyl shifts up the corresponding nN and nO chain states by more than 0.3 eV (see Figure 6b): Charge transfer is now exoenergetic and the chain orbitals form a downward leading staircase (Table 1). This energetic situation agrees well with the fact that for the peptide Leu-Leu-Leu-Trp our two-color laser excitation spectra indicate charge transfer. Due to a strong coupling of the isoenergetic chain states this charge transfer can be assumed to be mediated to the N-terminal end (see section V.III). Because the N-terminal nitrogen lonepair state is somewhat lower than the chain nN orbital the hole

Figure 6. Zero-order molecular orbital scheme for peptides: (a-c) Gly-Gly-Trp (a), Leu-Leu-Trp (b), and Phe-Leu-Leu-Tyr (c). For energetic positions see text and Table 1. Note that the orbitals of the CH2-CH2 and CH2-C bridges are low in energy and not shown here. Ion photoactivation by a VIS photon results in transport of the hole into a lower chromophore orbital. Whereas electron transfer from the nitrogen chain state (nN) into the chromophore is energetically unfavorable in (a) it can take place in (b) and (c). The charge can be transferred to the other side of the molecule and stored there in the N-terminal amine group as shown in (b). This is in agreement with our experimental results in Figure 3b,c. Complete charge transfer can be detected in Phe-Leu-Leu-Tyr (see (c)) in agreement with experiments shown in Figure 4d.

could be trapped there (for further explanation see section V.IV). This charge trapping in a nonaromatic site of the peptide is an essential process for our observation of charge transfer for our excitation scheme with ns laser pulses. As our scheme shows charge transfer in Leu-Leu-Trp is a process involving energetically accessible intermediate electronic levels in the chain spaced by short saturated carbon bridges. This is a rather untypical situation for charge transfer systems up to now. The role of the CH2-CH2 bridge is discussed in detail below. By MO considerations in biaromatic peptides as for Phe-LeuLeu-Tyr (see Figure 6c) it can be expected that after activation

Charge Transfer in Peptide Cations by VIS light in tyrosine, charge now would flow to the benzyl ring of the N-terminal phenylalanine. Note that the HOMO orbital of Phe is situated above the N-terminal nN orbital. Therefore, after VIS photoactivation (VIS1) in Tyr charge transfer can be completed by ending up in the Phe side chain. In the now charged aromatic benzyl ring a second VIS photon could be absorbed. This energetic scheme is in complete agreement with our experimental finding (Figure 4d), and hence we believe we have a self-consistent and plausible mechanism. The local molecular orbital picture, just looking at energetics, nicely explains the effects observed for this and other peptides including barrier effects. So far, however, strong couplings between local states in the peptides have been neglected, and only states corresponding to localized hole positions have been considered. V.II.II. Extended Coulomb States and Couplings between Localized Electronic States. Electronic coupling between electronic states have to be classified into weak and strong couplings. Strong electronic coupling causes large measurable energetic shifts and energetic splittings. Most of our local picture above is based on the observation that shifts and splittings of localized electronic states in peptides are small and therefore not observed in HeI photoelectron spectra. For determination of the energetic positions of the relevant states, in zero-order approximation we assume electronic states to be weakly coupled. A weak electronic coupling, however, can also result in fast and ultrafast radiationless relaxation rates. This can be due to the high isoenergetic density of vibrational states or/and due to strong vibronic coupling in one or several reaction coordinates. In this case despite the weak electronic coupling one would talk about a “strongly coupled case” in theory of relaxation dynamics. Couplings between Electronic States in Neutrals. In neutral molecules coupling between degenerate electronic states even when spaced by two saturated carbon atoms can be relatively large as calculations of Campbell et al. show.78 This is induced by “through-bond” coupling of the local electronic states via σ orbitals of the -CH2-CH2- bridge.79 In the case of our localized nitrogen lone-pair states in the peptides this model, however, is strongly overestimating the electronic coupling: The coupling between lone-pair orbitals and bridge orbitals we have is much smaller than for a σ orbital directly linking with the bridge. We therefore assume the direct electronic coupling of Hu¨ckel type between chain states and chromophore states to be weak. “Through-space” coupling via direct orbital overlap is an other mechanism which has to be discussed. It is clearly dependent on relative molecular orbital orientation and therefore strongly varies with bridge geometry.80 We believe that such coupling does not apply for the kind of small localized lone-pair orbitals in peptides. For radical cations couplings between localized states as the lone pair and the chromophore states are expected to be larger than in neutrals. Beside couplings of type as discussed for neutrals, effects characteristic for radical cations such as charge resonance and shakeup states have to be taken into account. Charge Resonance in Radical Cations. Charge resonance effects have been discussed for degenerate chromophores in molecular anions81 and cluster cations.82,83 The couplings between degenerate local states does not influence strongly energetic positions of electronic states as observed in photoelectron spectra of ethylenediamine.44 Also in peptides some delocalization of isoenergetic orbitals of different peptide bond sites can be qualitatively seen by population analysis in our PM3 calculations.

J. Phys. Chem., Vol. 100, No. 47, 1996 18579 Coupling by Extended Coulomb States (EC States). We believe, however, that besides the Hu¨ckel bridge coupling and charge resonance there is a special mechanism active in peptide cations which is strongly influencing the dynamics of the chargetransfer process. This mechanism is involving electronic states not considered up to now. These electronic states correspond to a molecular orbital electron population with three open orbitals. The formation of such states from neutral molecules would require emission of an electron and a simultaneous excitation of a second electron. Therefore these states are termed shakeup states64 or non-Koopmans states49 and are usually not found in HeI photoelectron spectra. Only in few molecules such states have been observed as small satellites.49 In principle, however, they can be excited by photoabsorption of the radical cation and can participate efficiently in all kind of relaxation processes. As HeI photoelectron spectra of ethylenediamine44 and substituted phenylethylamines58 show, the electronic coupling of EC states with local hole states seems to be weak in a sense that the hole states keep their normal energetic positions. This fact strongly supports our local picture. The existence of low energetic EC states seems to be a characteristic for extended linear radical cations: the displacement of charges, e.g., the extension is necessary for the strong energetic lowering of some of these states by coulomb effects. We therefore terme these states “extended coulomb states” (EC states). One of such shakeup states corresponds to electron transfer between two localized chromophores through a saturated -CH2-CH2- carbon bridge. The corresponding molecular orbital population for such a process is shown in Figure 7a-c for N-methylethyldiamine. Suppose that the positive charge initially can be located at the nonmethylated amine (see Figure 7a). This positive charge then be transferred by electron transfer through σ* orbitals of the -CH2-CH2- bridge (Figure 7b). The final state corresponds to an electron population where the charge is situated in the methylated amine group (Figure 7c). The shakeup state as shown in Figure 7b is characterized by two positive charges on both sides and the surplus electron in a σ* orbital of the bridge. Normally one would exclude contributions of such a shakeup state to charge transfer in cations, because the σ* orbitals of N-C and C-C bonds are high in energy. However, the electron in the CH2-CH2 bridge is attracted by two positive charges on both sides. This situation causes a strong reorganization of the nearby valence electrons and as a result lowers the energy of the shakeup state. We therefore term this state the extended coulomb state (EC state). The electronic state energies have been determined by PM3 calculations including CI.36 The energetic position of the EC state is calculated to be between the initial excited state (IP ) 8.7 eV, Figure 7a) and the final state (ground state, IP ) 7.8 eV, Figure 7c). This holds as well for other EC states situated in the N-C bond. It must be considered that such calculations are quite difficult and can have considerable errors, but we believe that the qualitative conclusion is sound that the EC state is energetically situated between both hole states where the charge is either at the left or right amine group. The intermediate energetic position for the EC states in N-methylethyldiamine clearly shows that charge transfer through short saturated carbon bridges probably is nearly or completely activationless and therefore is presumed to be extremely fast. This makes a conductive molecular wire out of certain peptide cations. Note that during the charge-transfer process at no time is the hole in the saturated carbon bridge. The strong shifting of antibonding bridge states in EC states seems to be a general feature of short saturated carbon bridges

18580 J. Phys. Chem., Vol. 100, No. 47, 1996

Weinkauf et al.

Figure 7. Role of extended coulomb states in charge transfer demonstrated in the MO scheme for N-methylethyldiamine. The electronic state energies have been calculated by PM3 taking configuration interaction into account (see text). (a) Suppose the positive charge can be initially localized in the nonmethylated anime group. (b) Transfer of the positive charge can be mediated by electron transfer via nonbonding σ* orbitals of the C-C and C-N bonds. Due to the special configuration in radical cations the electron in the bridge is attracted by two positive charges, resulting in a strong lowering of the total state energy. (c) In the energetically optimized state the positive charge is situated at the methylated amine group.

in extended linear cations. For this modified situation the question of strong coupling between initial, EC, and final state needs further investigation. It is however plausible and shown by our own calculations, that the local C-C stretch reaction coordinate is strongly changed in the EC state. Therefore vibronic couplings and conical intersections47,48 are interesting effects that have to be expected. The energetic position of the EC states clearly becomes strongly dependent on the length of the saturated carbon bridge. Mediating charge transfer through long nonconducting bridges therefore should become inefficient. Due to the special structure of peptides consisting of short saturated carbon bridges and repetitive localized electronic states of functional groups, we believe that the interplay of energetically accessible chain states (accessible hole positions) and EC states plays the predominant role in charge transfer in peptide cations. The molecular orbital scheme is no longer appropriate to describe this situation because EC states must be taken into account. Therefore in the following we use a picture of localized ionic states. These consist of localized ionization potentials and localized EC states. V.II.III. Landscape of Localized Electronic States Consisting of Local Ionization Potentials and Localized Extended Coulomb States. It is common usage that a molecule has a single ionization energy. For large chainlike molecules, however, we have a situation of states localized at various sites of the peptide which are characterized mostly by the local environment. Their ionization leads to energies which we attribute to local sites in peptides and therefore define as local ionization potentials. This local ionization potential agrees with the energetic positions of the positive charge in various sites of the peptide. The ensemble of all hole positions defines a “landscape” of localized ionization potentials showing valleys and barriers. In Figure 8a the landscape of local ionization potentials of the peptide Leu-Leu-Tyr is shown. Excited electronic states and EC states (dotted lines) are included. Note that in this picture each electronic state corresponds to an electron configuration where open molecular orbitals and hence charges are localized. The rest of the molecule is always neutral. The coordinate in Figure 8 is an electronic coordinate corresponding to the displacement of the hole along the peptide chain. The position of the EC states is energetically and spatially between the local hole states and hence acts to efficiently mediate charge transfer in peptides. The staircase-like arrangement of the electronic states along the peptide chain to the N-terminus becomes evident in Figure 8a. Note as well the

Figure 8. (a) Landscape of local electronic states in peptide cations: local ionization potentials, local excited states and extended coulomb states fo the peptide Leu-Leu-Trp. (b) Discussion of charge-transfer rates. After photoexcitation, a staircaselike situation mediates charge transfer to the N-terminus. Charge back transfer to the chromophore is an improbable tunneling process along the entire peptide chain.

high density of electronic states at energies larger than 1 eV above the chromophore ground state, which resembles a band structure. The electronic states corresponding to the hole to be in σ orbitals of the saturated carbon bridge are high in energy and therefore should not contribute substantially to the charge-

Charge Transfer in Peptide Cations transport mechanism. In the following we discuss the mechanism and the rates of charge transfer in peptides in the landscape of local electronic states. V.III. Order of Time Scales. In our nanosecond experiment we intrinsically cannot detect fast molecular processes directly. However, optical pumping rates, rates of intrachromophore relaxation processes as well as the storage of the positive charge for more than 5 ns in the nonaromatic part of the peptides gives us an order of magnitude of the time required for the processes involved. In the following we discuss these processes, their mechanisms, and their theoretical description. Several processes can compete in photoactivated peptide cations. The relevant rates are shown in Figure 8b again for the tripeptide Leu-Leu-Trp. We distinguish between intrachromophore (internal conversion, optical pumping rate) and interchromophore processes (charge transfer). In the chromophore states and the charge-transfer state (CT) the available global vibrational state densities are shown. In peptide cations the density of electronic states at the one photon energy level is very high and resembles a band structure. The local hole states form a staircase-like path down to the N-terminus. Note that the situation for photoactivated charge transfer out of the chromophore is naturally different than the charge back transfer to the chromophore. First we discuss “through-bond” mechanisms only since we believe our experiments provide strong evidence for this approach here. In the following we distinguish between (i) intrachromophore processes and (ii) interchromophore processes. The cation is activated for charge transfer by one-photon VIS absorption. At this energy level, in the chromophore B state, intrachromophore rates are competing with the interchromophore charge transfer rate kCT1, thus providing an internal clock. (i) Intrachromophore Processes: Intrachromophore depopulation processes of the chromophore B state of the cation are internal conversion and optical pumping rate. The optical pumping rate kopt can be varied over some range by varying laser intensity and was estimated to be at maximum 1/(100 ps). The intrachromophore internal conversion rate to the A or subsequent to the X state is an irreversible loss mechanism for charge transfer. Its exact rate is unknown and can be only estimated. For fluorobenzene the observed line width gives an upper limit of kIC < 1/(1 ps).38 The line width for the benzene B-state gives a rate kIC < 1/(100 fs). However, in this spectrum we believe the line width mostly to be due to saturation and power broadening by the high VIS laser power necessary for the three-photon step needed for detection by dissociation. Very large internal conversion rates have been calculated for the C state and high vibrational levels of the B state in benzene47,48). This energy range is, however, out of the range of our investigation. Nevertheless the intrachromophore internal conversion rate can be assumed to be large (1/(100 fs) to 1/(10 ps)). In good approximation the optical pumping rate can be neglected. The intrachromophore internal conversion rate is competing with the interchromophore charge transfer rate kCT1, thus providing an internal clock. As shown in Figure 2b the internal conversion process moves an electron down to fill the lower hole. Again the hole is in the HOMO orbital and the second VIS2 excitation is resonant (for details see section III). The intrachromophore relaxation path leads as well to two-VIS-photon dissociation (see Figure 8b, right side). Intrachromophore rates such as photoabsorption or internal conversion are taken not to change substantially by embedding the amino acid into different peptides. Note that in all peptides, more than six σ bonds act as spacer between the chromophore

J. Phys. Chem., Vol. 100, No. 47, 1996 18581 and the next substitution site. No changes can be especially in cases where nonneighboring amino acids are substituted as in Figure 3c,d and 4c,d. (ii) Interchromophore Processes: Interchromophore electronic relaxation rates in cations intrinsically involve charge transfer. After photoexcitation of the chromophore to the B state the charge-transfer rate is in direct competition with the intrachromophore decay rates. The first step of charge transfer CT1, therefore, has to be very fast in comparison to intrachromophoric rates in order to explain the total lack of further photoabsorption and fragmentation. So far we are not able to measure the first charge-transfer rate KCT1 directly. We can distinguish only two situations in which either the charge transfer rate is much faster or much slower than the intrachromophore rates. For some peptides we find an energetic situation where charge transfer out of the chromophore into the peptide chain is barrierless. It is evident that such a process must be very fast. Time Scale of Charge Transfer. We here do not refer to classical charge-transfer theories developed for weakly coupled systems and for neutrals in solution.11 We here stress the intramolecular and the strong coupled character of charge transfer in cations in the gas phase. In typical “through-bond” charge-transfer systems charge transfer is seen as a bridge-assisted process.84-88 The coupling between initial and charge transfer state is mediated by electronic orbitals of the nonconducting bridge and usually is weak. The bridge on one hand acts as a barrier but on the other hand induces state coupling. The tunneling rate for the hole or the electron through a square potential displays an exponential dependence of the CT rate on the barrier length L and its height EBarr:89,90

k ) C exp(-R(EBarr)1/2L)

(1)

Such exponential length dependence has been confirmed for electron-transfer rates in neutral molecules in solvents.84-88 Jortner and Bixon14,15 and others12,13 considered charge transfer in isolated supermolecules. A relaxation from a local state to a charge-transfer state as a two-state process, similar to an internal conversion process, was assumed. For a weak coupling case Jortner and Bixon find that for vanishing vibrational energy in the initial state (Evib ) 0) in good approximation the chargetransfer rate becomes exponentially dependent on the energy gap ∆Eif between initial and final electronic state:14,15

k ) (4π2V2/h)A exp(-γ|∆Eif|)

(2)

Here V is the electronic coupling constant which can be assumed to be exponentially dependent on length and energy of the barrier (see eq 1). The constants A and γ vary slightly with energy. This expression is similar to the energy gap law found experimentally16 and theoretically17 for the internal conversion rate. Equation 3 may be applicable to charge back transfer as we discuss later but not for photoactivated charge transfer to the N-terminus. If we look at the situation for charge transfer in Leu-LeuTrp (see Figure 8b) we note that charge transfer in our peptide cations is much different for several reasons and is not amenable to a simple two state model: (i) Several intermediate hole states chain1 and chain2 are situated along the charge migration path. If they are energetically accessible they can accept the positive charge and charge transfer in peptide cations becomes a multistep mechanism.

18582 J. Phys. Chem., Vol. 100, No. 47, 1996 (ii) The hole states are efficiently coupled by energetically and spatially close EC states. Probably intersections of potentials occur and no barrier exists for charge flow. (iii) Due to the close spatial and energetic position of EC and local hole states, a perturbative description might not be useful. As shown for Leu-Leu-Trp in Figure 8a,b at the one-photon level, the electronic states are very close in energy and therefore can be assumed to be strongly coupled. In the case of peptides we believe the potentials to be nested in the N-terminuschromophore distance (see section V.IV). However, we can assume that EC states, which can mediate efficiently the charge transfer, are strongly distorted in the local C-C coordinate of the bridge. Large differences in C-C bond length between local hole states and EC states could cause conical intersections of potentials in this reaction coordinate. Hence, the question arises if charge transfer here can be described by a perturbative approach as in refs 14 and 15. Probably exact time-dependent quantum calculations with a restricted ensemble of vibrational degrees of freedom are more appropriate here. Such calculations have been previously applied to ultra-fast relaxation dynamics of some smaller molecules.47,48,91-95 As for Leu-Leu-Trp, in the manifold of energetically and spatially close electronic states the charge-transfer process can be assumed to be an ultrafast process, much faster than picoseconds. Such an ultrafast charge transfer to nonaromatic parts of the peptide would nicely explain the complete switch off of the intrachromophore VIS photon absorption. Interplay of Local Hole States and Extended Coulomb States. An essential point of our model is that the hole states in the peptide chain are energetically accessible. This is due to the strongly energetic decrease of the EC states in the saturated carbon bridges. It is obvious that decrease strongly depends on the barrier length. In a long barrier the energy lowering of EC states is smaller, and again a barrier is formed. As an example by shifting a single peptide hole state in one amino acid to higher energy, as done in Leu-Gly-Leu-Trp, this chain hole state of Gly is no longer energetically accessible after one-photon VIS absorption. As a result the length of the entire amino acid becomes a barrier. Our experimental result for LeuGly-Leu-Trp, shown in Figure 3d, demonstrates the persistent presence of the charge in the chromophore. Obviously the fact that one intermediate chain state becomes a virtual state is strongly affecting the efficiency of the process. This can be understood by a tunneling process through the long barrier of six σ bonds. Obviously in Leu-Gly-Leu-Trp tunneling cannot compete with relaxation of the internal conversion type. Charge Back Transfer into the Chromophore. Storing of the charge in the N-terminal state can then be explained as due to a low back-charge-transfer rate. In the nonaromatic part of the peptide chain the N-terminal nitrogen lone-pair state is energetically the most favored site for the charge. If charge is situated here the electronic excess energy has been transformed into vibration. This process is irreversible on a short time scale of 5 ns because of the large number of internal degrees of freedom in peptides. Fast back charge transfer then can be only a tunneling process of the positive charge through the entire peptide chain as a barrier (nine σ bonds). In a two-state model for tunneling, the landscape of barrier heights would have to be considered. In the case of tunneling through nine σ bonds as in tripeptides this is expected to lead to a small rate due to the exponential length dependence (see eq 1). This is in agreement with our experimental observation which demonstrates that charge does not reenter the chromophore within the 5 ns window of our laser pulse width.

Weinkauf et al. For experiments with small peptides such as Leu-Tyr (through bond barrier length six σ bonds), on a time scale of 5 ns it is not surprising to find that the charge is again in the chromophore. A detailed analysis of the energetics of some small molecules by our model shows that charge transfer can indeed occur in amino acids and even directly after ionization. This has been found for N,N-dimethylphenylethylamine34 and other molecules.33 However, such processes are by far faster than our time resolution, and fast laser pulses are required. Although we have evidence for a very fast charge-transfer “through bond” we consider here the possibility of a chargetransfer “through space”. If the electron is assumed to be between two peptide sites, the coulomb attraction of the two positive charges would lower the potential barrier for “throughspace” transfer considerably. The point charge picture, however, is not appropriate here. Changes in electron density distributions of both positive peptides sites during charge transfer through space has to be taken into account (shielding of the positive charge by electron correlation effects). Therefore we cannot predict rates for “through-space” charge-transfer dynamics. From our experiments we exclude a through space mechanism for charge transfer in peptides due to the following reasons: (i) Through-space charge transfer usually is slower than through-bond charge transfer. It is doubtful if such a rate could compete efficiently with our fast intrachromophore transfer rate. (ii) We do not observe charge transfer in systems with blocking groups (Leu-Gly-Gly-Leu-Tyr and Leu-Gly-Leu-Tyr). For a through-space mechanism the presence of the barrier in the chain would not prevent charge transfer. (iii) For peptides of a size larger than two amino acids we do not observe back charge transfer into the chromophore during 5 ns. The situation is different for back charge transfer because there is no competing rate. “Through-space” transfer then can compete with “through-bond” transfer through a long barrier. Our charge-transfer model justifies a scenario of intramolecular rates that are in good agreement with our observations. Hence we believe our explanation to be reasonable. Our local electronic state picture for peptide cations uses the energetics of the electronic states involved in charge transfer and gives an understanding for photoexcitation and electron flow processes in these systems. Our scheme is suitable for understanding of the time scale of processes occurring in peptide cations, the coupling between neighboring states, the barriers and the irreversibility of processes on a fast time scale. In this descriptions “local hole states” and EC states are used which are characterized by charges to be in certain electronic states and a certain site of the molecule. In this local description the suggested coordinate is the motion along the peptide bond for either electron or hole transport. V.IV. A Global Description of the Charge Transfer in Peptide Cations. In the following we want to show how the charge-transfer process in peptide cations can be described in a global picture. A convenient reaction coordinate for the charge transfer would be the distance between N-terminus and chromophore (donor-acceptor distance). For a rough estimate of the changing in donor acceptor distances in excited peptide cations, we calculated the conformation for the tripeptide GlyGly-Tyr on a PM336 level taking configuration interaction into account. In Figure 9 the calculated conformation of Gly-GlyTyr is shown for the ground hole state (9a: positive charge in the aromatic chromophore) and an excited hole state (9b: positive charge in the amino group). Both structures are similar and form the beginning of a bent structure. The change in the donor acceptor distance between both structures is 0.31 Å, about 5% of the total distance. This small change in donor-acceptor

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J. Phys. Chem., Vol. 100, No. 47, 1996 18583

Figure 10. Global scheme of electronic states in the peptide LeuLeu-Trp. Note the flat potentials and the small shifts in the donoracceptor reaction coordinate between charge transfer (N-terminus) and local aromatic chromophore states (X, A, B, C, D). Excited electronic states corresponding to charge localization in the chromophore (B), the chain (chain 1, 2) and the N-terminus are assumed to be strongly coupled via extended coulomb states (dotted lines, see text). Their high density creates a structure similar to a band structure. In the global description the information of strong next neighbor state coupling and weak coupling between N-terminus and X state is lost.

Figure 9. Structures of the tripeptide Gly-Gly-Tyr calculated at a PM3 level36 including configuration interaction. (a) Cation ground state: positive charge in the aromatic chromophore. (b) Excited cation state: positive charge localized at the N-terminal amino group. Note the small geometry change in the donor-acceptor ((N-terminus aromatic ring) coordinate.

distance is due to the lack of coulomb attraction since there are no opposite charges and the fact that mostly nonbonding orbitals are involved in charge transfer. For better understanding we calculated the depth of the potential and the force constant in this reaction coordinate. The result is a flat and anharmonic potential which we schematically approximate in Figure 10 by a smooth potential. Other conformers have been found to exist at higher energies. Calculations at a MNDO level for several peptides containing leucyl, glycyl, tryptophan, and tyrosine showed very similar structures to Figure 9. We assume that the bent structure is a typical conformation for the type of peptides we investigate and that the situation of Gly-Gly-Tyr can be seen as a model case. In Figure 10 a global scheme for electronic peptide cation states is shown for Leu-Leu-Trp (compare Figure 8a,b). The reaction coordinate is the distance between N-terminus and chromophore. The electronic states drawn as solid lines correspond to positive hole positions in the aromatic chromophore (X, A, B, C, D), the peptide chain (chain1, chain2) or the N-terminal nitrogen lone pair state (CT). EC states are shown by dotted lines. Their position is energetically and spatially between the neighboring hole states. Their exact position and their displacement in the C-C reaction coordinate is, however, unknown. The EC states enhance coupling between neighboring states and increase electron state density. At the one-photon VIS energy level the situation becomes similar to a

band structure. All potential curves have only small displacements in the donor-acceptor reaction coordinate (nested states) according to the considerations given. The total charge transfer is shown to be a multistep internal conversion mechanism. The global picture, however, is unfavorable here because information about nearest-neighbor coupling is lost, a information important for the description of charge-transfer mechanisms in cases of blocking groups and bichromophoric peptides. For the description of charge transfer and positive charge mobility in peptide cations, we therefore favor the hole-transfer description as shown in Figure 8b. Our model is a zero-order model founded on the interaction between hole states at local sites in the peptides that are coupled by intermediate EC states. The resulting picture of a strongly positive charge conducting medium is an important peculiarity of proteins. It results from the interplay of the repetitive structure in peptides, energetic accessible hole states, extended coulomb states, and short saturated carbon bridges. It nicely explains the energetics and qualitatively the rates of the processes involved. Further consequences of our results and our model have to be discussed separately. Investigations of analogous molecules and peptides are planned. Short-pulse experiments are now being set up to determine the rates of the individual charge-transfer steps. VI. Conclusion Since much of what is presented here are new experiments together with a new theoretical model, we indulge in some detail in order to provide some background and to show specialties for these highly efficient charge-transfer processes in peptide cations. Our work contains the following points: We confirm that positive charge migration can occur in peptides in the gas phase at high internal energies above 4 eV. By resonant VIS photodissociation experiments in the cation,

18584 J. Phys. Chem., Vol. 100, No. 47, 1996 we find threshold behavior for charge transfer and show that after activation in some peptides the positive charge is stored outside the aromatic chromophore for the duration of the laser pulse. This can be explained only if a very fast charge-transfer rate is assumed which can efficiently compete with fast intrachromophoric rates. We can show that charge transfer is directly correlated with the properties of individual amino acids. Barriers of a complete width of a single amino acid in the chain are found to block charge transfer efficiently. In experiments with biaromatic peptides we can show that we move charge by laser activation to the other side of the peptide. No charge back transfer has been observed in peptides of size larger three amino acids. We present a local molecular orbital model for peptides similar to molecules in a molecule picture. Peptides are described as a chain of individual amino acids. Their local states are found by data from HeI photoelectron spectra of amino acids and analogous molecules. Calculations have been performed in order to gain insights concerning the reliability of our local model especially concerning the localization of electronic states in peptides. We find a new charge-transfer mechanism for linear extended radical cations which efficiently can mediate charge transfer via extended coulomb states through short saturated carbon bridges. The energetic positions of these extended coulomb states and the local hole states gives us an energy landscape in a chain of local electronic states. In this landscape energetic hole positions and extended coulomb states are shown in relation to their distance from the chromophore. This energy landscape is useful to determine the energetics and the electronic couplings of the charge-transfer processes. In this picture next neighbor state couplings are evident and charge-transfer rates can be discussed. We find that charge transfer occurs only in peptides where a sequence of energetic accessible hole states are found along the charge-transfer path. A multistep “through-bond” chargetransfer mechanism is proposed that involves an interplay between localized hole positions and extended coulomb states: a mechanism possible only in repetitive structures as in peptides. Time scales and rates can be qualitatively understood and reproduced by this model. In particular our model for charge transfer in peptides relies heavily on a good zero-order model. The conventional global molecular picture would lead us to adopt a picture as a supermolecule for a peptide. Such a picture is useful for describing the overall radiationless process and its energetics but is not useful for the discussion of electronic next-neighbor couplings and charge-transfer rates. A local energy landscape picture seems to be the most convenient model to describe the multistep charge-transfer process in peptide cations. We clearly see the manifestations of a many-molecules behavior in the peptide more analogous to a “chain of pearls” rather than to a supermolecule. This distinction becomes quite evident in the treatment presented here and affords an easy understanding of the basic physics involved, as a coupling of local electronic properties mediated by the newly found extended coulomb states. The mechanism of such a conductivity in peptides may be relevant to many experiments involving positive charge flow such as for scanning tunneling microscopy of proteins.96 Our results and our model may be also important for hole transfer in solution because the same orbitals and couplings would be involved. It is of interest to speculate now on hole transfer in a highly polar solvent where after the first step of charge separation the charges are completely shielded is a situation

Weinkauf et al. closest to the charge transfer in cations. Here we expect the intramolecular electronic coupling effects to be very similar. At high internal energies in peptides positive charge can be conducted efficiently by the close energetic spacing of electronic states and their efficient coupling. Hence highly excited peptides can be seen as having unique properties as conductive molecular wires in which the positive charge can move very fast. A molecular wire descriptions have been theoretically discussed by Ratner and co-workers10 and recent experimental results on DNA deliver evidence for such a molecular wire behavior.97 A consequence of the local molecular orbital model is a new picture to look at peptide cations is an energy landscape of local electronic states. The role of charge migration for energy transport and dissociation in peptide cations in this landscape has to be discussed separately. Here questions of energetics and time scales remain for the future and will address the question of how electron flow will be correlated with energy flow and reactivity. This will be one of the most fundamental questions for all processes driven by charge transfer. First conclusions from our results indicate, that density of molecular orbitals, charge hopping, and conductivity could be directly related to reactivity. This is especially assumed to be so for the “ac conductivity”.98 Acknowledgment. Funding by the Volkswagen-Stiftung is gratefully acknowledged. We thank Prof. Sieghart F. Fischer for his fruitful discussion and his remarks about direct photoinduced charge-transfer processes. We thank Dr. Leonid Y. Baranov for his discussion about coulomb barriers. We thank Dr. Heinrich Selzle for the “ab initio” calculations of glycine and Gerhard Gilch for sample preparation and his experimental help. A.M. thanks the Deutsche Akademische Austauschdienst for his grant. References and Notes (1) Photoinduced Electron Transfer; Fox, M. A., Chanon, M., Eds.; Elsevier: Amsterdam, 1988; Parts A-D. (2) Fundamentals of Photoinduced Electron Transfer; Kavernos, G. J., Ed.; VCH Publishers, Inc.: New York, 1993. (3) Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle, M.E., Eds.; Springer Series in Biophysics 6; Springer-Verlag: Berlin, 1990. (4) Regan, J. J.; Risser, S. M.; Beratan, D. N.; Onuchic, J. N. J. Phys. Chem. 1993, 97, 13083 and references therein. (5) Onuchic, J. N.; Beratan, D. N. J. Chem. Phys. 1990, 92, 722. (6) Socci, N. D.; Onuchic, J. N.; Wolynes, P. G. J. Chem. Phys. 1996, 104, 5860. (7) Isied, S. S.; Vassilian, A. J. Am. Chem. Soc. 1984, 106, 1726. (8) Isied, S. S.; Vassilian, A. J. Am. Chem. Soc. 1984, 106, 1732. (9) Schanze, K. S., Sauer, K. J. Am. Chem. Soc. 1988, 110, 1180. (10) Kemp, M.; Roitberg, A.; Mujica, V.; Wanta, T.; Ratner, M. A. J. Phys. Chem. 1996, 100, 8349. (11) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (12) Jortner, J.; Bixon, M.; Heitele, H.; Michel-Beyerle, M. E. Chem. Phys. Lett. 1992, 197, 131. (13) Jortner, J.; Bixon, M.; Wegewijs, B.; Verhoeven, J. W.; Rettschnick, R. P. H. Chem. Phys. Lett. 1993, 205, 451. (14) Jortner, J.; Bixon, M. J. Photochem. Photobiol. A: Chem. 1994, 82, 5. (15) Bixon, M.; Jortner, J. J. Phys. Chem. 1993, 97, 13061. (16) Schlag, E. W.; von Weysenhoff, H. J. Chem. Phys. 1969, 51, 2508. (17) Englman, R.; Jortner, J. Mol. Phys. 1970, 18, 145. (18) Felker, P. M.; Syage, J. A.; Lambert, W. R.; Zewail, A. H. Chem. Phys. Lett. 1982, 92, 1. (19) Shou, H.; Alfano, J.; van Dantzig, N. A.; Levy, D. H.; Yang, N. C. J. Chem. Phys. 1991, 95, 711. (20) Wegewijis, B.; Scherer, T.; Rettschnick, R. P. H.; Verhoeven, J. W. Chem. Phys. 1993, 176, 349 and references therein. (21) Castella, M.; Tramer, A.; Piuzzi, F. Chem. Phys. Lett. 1986, 129, 105. (22) Piuzzi, F.; Tramer, A. Chem. Phys. Lett. 1990, 503. (23) Piuzzi, F. Proc. Indian Acad. Sci. 1991, 103, 477. (24) Pru¨tz, W. A. Biochim. Acta 1982, 705, 139.

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