Radicals and Radical Ions As Intermediates of Electron Transfer Processes Through Peptides

364 CHIMIA 2012, 66, No. 6 Organic Free radicals doi:10.2533/chimia.2012.364 Chimia 66 (2012) 364–367 © Schweizerische Chemische Gesellschaft Radicals and Radical Ions as Intermediates of Electron Transfer Processes through Peptides Bernd Giese*, Sonja Eckhardt, Miriam Lauz, Jian Gao, and Min Wang Abstract: Electron transfer (ET) through peptides and proteins is a key biochemical process, which involves radicals and radical ions as reactive intermediates. We have developed an assay that allows us to study this fundamental chemical reaction. Keywords: Electron transfer · Hopping · Peptide · Radical ion 1. Assay radical cation like 4 by chemically induced tween the electron acceptor and the elec- dynamic nuclear polarization (CIDNP).[6] tron donor.[4] Long-distance electron transfer (ET) Monte-Carlo simulations showed that the As shown by CD-spectroscopy the two through peptides, proteins and enzymes heterolytic cleavage 3➝4 is only possible triproline spacers induce the conforma- plays an important role in living organ- in the presence of polar solvents.[5b] The tion of a polyproline II (PPII) helix into isms.[1] More than a decade ago we studied last step of the reaction sequence is the the peptide, which does not change up to related reactions in DNA and concluded oxidation of the aromatic ring to radical 80 °C. The NMR spectra demonstrate that that long-distance ET through DNA can cation 5, which can be detected by UV/VIS 80% of the proline amide bonds are pres- occur only as a multistep hopping reac- spectroscopy (λ = 450 nm).[4] ent in trans-conformation, and molecular max tion.[2] To answer the question whether According to the mechanism shown in dynamic calculations predict the stretched an analogous hopping process also occurs Scheme 1, laser flash photolysis of 1 yields conformation 1b (Fig. 1) as the major one in peptides,[3] we have developed assay peptide 6, which contains the active elec- of this assay.[4,7] In such a system the dis- 1a that is suitable to address this issue.[4] tron acceptor at the C-terminal amino acid. tance between the electron donor and the Furthermore it allows the determination If ET occurs through peptide 6, tyrosine at electron acceptor is approximately 20 Å. of the influence of side chains, charges the N-terminal end will be oxidized to the Fluorescence quenching experiments of a and conformations on the ET processes of tyrosyl radical (6➝7). This process can be hexapeptide containing four prolines have these biopolymers. Peptide 1a consists of followed by transient absorption spectros- shown that the conformational rigidity of nine amino acids (Fig. 1): tyrosine at the copy, observing the decreasing absorption these PPII helices is so high that an encoun- N-terminal end acts as electron donor, the of the dialkyl radical cation (λ = 450 nm) ter of the N- with the C-terminal amino ac- max dialkoxyphenylalanine at the C-terminal as well as the increasing signal of the ty- id is slower than 2×105 s–1.[8] Therefore an end is the precursor of the electron ac- rosyl radical (λ = 408 nm). The driving experimentally detected ET of 6➝7, which max ceptor, and the amino acid with the side force for this ET step is the difference of is faster than about 5×105 s–1,can occur chain X in the center of the peptide can be the oxidation potentials (about 0.3 eV) be- through the stretched peptide backbone. varied in order to detect the influence of side chains on the ET reaction. These three Fig. 1. Peptide assay amino acids are separated from each other used for the ET ex- by triproline spacers. periments. 1b shows The dialkoxyphenyl radical cation 5, a polyproline (PP) II which acts as electron acceptor, is gener- helix, which is the ated from its precursor 2 by laser flash dominant conforma- photolysis.[5] The first ground state inter- tion of 1a. mediate is radical 3, whose β-C–O bond is cleaved in a heterolytic fashion, yielding 1a radical cation 4. We have demonstrated the generation of radicals like 3 by ESR- spectroscopy,[5b] and the formation of a *Correspondence: Prof. Dr. B. Giese Université de Fribourg Département de chimie Chemin du Musée 9 CH-1700 Fribourg Tel.: +41 26 300 8701 1b E-mail: [email protected] Organic Free radicals CHIMIA 2012, 66, No. 6 365 Scheme 1. a) [4] �� thoxyphenylalanine are nearly the same. Formation of the Therefore the radical cation in 9 should be electron acceptor. b) able to oxidize the central amino acid and Formation of the tyro- to generate the aromatic radical cation 10. syl radical during ET This process can be followed spectroscop- through peptide 6. ically as radical cation 10 has its λ at max 550 nm. The subsequent ET step is the oxidation of tyrosine that generates the tyrosyl radical (λ = 408 nm) after deprotonation max 5 (10➝11). The experiments were carried out by �� laser flash photolysis (excimer laser, 100– 150 mJ per pulse) of 2–5 mm solutions of 8 in CH CN:H O = 3:1 at 20 °C and neutral 3 2 pH. Snapshot experiments 40 ns after the laser flash have demonstrated that all three reactive intermediates of 9, 10 and 11 are observable[4a] and we could measure their concentration at different times.[11] The ex- perimental results are shown in Fig. 2. From the data of Fig. 2 it is obvious that ET (9➝11) occurs as a consecutive reaction: by increase of the reaction time the concentration of the radical acceptor 9 decreases, the oxidized intermediate 10 is formed in a small, nearly steady state concentration, and the amount of the oxidized electron donor 11 increases. Obviously the side chain of the central aromatic amino But the time window is not only lim- (radical cation of the dialkoxyphenylala- acid is oxidized during ET (9➝10). We ited by the conformational stability of the nine) at the C-terminal end of peptide 6, have referred to amino acids, which act as peptide backbone but also by the forma- which are about 20 Å apart, is expected to stepping stones in an ET hopping process tion time of the electron acceptor (1a➝6), be slower than 106 s–1 because of the dis- as relay amino acids.[4a] The absolute rates the lifetime of the reactive intermediates, tance dependence of ET rates described of the individual ET steps differ from each and the intermolecular ET rates (reaction by Eqn. (1), where k is the ET rate be- other by a factor of 10, which is caused by ET between 6 and remaining 1a). Under the tween donor and acceptor, k the rate at the difference of the free reaction enthal- 0 reaction conditions of our ET experiments van der Waals contact distance, β the at- pies.[11] The thermodynamic energy change (solvent: CH CN:H O = 3:1, tempera- tenuation factor and r the donor/acceptor of the ET step between the trialkoxy- and 3 2 DA ture = 20 °C, neutral pH) we have mea- distance.[12] Typical β-values for proteins the dialkoxyphenylalanine is close to zero sured a formation rate 1a➝6 of 3×108 s–1 are between 1.0–1.5, and k is in the order and the rate k of the first hopping step is 0 1 by laser flash irradiation.[9] Under the 1013 s–1.[13] ten times slower than the exergonic (about same reaction conditions the life time of 0.3 eV) ET between tyrosine and the radi- the aromatic radical cation 5 (trapping by cal cation of the trialkoxylated phenylala- k = k exp(–β r )(1) the solvent) and of tyrosyl radical 7 (di- ET 0 DA nine (k ). The absolute rate of 3×107 s–1 for 2 merization) was measured to be longer the exergonic ET step via three prolines is than 0.1 ms.[10] However, the bimolecular According to these data one would ex- comparable with measurements of Isied ET reaction between radical cation 6 and pect rates in the order of 105 s–1 or less for et al.,[14] who attached ligand/metal com- the starting material 1a, which also gen- single step (superexchange) ET in peptide plexes as donor/acceptor systems to the erates tyrosyl radicals, is about 4×105 s–1 6, where the donor/acceptor distance is N- and the C-terminal end of PPII helices. (pseudo first order) for a 1 mm solution about 20 Å. It is therefore not surprising If we increased the number n of prolines of the starting material.[11] We need these that our experiments with peptides car- between tyrosine as electron donor and the relatively high concentrations for our ex- rying an alkyl side chain X at the central aromatic radical cation as electron accep- periments even with a powerful excimer la- amino acid of 6, which cannot be oxidized tor the ET rate decreased from 3×107 for ser (100–150 mJ per pulse) because of the by the aromatic radical cation and cannot n = 3 to 4×106 s–1 for n = 4 and 6×105 s–1 for weak extinction coefficients of the starting induce a two-step hopping mechanism, n = 5.[15] This shows the distance effect on ketone group of 1a, as well as the reactive was too slow to be measured exactly by the ET rate, and is in accord with measure- intermediates in 6 and 7. Therefore the our assay: the intermolecular ET was as ments of Isied et al.[14] time window of our assay for exact mea- fast as the ET through the peptide bonds.[4a] surements of intramolecular ET through This is in accord with experiments of Isied peptides is about 106 to 108 s–1.

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