J. Phys. Chem. B 2001, 105, 10407-10415
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Distance Dependence of Photoinduced Electron Transfer along r-Helical Polypeptides Masahiko Sisido,*,† Satoshi Hoshino,‡ Hajime Kusano,† Masahiro Kuragaki,† Maki Makino,† Hiroshi Sasaki,† Trevor A. Smith,§ and Kenneth P. Ghiggino§ Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama UniVersity, 3-1-1 Tsushimanaka, Okayama, 700-8530, Japan, Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan, and School of Chemistry, UniVersity of Melbourne, Victoria 3010, Australia ReceiVed: March 28, 2001
R-Helical polypeptides containing a pair of L-1-pyrenylalanine and L-p-nitrophenylalanine that are separated by 0-8 amino acid units were synthesized. The rates of photoinduced electron transfer (ET) from the pyrenyl group to the nitrophenyl group were evaluated from the decay curves of pyrenyl fluorescence recorded at different temperatures from -58 to +30 °C. The rate constants showed a complex dependence on the number of spacer amino acids. In particular, recoveries of the ET rates with increasing the number of spacer amino acids from 1 to 2 and from 5 to 6 were found. The ET rate constants, however, exhibited a simple exponential dependence on the edge-to-edge distance between the two chromophores, with a distance decay factor β ) 0.66 ( 0.1 (Å-1). The ET data on the R-helical polypeptides were analyzed on the basis of the tunneling pathway model. The optimum ET pathways from the pyrenyl group to the nitrophenyl group were searched, and the relative values of the ET matrix elements were evaluated for each polypeptide with different number of the spacer units. The calculated distance dependence was in reasonable agreement with the experimental one when jumps through hydrogen bonds were taken into account.
Introduction Photoinduced electron transfer (ET) in proteins has been studied from the biological viewpoint as the model system for the primary processes of photosynthesis and from the physicochemical viewpoint as a simple system in which chromophores are positioned with known interchromophore distances and orientations along the polypeptide chain.1,2 However, due to the complex structure of proteins in which the polypeptide main chains with different conformations are compactly folded and the side chains with a variety of functional groups are densely packed, the interpretation of protein ET is not straightforward. Beratan et al.3 proposed the tunneling pathway model for protein ET. In their theory, the ET tunneling matrix element HDA is evaluated as a product of the components for jumps through σ bonds, for jumps through space, and for jumps through hydrogen bonds (HBs). Although the tunneling pathway model seems to interpret the ET processes in several proteins successfully, further examinations on model polypeptide systems that have simple R-helical conformations or β-sheet structures are needed. Isied4 and other workers5-7 studied end-to-end ET on oligoprolines with metal complexes linked at the two ends. The end-labeled oligoprolines may be the simplest model peptide systems so far investigated. However, they were not the best models for proteins because of the difference in main chain conformations and the lack of intrachain hydrogen bonds. Polyproline chains take either polyproline I- or II-type conformation that is more extended than the standard R-helix. Furthermore, possible conformational fluctuations at the terminal donor and acceptor groups must be taken into consideration. * Corresponding author: Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan. † Okayama University. ‡ Tokyo Institute of Technology. § University of Melbourne.
Our group8-10 and Fox’s group11 have been studying ETs on R-helical polypeptides that contained nonnatural amino acids carrying chromophores on the side chains. The polypeptide samples have been synthesized by a stepwise method and leave no uncertainty in the order of amino acids and, therefore, in the order of chromophores. In these studies, β-arylalanine-type nonnatural amino acids have been employed to minimize orientational freedom of the chromophores and to keep a stable R-helical conformation. Other amino acids are carefully selected to maintain the stable R-helical conformation that guarantees proper spatial arrangement of the chromophores. Furthermore, the chromophoric amino acids are incorporated in the middle of the R-helix to ensure that the chromophores are built into the helix scaffold. In our previous study, the rigidity of the R-helical conformation has been confirmed from the small intensity of intramolecular excimers on the R-helical polypeptides that contain two pyrenylalanine units.12 The constrained orientations of the side-chain pyrenyl groups have been supported from a very strong circular polarization of the excimer fluorescence.13 The stability of the R-helical conformation in supporting the side-chain chromophores has been confirmed from the Fo¨rster-type energy transfer data.14 In this study, we have incorporated a pyrenyl group as a photosensitizer and a p-nitrophenyl group as an electron acceptor into a single R-helical polypeptide with different numbers of spacer amino acids (m) from 0 to 8. The general structure of the polypeptides is shown below, where abbreviations are Glu(OBzl) ) γ-benzyl L-glutamate, pyrAla ) L-1-pyrenylalanine (P), Glu(OChx) ) γ-cyclohexyl L-glutamate, ntrPhe ) L-pnitrophenylalanine (T):
H-Glu(OBzl)n-pyrAla-Glu(OChx)m-ntrPheGlu(OBzl)4-NH2 (PmT) (m ) 0-8) A polypeptide sample that contains only a pyrenyl group,
10.1021/jp011180h CCC: $20.00 © 2001 American Chemical Society Published on Web 10/03/2001
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TABLE 1: Characterization of the Fmoc-Oligopeptides and the Polypeptides no. of spacer units (m)
exact mass (M + Na)
observed mass
average no. of Glu(OBzl) units (n)a
∆222 in TMP
Pb 0 1 2 3 4 5 6 7 8
1409.54 1601.60 1812.72 2023.84 2234.96 2446.08 2657.20 2868.32 3079.44 3268 (M + H)
1409.30 1601.77 1812.91 2024.01 2234.89 2446.33 2657.48 2868.66 3079.67 3278c
48 39 32 48 54 37 47 36 51 40
-12.1 -11.3 -11.0 -12.2 -11.3 -11.7 -10.1 -11.6 -12.4 -11.3
a Determined from UV spectrum in TMP b Fmoc-pyrAla-Glu(OBzl)4-NH2 c Measured with different instrument under different conditions.
H-Glu(OBzl)n-pyrAla-Glu(OBzl)4-NH2 (P), was also prepared as a reference. Since the electron transfer from the LUMO of the pyrenyl group to the LUMO of the nitrophenyl group is nearly optimized for the nuclear factor and the fluorescence lifetime of the pyrenyl group is very long (252 ns at -58 °C), ET rate constants over a wide range of interchromophore distances can be studied on the polypeptide samples. The goal of this study is to clarify the dependence of ET rates on the number of spacer units (m) and on the edge-to-edge distances between the pyrenyl and nitrophenyl groups up to about 16 Å. The distance dependence will be compared with that predicted from the tunneling pathway model and the applicability of the latter model will be examined.
Figure 1. CD spectra of the P, P0T, P1T, P2T, P3T, and P4T polypeptides in the amide absorption region (inset) and in the pyrenyl absorption region measured in TMP at 25 °C.
Experimental Section General Strategy for the Synthesis of the Polypeptides Carrying a Single Pyrenyl-p-Nitrophenyl Pair. Oligopeptides pyrAla-Glu(OChx)m-ntrPhe-Glu(OBzl)4-NH2 (m ) 0-8) were synthesized through solid-phase synthesis according to the Fmoc/PyBOP/HOBt strategy. After the peptide was cleaved off from the resin, the Fmoc group was removed, and the peptides with free amino termini were elongated by the polymerization of Glu(OBzl) N-carboxyanhydride (NCA). The resulting polypeptides contain a single pyrenyl-nitrophenyl pair inside an R-helix of poly(γ-benzyl L-glutamate) chain. Solid-Phase Synthesis of Fmoc-pyrAla-Glu(OChx)mntrPhe-Glu(OBzl)4-NH2 (m ) 0-8). A superacid-labile resin [4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin] (Watanabe Chemicals, Hiroshima, Japan) was used as the supporting resin, and the peptide was elongated step-by-step by using a 3-fold excess of Fmoc-amino acid, benzotriazole1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP), and 1-hydroxybenzotriazole hydrate (HOBt). Before each elongation step, the Fmoc group was removed with 20% piperidine in DMF. Finally, the oligopeptides were cleaved off from the resin by treating with 2% trifluoroacetic acid (TFA) in dichloromethane several times until no absorption of pyrenyl group was detected. Under this condition, the benzyl and cyclohexyl ester of glutamates suffered no damage. The crude peptide solution was concentrated and precipitated in ether. The crude peptides were purified on Sephadex LH-20/DMF column to give a single peak in HPLC (C4 column in 0.1% TFA in pure H2O/acetonitrile). The Fmoc-peptides were characterized by a TOF mass spectroscopy (PE Biosystems, Voyager DE Pro), and the results are collected in Table 1. Elongation of the Peptide Chain by the Polymerization of Glu(OBzl) NCA. The Fmoc-protected peptide was dissolved
Figure 2. CD intensities of the PmT polypeptides at 278 nm (pyrenyl absorbance) in TMP and in DMF.
in 20% piperidine in DMF for 10 min, and the mixture was evaporated in vacuo. The residual oil was precipitated in ether and washed with ether several times. The oligopeptides with the free amino terminus was dissolved in DMF, and a 30-fold quantity of Glu(OBzl) NCA was added. The polymerization was completed within 2 days, and the polypeptides were purified on a Sephadex LH60 column in DMF to remove unreacted peptides. A small portion of the deprotected oligopeptide was acetylated with an excess amount of acetic anhydride/pyridine (1/3) mixture in DMF. The reaction mixture was concentrated and purified on a LH20 column in DMF. The acetylated peptides were used as reference compounds as follwos. The average number of Glu(OBzl) units n, was determined from the absorption spectrum of the PmT polypeptide in trimethyl phosphate (TMP). The latter spectrum was subtracted by the spectrum of Ac-pyrAla-Glu(OChx)m-ntrPhe-Glu(OBzl)4-NH2 and that of the Glu(OChx)m unit. The molar absorption coefficient of the Glu(OBzl) unit was determined from the spectrum of the helical polyGlu(OBzl) with the number-average degree of polymerization of 40. The results are also collected in Table 1. Spectroscopic Measurements. Absorption and CD spectra were recorded on a Jasco U-best 560 and a Jasco J720WI instrument, respectively, in DMF and in TMP. Fluorescence spectra were recorded on a Jasco FP777 instrument in argonsaturated DMF solution. The concentration of the pyrenyl group
Electron Transfer along Helical Polypeptides
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Figure 3. Computer-predicted conformations of the PmT polypeptides. The main chain conformation was assumed to be regular R-helix, and only the side-chain rotational angles of the pyrenyl and nitrophenyl groups were varied to find the minimum-energy orientations. The predicted edgeto-edge distances are indicated in the figure.
was 2.4 × 10-6 M. Fluorescence decay curves were measured on a time-correlated single-photon counting machine (Horiba NAES550), equipped with a hydrogen discharge lamp (pulse width ) about 2.5 ns) in argon-saturated DMF. For the P0T, P2T, and P3T polypeptides that showed fast fluorescence decays, a picosecond photon counting system was used. This consisted of a frequency-doubled, cavity-dumped DCM dye laser (Spectra Physics 3500), synchronously pumped by a mode-locked Argon ion laser (Spectra Physics 2030). Excitation was at 345 nm and emission was monitored at ∼375 nm following a polarization analyzer (set at the magic angle), a filter (WG360) and monochromator (Jobin Yvon H10) combination, and detected by a microchannel plate photomultiplier (Hamamatsu R1564U-01). The laser repetition rate was reduced to 800 or 400 kHz, in cases where residual (long-lived) emission was observed. The observed decay curves were deconvoluted into multicomponent exponential functions by using a repeated reconvolution program.8,9 The fluorescence
spectra and the decay curves were measured at -58°, -30°, 0°, and +30 °C, by using an Oxford DN1704 or Oxford Optistat liquid nitrogen cryostat. Transient absorption spectra were measured on a picosecond Nd3+:YAG laser system of Professor Okada’s laboratory, Osaka University.15 Results and Discussion Absorption and CD Spectra of the Polypeptides. Absorption spectra of the polypeptides were virtually the same as the sum of the spectra of the pyrenyl and nitrophenyl components, indicating very small ground-state interaction between the pyrenyl and nitrophenyl group. CD spectra were measured in TMP down to 195 nm. As examples, CD spectra of the P, P0T, P1T, P2T, P3T, and P4T polypeptides are shown in Figure 1. The CD profiles at the amide absorption region showed a typical pattern of right-handed R-helix with 100% helicity. The ∆222 values as the measure of the helix content are listed in Table 1.
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TABLE 2: Edge-to-Edge Distances (ree) and ET Rate Constants (kET) in DMF at Different Temperatures on Glu(OBzl)n-pyrAla-Glu(OChx)m-ntrPhe-Glu(OBzl)4-NH2 kET (s-1)
ree m 0 1 2 3 4 5 6 7 8
(Å)
-58 °C
-30 °C
0 °C
30 °C
∆Hq a
∆Sq b
ΠeCHBTSc
σL (Å)d
6.5 9.3 3.9 5.0 10.9 11.1 8.9 12.3 15.9
4.6 × 2.2 × 107 3.5 × 108 e 7.2 × 106 1.8 × 106 2.3 × 107 4.0 × 106 4.3 × 105
108
4.7 × 2.8 × 107 4.3 × 108
108
5.1 × 3.6 × 107 3.9 × 108
108
6.9 × 4.0 × 107 3.3 × 108
0.1 0.5 0.1
-18 -22 -19
1.2 × 107 2.3 × 106 3.4 × 107 1.2 × 107 4.3 × 105
2.8 × 107 2.8 × 106 3.5 × 107 2.4 × 107 6.0 × 105
4.9 × 107 3.8 × 106 2.7 × 107 3.4 × 107 8.9 × 105
2.3 0.5 0.2 3.0 0.2
-16 -27 -23 -14 -31
0.28 × 0.60 × 10-2 0.96 × 10-2 0.97 × 10-2 0.42 × 10-2 0.14 × 10-2 0.12 × 10-2 0.73 × 10-3 0.47 × 10-3
9.8 14.0 12.7 12.7 15.0 18.1 18.4 19.8 21.0
108
10-1
a In kcal mol-1. b In cal mol-1 deg-1. c Factor that is proportional to the electronic matrix element calculated according to the tunneling pathway model d Equivalent tunneling distance along σ bonds e The ET rate constant could not be determined due to the formation of the exciplex-type species.
The somewhat large scattering of the ∆222 values around the value for the 100% helix (-12.0) may be due to the uncertainty in the estimation of the number of Glu(OBzl) units n. The positive CD peak around 278 nm originates from the La absorption band of the pyrenyl group that is asymmetrically perturbed by the R-helical polypeptide chain and by the excitontype interaction with the nitrophenyl group. Except for the P2T polypeptide, all the polypeptides show positive CD profiles for the La band. The CD intensities, however, depend sharply on the number of spacer units m as shown in Figure 2. Except for the P1T and P2T polypeptides, the peak intensity decreases with the number of spacer units and approaches the value for an isolated pyrenyl group in the R-helix. The spacer-dependent CD intensity indicates that a very weak electronic coupling is operating between the pyrenyl and nitrophenyl group even up to a distance of 16 Å. This weak coupling could not be observed in the absorption spectrum. The nature of the electronic coupling may be an exciton-type dipole-dipole coupling between the transition moments of the pyrenyl and nitrophenyl groups. Since the CD intensity is a complex function of the electronic coupling and the molecular geometry, it is not easy to correlate the CD intensity with the pyrenyl-nitrophenyl distance. However, a close look at Figure 2 reveals that the CD intensity is larger when the pyrenyl-nitrophenyl distance is shorter and the plots of the ∆278 values against the edge-to-edge distances show a linear decreasing function, except for the P2T polypeptide. These results indicate that the strength of the weak interaction is proportional to the pyrenyl-nitrophenyl distance and the latter distance is correctly predicted from the helix geometry. The small and split CD pattern of the P2T polypeptide is an exceptional case. In the P2T polypeptide, the two chromophores are very close to each other, and this may cause strong excitontype interaction that is different from the interactions in other cases. In DMF solution, CD spectra at the amide band could not be measured, and the helical content could not be evaluated, due to strong absorption of the solvent. However, similar spacer dependence of the CD intensity at the La band as shown in Figure 2 strongly supports R-helical conformation in DMF solution. R-Helical conformation of polyGlu(OBzl) in DMF has been confirmed from optical rotatory dispersion and from other physicochemical behavior.16 Prediction of the Chromophore Orientations on the r-Helical Polypeptide Chain and the Estimation of the Edgeto-Edge Distances Between Pyrenyl and p-Nitrophenyl Group. Molecular mechanics calculations were carried out for the side-chain orientations (χ1,χ2) of the pyrenyl and pnitrophenyl group.17,18 The polypeptide main chain was fixed
Figure 4. Fluorescence spectra of the PmT polypeptides in DMF at -58 °C. λex ) 346 nm. The inset shows normalized spectra of the P3T, P2T, P0T, and P polypeptides. For the latter three samples, the fluorescence spectra were too small to display in the main figure.
to a right-handed R-helical conformation with φ(N-CR) ) -62.5°, ψ (CR-C′) ) -42.3°, and ω(C′-N) ) 180°. The side groups of the Glu(OBzl) and Glu(OChx) units were taken to be a methyl group for simplicity. In the preliminary work, replacement of Glu(OChx) units by Ala units did not affect the ET behavior, although the solubility of the Ala oligopeptides was reduced significantly. The energy contour map for the side-chain orientation of pyrenylalanine unit in the R-helix showed a single minimum. The minimum point and the width of the low-energy area ( 5. Even in the
Sisido et al.
Figure 11. ET rate constants of the PmT polypeptides in DMF at -58 °C plotted against the equivalent σ-distance.
P2T polypeptide in which the edge-to-edge distance is 3.9 Å, the optimum pathway is a through-HB one. This indicates that the tunneling pathway model with Beratan’s parameters strongly favors through-bond and through-HB pathways and the direct through-space jumps are virtually ignored. As shown in Figure 10, there are several equivalent or nearly equivalent ET pathways that give the same or almost the same coupling terms in some polypeptides. In the case of P2T polypeptide, the optimum pathway is a through-HB pathway that gives the product of the coupling terms ΠCHBTS of 0.55 × 10-2. The second pathway is a direct through-space process across 3.9 Å, that gives the product of 0.42 × 10-2. In this case, it is appropriate to take a sum of the two products as the coupling term. In the case of the P4T and P5T polypeptides, there are two equivalent pathways that involve different through-HB jumps, as shown in Figure 10. Furthermore, there are three equivalent pathways for the P8T polypeptide in which two HBs are selected from the four HBs positioned between the pyrenyl and the nitrophenyl groups. In these cases, the products were doubled or tripled for the evaluation of the coupling terms. The total coupling terms are listed in Table 2. The total coupling terms were converted to a nonintegral number (n) of σ bonds that gives the same coupling terms
ΠCHBTS ) (C)n ) (0.6)n
(3)
If one takes 1.4 Å as the standard bond length for a σ bond, the equivalent number of σ bonds may be converted to an equivalent σ-distance σL, which gives the same coupling term
σL ) 1.4n
(4)
The σL distances for the optimum ET pathway are also listed in Table 2. The logarithms of the maximum ET rate constants kET(max)’s, are plotted against the equivalent σL distances in Figure 11. The expected distance dependence, kET(max) ) 1013 exp{-0.73(σL - 3)} (s-1, σL in Å), is indicated by a solid line. Although the data points are smaller than the expected values, the slope of the data points appears close to the expected dependence. The fairly good agreement suggests that the ET process on the R-helical polypeptides may be well explained in terms of the tunneling pathway model with through-HB jumps taken into consideration. If the through-HB jumps were ignored, the agreement was much worse, and the recoveries of the ET rates on going from m ) 1 to 2 and from m ) 5 to 6 could not be predicted.
Electron Transfer along Helical Polypeptides For further improvement of the tunneling model, the coupling parameters must be optimized. For the optimization of the coupling parameters, however, more ET data on simple model peptides, especially those on β-sheet peptides, are necessary. The latter experiment is now in progress. Conclusion ET rate constants on R-helical polypeptides were determined as the function of the number of spacer amino acids between the two nonnatural amino acids that carry pyrenyl and nitrophenyl group, respectively. The ET rate constants showed a complex dependence on the number of spacer units, and this ruled out the possibility of simple through-bond ET mechanism. The rate constants showed a relatively simple exponential dependence on the edge-to-edge distance between the two chromophores with a distance decaying factor of -0.66 (Å-1). The tunneling pathway model, however, could predict a major part of the distance dependence, when through-hydrogen-bond jumps were taken into consideration. Acknowledgment. The authors wish to thank professor Tadashi Okada of Osaka University for measuring picosecond transient absorption spectra of the polypeptide samples. This work has been supported by a Grand-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports and Culture, Japan (No.11102003). References and Notes (1) (a) Langen, R.; Colon, J. L.; Casimiro, D. R.; Karpishin, T. B.; Winkler, J. R.; Gray, H. B. JBIC 1996, 1, 221. (b) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537. (c) Wuttke, D. S.; Bjerrum, M. J.; Winkler, J. R.; Gray, H. B. Science 1992, 256, 1007. (d) Beratan, D. N.; Onuchic, J. N.; Winkler, J. R.; Gray, H. B. Science 1992, 258, 1740. (e) Wuttke, D. S.; Bjerrum, M. J.; Chang, I.-Jy; Winkler, J. R.; Gray, H. B. Biochim. Biophys. Acta 1992, 1101, 168. (f) Casimiro, D. R.; Richards, J. H.; Winkler, J. R.; Gray, H. B. J. Phys. Chem. 1993, 97, 13073. (g) Langen, R.; Chang, I.-Jy.; Germanas, J. P.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science 1995, 268, 1733. (h) Lopez-Castillo, J-.M.; Filali-Mouhim, A.; Nguyen, E.; Binh-Otten, V.; Jay-Gerin, J-.P. J. Am. Chem. Soc. 1997, 119, 1978.
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