J. Phys. Chem. 1993,97, 11456-1 1463
11456
Distance Dependence of Intramolecular Electron Transfer across Oligoprolines in [(bpy)zR~~L’-(Pro),-Co~(NH3)5]~+, n = 1-6: Different Effects for Helical and Nonhelical
Polyproline I1 Structures Michael Y. Ogawa,t**James F, Wishart,$ Zuyung Young,? John R. MiUer,ll and Stephan S. Isied’J Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855, Department of Chemistry, Brookhaven National Laboratory, Upton, New York 1 1 973, and Department of Chemistry, Argonne National Laboratory, Argonne, Illinois 60439 Received: March 1, 1993; In Final Form: August 17, 1993’
A series of complexes of the type [(bpy)2R~~~L-(Pro),-Co~~I(NH3)5]~+, n = 1-6, where L = 4-carboxy-4’methyL2,2’-bipyridine, bpy = 4,4’-bipyridine, and Pro = l-proline, have been synthesized from the corresponding [ (bpy)zRuIIL] and [(NH3)5Co1I1(Pro),] components. The compounds were characterized by metal analyses, electrochemical measurements, and absorption spectroscopy. For n = 4-6 prolines, the C D spectra of the complexes show a polyproline I1 helical structure. Intramolecular electron transfer within these complexes was studied by generating the [(bpy)2R~~IL*-(Pro),-Co~~~(NH3)5] intermediate by the reaction of e,, (generated by pulse radiolysis) with the [(bpy)2R~~~L-(Pro),-Co~~~(NH3)5] molecules. The driving force for this reaction is estimated to be lAGol -1.1 V. The intramolecular electron transfer rates (k)and activation parameters (AH*(kcal/mol), AS* (eu)) found for these studies were (1.6 f 0.1) X lo7 s-l, 6.0 f 0.6, -6 f 2; (2.3 f 0.2) X los s-l, 9.2 f 0.4, -3 f 1; (5.1 f 0.4) X lo4 s-l, 9.4 f 0.2, -5.5 f 0.8; (1.8 f 0.1) X lo4 s-l, 9.0 f 0.4, -9 f 1; and (8.9 f 0.6) X lo3 s-l, 8.8 f 0.4, -1 1 f 1 for n = 2-6, respectively. For n = 1 proline, k is >5 X lo8 s-* and no temperature dependence could be determined. The rates of intramolecular electron transfer decrease rapidly with distance for n = 1-3 prolines but show a surprisingly weak decrease with distance for the n = 4, 5, and 6 prolines, which exhibit the polyproline I1 helical structure. The electron-transfer pathways within these molecules and the relationship of the electron-transfer rates to the helical polyproline I1 structure are discussed. N
Introduction Electron-transfer (ET) reactions have been observed over long distances ( > l o A) in a variety of synthetic peptides,’“ modified proteins,1@-14 and protein-protein complexes.lsJ6 These observations have stimulated interest in the study of long-range electron transfer in synthetically designed peptide Thoughtful and comprehensive analyses of these diverse experiments have been carried out by several theoretical group^.^^-^^ The primary goal of this work is to understand the role of the intervening peptide in mediating long-range electron-transfer reactions. In order to examine long-range electron-transfer pathways through peptides, we have synthesized and studied several series of transition metal donor-acceptor complexes bridged by polypeptide ligands.’ The most thorough studies were those using rigid proline oligomers to separate the donor and acceptor sites. These studies have demonstrated that rapid electron transfer can occur between donor and acceptor sites that are separated from one another by as many as four proline residues, even in cases where the driving force of the reaction (-AGO) is relatively modest.’c A significant limitation of these early studies is that the distance between the donor and acceptor sites could not be increased beyond four amino acid residues, since the rates of the intramolecular electron-transfer reactions became slow enough for intermolecular reactions to dominate the observed kinetics, even at the low concentrations used in the studies (ca. 5 X 1od M). In this paper we extend the study of long-range electron transfer to a similar oligoproline series with a high driving force (IAGOl 7 Rutgers.
t Present address: Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403. Brookhaven National Laboratory. 1 Argonne National Laboratory. * Abstract published in Aduance ACS Absrracts, October 1, 1993.
> 1V). We report our detailed study28of rates of long-range electron transfer that proceed at metal-to-metal distances ranging from 10 to 30 A in a series of complexes, [(bpy)2RuIIL-(Pro),C O ~ ~ I ( N H ~ n) ~=] ~1-6, + , where bpy = 2,2’-bipyridine and L = 4-carboxy-4’-methyl-2,2’-bipyridine.Surprisingly rapid rates of electron transfer are observed for the compounds having longer proline bridges. These results are discussed in terms of the conformational properties of the intervening peptide bridge. Experimental Section Synthesis of Potassium 4-Carboxy-4’-methyl-2,2’-bipyridme. The ligand, 4-carboxy-4’-methyl-2,2’-bipyridine (L) was prepared by a modification of the procedure reported in the literature.29 The 4,4’-dimethyL2,2’-bipyridine (Fluka, 2.0 g, 11 mmol) and KMn04 (1.7 g, 1 1 mmol) were dissolved in water (100 mL) and refluxed for 2 h. The reaction mixture was then allowed to cool, and excess KMn04 was destroyed by the addition of ethanol (1 5 mL). The solution was then filtered and extracted three times with diethyl ether to remove any unreacted starting material. Crude separation of the desired monoacid product as a potassium salt, potassium 4-carboxy-4’-methyl-2,2’-bipyridine (KL), from the diacid was achieved by reducing the solvent volume by rotary evaporation and precipitating the diacid with the addition of ethanol. Further purification was achieved by reverse-phase cl8 column chromatography (Yamamura Chemical Laboratories Co., Ltd) using aqueous solvent to yield 0.23 g of product (0.91 mmol, 12%). The identity of the monoacid product was confirmed by observing the lH NMR signal of the single methyl group at 2.29 ~ p m . *Anal. ~ Calcd for C12H9N2K02-2H20: C, 49.99; H, 4.54; N, 9.72. Found: C, 50.69; H, 4.05; N, 9.63. Synthesis of [(bpy)zRuL](PFs)rH20 (bpy = 2,2’-Bipyridme, L = 4-Carboxy-4’-methyl-2,2’-bipyridine).The (bpy)2RuC12 (Strem Chemicals, 0.350 g, 0.673 mmol) and L (0.176 g, 0.698 mmol) were refluxed in 1:l methanol/water (100 mL) for 4 h.
0022-3654/93/2097-11456$04.00/0 0 1993 American Chemical Society
Electron Transfer across Oligoprolines
The Journal of Physical Chemistry, Vol. 97, No. 44, 1993 11457
1
Methanol was removed by rotary evaporation, and the product, [Ru(bpy)zL] (PF&H20, was precipitated as a dark-red powder 16 with HPF6and washed thoroughly with ethanol (0.48 g, 5.2 mmol, 76%). Anal. Calcd for C ~ ~ H ~ ~ N ~ F I ~ O ~ PC,~41.08; R U 'H, H~O: 3.02; N, 8.98. Found: C, 41.59; H, 3.06; N, 8.93. Synthesis of [(NH3)&o(Pro),,](BF& n = 1-7. The prolylcobalt complexes (n = 1-4) were prepared using solution-phase peptide synthesis methods developed in this laboratory.l*.b930 For n = 5 , 6, and 7 prolines, the [(tert-butyloxy)carbonyl]tetra-l1 proline (Sigma Chemicals) was coupled with [(NH3)&o(Pro),,](BF4)3 (n = 1, 2, 3). Synthesisof [(bpy)lRunL(Pr0)~Cfl(NH3)s](BF4)an = 1-7. All the binuclear complexes [(~~~)~RU~IL-(P~~),,-CO~I~(NH~)~]- 350 450 550 (BF4)4,n = 1-7, werepreparedbythesamemethod. Thesynthesis h,nm of the complex with n = 1 proline is described as a sample protocol. The [(bpy)2RuL](PF6)2 (50 mg, 54 pmol) and l-hydroxybenFigure 1. Absorption spectraoftheintermediate[((bpy)~RuIIL]*-(Pro),Co1I1(NH&I4+(circles) and its parent complex [(bpy)ZRu*IL-(Pro),zotriazole (Aldrich Chemicals, 7.4 mg, 54 pmol) were dissolved Co111(NH3)51 4+ (squares). in dry CHjCN/DMF (1:l ratio) (400 pL). Diisopropylcarbodiimide (10 pL, 64 pmol) was added, and the mixture was stirred spectrum of the ligand-centered radical intermediate, which was at room temperature for several minutes. The [(NH3)5Cc-(Pro)]obtained by addition of the dose-normalized absorbance changes (BF& (42 mg, 81 bmol) and 4-methylmorpholine (Aldrich (At = AA/(dose.path)) at various wavelengths to the spectrum Chemicals, 10 pL, 91 pmol) were added to initiate the coupling of the Ru(I1) starting material. Pulsedand CW xenon arc sources reaction which was monitored by observing the reverse-phase were used as the probe light. Due to the photosensitivityof some HPLC profile of thereaction mixture. Thereaction wasquenched of the complexes, a 325-nm long-pass filter and a Hoya U-380 with water, and the crude product was precipitated with diethyl blue filter were combined for the work at 350 nm, and a 495-nm ether. The unreacted ruthenium complex was separated from long-pass filter was used for work at 500 nm. The kinetic data the desired binuclear complex by ion-exchange chromatography were collected and analyzed using a Fortran program written by (SP-Sephadex C-25) using HTFA eluents (0.2 M, 0.6 M). The Dr. Harold Schwarz of BNL. The kinetic fits were calculated product was concentrated by rotary evaporation and converted using a standard nonlinear least squares procedure. Errors in to the BF4 salt by addition of HBF4. Final purification was individual rate constants determined were always < 10%. Error achieved by reverse-phase (CIS)column chromatography to yield limits for rate constants obtained from several experiments are 27 mg (22 pmol, 41%). Anal. Calcd for C37H47N&oF1603B4given in Table 111. The analysis for determining the contribution Ru: C, 36.58; H, 3.90; N, 13.83. Found: C, 36.80; H, 4.08; N, of the intermolecular reaction to the intramolecular rate is given 13.62. in the Results section. Circular Dichroism Measurements. Circular dichroism (CD) spectra of the binuclear compounds [ (bpy)2R~~~L(Pro),,-Co~~I- The enthalpy and entropy of activation for the rates of intramolecular electron transfer were determined over a tem(NH3)5](BF4)3,n = 1-7, were obtained at ambient temperatures perature range 277-313 K using the Eyring rate equation k = with an Aviv Model 60DS CD spectropolarimeter. Sample (keT/h) exp(-AH*/RT) exp(dS*/R). Errors in the activation concentrations were about M in water. For [(NH3)5Coparameters were determined by treating each experimental rate (Pro)513+ CD spectra were also obtained as a function of determination as a separate observation in the Eyring plots. The temperature from 25 to 60 OC. slope and intercept of the plots of In k / T vs 1/ T were obtained Electrochemical Measurements. The reduction potential for using linear regression analysis. Standard methods of error [(bpy)2RuIIL-(Pro) ~-COIII(NH~)S] was obtained by differential analysis were used to estimate the error in the data. Eyring plots pulse polarography in a solution containing 76 pM complex in of the data are provided as supplementary material. 0.1 M HTFA. A PAR Model 175 potentiostat with a dropping Pulse radiolysis experimentswere conducted for the compounds mercury electrode, a platinum wire counter electrode, and a Ag/ [ ( ~ ~ ~ ) ~ R u ~ ~ L - ( P ~ o ) , , - [ ( N H ~ ) $n ~=o ~1,~2,~ using ] ( B Fthe ~)~, AgCl reference electrode was used. 30-ps pulsewidth, 20-MeV linear accelerator facility at Argonne Metal Analysis. Cobalt analysis was carried out using directNational Laboratory. The solutions used were 1.0 X 10-3 M in coupled plasma emission spectra (DCP) at 238.892 nm. Ru0.13 M terr-butyl alcohol and 5.0 X 10-3 M sodium acetate buffer thenium analysis was carried out using the absorbance of the at pH 6.3. The intramolecular reaction was monitored at 520 MLCT band of the binuclear complex and comparing it to a pure nm. reference sample. This analysis gave a molar Co/Ru ratio of 1:1 (h0.05)for all compounds studied. ReSultS Kinetics Experiments. The kinetics of intramolecular electron Synthesis and Characterization of the Complexes. The binutransfer in thecomplexes [(bpy)2R~~~L-(Pro),,-Co(NH3)5] (BF4)4, clear complexes [(bpy)2R~~~L(Pro),,-Co~~~(NH~)5] (BF4)4, n = n = 3-6, were determined using 2-MeV electron pulse radiolysis 1-7 (1-7) were prepared by coupling the monomeric complexes techniques as previously described.1c The experiments were [(bpy)2RuI1L]2+and [(NH3)~CoI11(Pro),]2+ in acetonitrile. The performed under conditions of reduced light in 0.13 M tert-butyl complexes 1-7 were precipitated in diethyl ether and purified by alcohol, 40 mM NaC104, argon-saturated water at pH 6. ion-exchange and reverse-phase (CIS)column chromatography. Solutions of the binuclear complexes ranging from 6 X 10-5 M The HPLC elution behavior of the reaction mixtures were to 2 X 10-6 M were used (Table 111). Determination of the used to monitor the progress of the coupling reaction and was intermolecular electron-transfer rate constant was performed also used as a criterion of purity for the isolated products. The using 6.55 X 10-5 M [(bpy)zRuIIL]+ and 1.3, 2.7, 4.2, 5.8, and retention times for compounds 1-6 increase with the number of 7.7 X 10-5 M [(NH3)5Co*11(Pro2)]3+.Reactions of the aqueous proline residues in the peptide chain (Table I). electron with the mononuclear and binuclear complexes were The absorption spectra of 1-7 in water are superimposable followed spectrophotometricallyat 650 nm; subsequent oxidation of the ruthenium(I1)-bipyridine ligand-centered radical was and are dominated by the spectral characteristics of the observed at 350 and 500 nm. Figure 1 shows the absorption [ (bpy)2Ru*IL]moiety. Thesespectra have a ligand-centeredband
Ogawa et al.
11458 The Journal of Physical Chemistry, Vol. 97, No. 44, 1993
I
TABLE I: HPLC Retention Times for the Series [(bpy)2RunL-(Pro)rC@(NH3)5p+, n = 0-6 (bpy = 2,2'-Bipyridine, L = 4-Carboxy-4'-methyl-2,2'-bipyridhe) no. of arolines 1
2
3
4
5
6
HPLCretention tim@ 4.4 4.7 5.4 6.3 7.9 9.2 a HPLC retention times on a 3.9 X 300 mm reverse-phase HPLC column (35%CHjCN, 0.2% HTFA, pH 3.2). 0
-20
.*w
eXI04
I
I
I
I
I
I
I
0
100
200
goo
400
500
600
700
Time, psec
Figure 3. Pulse radiolysis transient absorption kinetic data at 350 nm for n = 4-6 prolines showing the decay of the [(bpy)2RuI1L-]' species.
SCHEME I
-20
V n - 7 2W
+ (bp~)2RPL(Prol,C~(NH3)5 220
140
260
180
3m
NANOLDTERS
Figure 2. CD spectra of [(bpy)2R~~~G(Pro),rCo~~~(NH,)lrl~+, n = 4-6 in water.
at 288 nm (E = 6.6 X IO4 M-l cm-I) and a metal-to-ligand chargetransfer (MLCT) band at 456 nm (c = 1.5 X 104 M-' cm-') (Figure 1). The expected d-d band from the [(NH3)SCo-Pro,] moiety (Amx = 502-505 nm, e = 80 M-1 cm-l) is masked by the tail of the more intense ruthenium MLCT band. Detailed high-field NMR analyses of the related [(NH3)5CoIII(Pro),] complexes confirm the structure and have been reported earlier in le. The value of the reduction potential for the ruthenium center in [(bpy)~R~~1L-(Pro)~-Co~~~(NH~)~] was found to be Eo = -1.2 V vs NHE. The redox potential for [(bpy)zRuIl/IllL] was determined to be 1.3 V vs N H E by cyclic voltammetry.4I Metal analysis of the binuclear complexes resulted in a molar Co/Ru ratio of 1:l (h0.05) for all the compounds studied. For n = 1, the identity of the reaction product was further confirmed by electrospray mass spectrometry. Circular Dichroism Spectra. The CD spectra of the binuclear compounds 1-7 in aqueous solution (Figure 2) show a negative Cotton effect at ca. 200 nm, which is characteristic of the trunspolyproline I1 structure; and the absence of a positive CD band at ca. 210 nm indicates that the amount of cis-helical polyproline I structure is 11egligible.31-~3The magnitude of ellipticity in Figure 2 corresponds with the number of proline residues in the complex. The positions of these maxima shift to longer wavelengths as the polypeptide chain completes its first helical turn (Le. for compounds with n 1 4). This CD behavior is identical to that observed for proline oligomers (n = 1-11 prolines) studied by Okabayashi et al. where formation of the helical polyproline 11 structure resulted in a red shift of both the CD and UV absorption spectra.31-33 For [(NH3)sCo(Pro)sl3+,CD spectra obtained between 25 and 60 OC, the temperature range used for the ET studies, showed no significant differences. Kinetics of Inter- and Intramolecular Electron Transfer. Scheme I shows the sequence of reactions used in this study to measure the rate of intramolecular electron transfer in the series of intermediates [(bpy)2Ru1IP(Pro),-CoI1I(NH~)~]4+, where n = 1-6. Irradiation of an aqueous solution with a pulse of 2-MeV (or 20-MeV) electrons generates aqueous electrons and other radicals whose chemistry can be controlled by the use of buffers and tert-butyl alcohol to scavenge the hydroxyl radical. Disappearance of the aqueous electrons was followed at 650 nm. The aqueous electrons, in the presence of excess [(bpy)zRuIIL(Pro),,-
A / CoIlI(NH3)s]4+, rapidly reduce ( k = 8 X 1010 M-1 s-I) the Ru1* and the ColI1 centers in the binuclear complex to produce the species shown in Scheme I. Direct formation of the reduced Coil complex results in the rapid ( t < 100 n ~ ) aquation 3~ of the cobalt complex and is of no further interest to this study. Reduction of the ruthenium center produces a ligand-centered radical species having absorption maxima at 340 nm (c = 1.5 X lo4 M-l cm-1) and 450 nm (e = 1.4 X 104 M-I cm-I). The spectra of the ligandcentered radical intermediate and the RuII parent complex are shown in Figure 1. The decay of the intermediate was followed at 350 and 500 nm (Figure 3), where the differential absorbance is the largest. The measured intramolecular electron-transfer rate constant (keJ corresponds to the rate at which the ligandcentered radical reduces the Coili site in the same molecule. The hydroxyl radicals produced in the radiolysis were scavenged from solution by reaction with 1% tert-butyl alcohol to form 'CH~C(CH~)BOH. The presenceof the tert-butyl alcohol radical, *CH2C(CH,)20H, does not interfere with the kinetics because the intramolecular electron-transfer reaction studied here occurs at shorter time scales than the rate at which the tert-butyl alcohol radical reacts with the [(bpy)2RuI1L'(Pro),] moiety." Experiments on the 1 and 2 proline complexes were done using the 30-ps pulse width, 20-MeV electron linear accelerator at Argonne National Laboratory (ANL). The data were fit to a double exponential by least squares fitting to a simulated absorbance transient (including convolution of the excitation pulse) using software developed at ANL. The intramolecular electron-transfer rate for [ (bpy)2Ru1IP(Pro) l-Colll( NH3)sl 4+ is still to fast to be measured by this technique. A lower limit for this rate is estimated as 25 X lo* s-l at 25 OC. The experiments on the complexes with n = 3-6 profiles were conducted with the 40-11sminimum pulse width, 2-MeV electron Van de Graaff accelerator at Brookhaven National Laboratory. In these cases, the intramolecular electron-transfer rates and activation parameters (Table 11) were obtained from the transient absorption data using nonlinear least squares curve fitting to two exponentials. The excitation pulse is short on the time scale of
The Journal of Physical Chemistry, Vol. 97,No. 44, 1993 11459
Electron Transfer across Oligoprolines
TABLE II: Rates, Activation Parameters, and Distances for Intramolecular Electron Transfer in [(bpy)&~~L'(ho)x~~(NH~)#+, n = 1-64' no. of AH*, AS*, M-M' prolines ET (25 'C), s-l kcal/mol cal/deg.mol dist, A 1
2 3
4 5 6
> 5 X lo8 (1.6 f 0.1) X lo7 (2.3 f 0.2) X lo5
(5.1 f 0.4)x 104
(1.8 f 0.1) X lo4 (8.9 f 0.6) X 103
6.0f 0.6 9 . 2 f 0.4 9.4f 0.2 9.0 f 0.4 8.8 f 0.4
-6 f 2 -3 f 1
12 15 18
-5.5
21
f 0.8
-9 f 1 -11 f 1
n=3
24
27
n=4
Intermoleculaf
n.5
(7.2 f 0.1) X lo* M-I s-' 3.63 f 0.06 -5.8 f 0.2 a bpy = 2,2'-bipyridine, L = 4-carboxy-4'-methyl-2,2'-bipyridine. 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037 Conditions: n = 1, 2, 0.13 M t-BuOH, 0.005 M NaCH3C02, Ar m saturated;n = 3-6 and intermolecular: 0.13 M t-BuOH, 0.04 M NaC104, Figure 4. Temperature dependence of intramolecular electron transfer Ar saturated. Reaction of [(bpy)2Ru11L']++ [(NH~)~CO~~~(O~C-P~Oacross polyproline bridges in the series [(bpy)2RuIIL'-(Pro),-CoIIIProH)]3+ at pH 6. (NH3)5I3+,n = 2-6. TABLE III: Rates of Intramolecular Electron Transfer as a An intermolecular electron-transfer rate constant was obtained Function of the Concentration of the Complexes for the reaction of 66 pM [(bpy)2RuI1L'-] with [(NH3),CoI11[(bpy)*Runc(ho),-Com(NH~)~r+, D = 1-6 at 25 OC (Pro)2l3+between 13and77pM. Therateconstant andactivation n IRu(pro).Col, LIM k,s-l wavelength. nm parameters determined for this reaction between 5 and 50 O C 1 2000 >5 x 108 520 were k25 = (7.2 f 0.1) X lo8M-l s-I, pH* = 3.6 f 0.1 kcal mol-', 2 890 1.6 x 107 520 and LW= -5.8 f 0.2 cal d e g l mol-'. In an additional experiment 3 21 500 (3.0 f 0.3) X los where 220 pM [ ( ~ ~ ~ ) ~ R u ~ ~ L ( P ~ o ) ~was - Creduced O~~~(NH~)~] 40 (2.6 f 0.3) X lo5 500 by eaq- (conditions that will not lead to the observation of (2.1 f 0.4) x 105 41 350 intramolecular electron transfer), the intermolecular electron63 (2.5 0.2) x 105 350 4 5 (5.7 f 0.4) X 104 350 transfer process could be clearly observed as the concentration 11 (5.1 f 0.4) X 104 350 of [(bpy)2RuI1L]*- was increased by repeated pulsing. The second19 (7.0 f 0.3) X lo4 500 order rate constant for this reaction is estimated as (6.6 f 0.4) 5 5.1 (1.8 f 0.1) X 104 350 X lo8 M-' s-l. In the above-mentioned reactions of binuclear 12 (1.9 0.2) x 104 500 complexes, the observed rates for the slower components corre6 2 (8.9 f 0.5) X lo3 350 sponding to the intermolecular electron process agreed well with 3.8 (9.2 f 1.3) X lo3 350 7.5 1.1 x 104 350 these explicitly measured intramolecular rate constants. these reactions and was therefore treated as an impulse function. Of the two exponentials, the faster decay term corresponds to the intramolecular electron-transfer reaction. The slower exponential decay is assigned to the intermolecular electron transfer between binuclear complexes and between mononuclear [ (bpy)2RuI1L'(Pro)"] and binuclear [(bpy)2R~~~L(Pro),-Co~~~(NH3)5]~+. The relative extent of the intermolecular component increased when additional radiolytic doses were applied to the sample. For each set of experimental conditions (Le. temperature and/or concentration) the slower intermolecular rate was independently determined by a single exponential fit on a longer time scale using a partially (5-15%) irradiated sample. The observed intermolecular rate was then fixed in the second term in the double exponential fit to determine the rate of intramolecular electron transfer presented in Table 111. The absorbance change for the intermolecular process was allowed to float, due to the dose dependence mentioned above. For n = 1-3 prolines, the relative extent of the intermolecular reaction also increased upon prior exposure of the sample to light. Therefore the compounds with n = 1-3 prolines were kept in the dark prior to the kinetic experiments. Concentrations of each of the binuclear complexes studied were adjusted to keep the intramolecular electron-transfer rate within the time window defined by the electron-addition rate (8 X 1010 M-1 s-1) and the composite intermolecular electron-transfer rate (7.2 X 108 M-I s-l at 25 "C), a factor of 100 between the two reactions. The range of concentrations studied for the proline series is as follows: for n = 1 proline, 2 mM (solubility limit of thecomplex); n = 2,890-1000 pM; n = 3 , 2 0 4 0 pM; n = 4,s-20 pM; n = 5,s-12 pM; n = 6,243 pM. The intramolecular electrontransfer rates and activation parameters for each complex are listed in Table 11, and the rates at various concentrations are listed in Table 111. The intramolecular rates a t various temperatures are given as supplementary material.
Discussion This work highlights several new aspects of long-range electron transfer acrass polypeptides. The driving force for the E T reaction (Scheme I) is high, estimated to be -1.1 V (RuI1/I is -1.2 V and the(NH3),Co1I1/*1isestimatedat -0.1 V,1*+ allvs NHE). Figure 4 shows that the rates of intramolecular electron transfer for [(bpy)~Ru~~L(Pro),-Co~~~(NH~)5] decrease very rapidly, by over 3 orders of magnitude, as the length of the proline bridge is increased from n = 1 to 3 proline residues. However, further additions to the bridge result in only a modest decrease in rate from k, = 5.3 X 104 s-1 for n = 4 to k,, = 1.0 x 104 s-1 for n = 6. The transition between the region of large and small decreases in rate with increasing bridge length occurs at the point where the polyproline structure first becomes helical ( n = 4). Therefore the discussion of the results of the intramolecular ET reactions in this series and comparisons to other similar studies will begin with a review of the factors influencing the distance dependence of rates. The compounds with n = 1-3 and n = 4-6 prolines will be discussed separately because of the different peptide conformations of the oligoproline bridge as the number of prolines increases beyond n = 3. Factors Influencing the Distance Dependence: Contributionof Nuclear and Electronic Effects. The rate of intramolecular electron transfer in the nonadiabatic regime, ket,can be expressed in terms of eqs 1-41' ket = KePnKn K,~Y,
= vel = 1013exp[-@(r - ro)]
(1) (2)
(2HDA2/h)[(.3/XRT)''2] (3) = exp(-(A + AGo)2/4ART) (4) where K,I and K~ in eq 1 are the electronic and nuclear factors and Y,, is the effective vibrational frequency of the nuclear modes. For Y,, K,
11460 The Journal of Physical Chemistry, Vol. 97, No. 44, 1993 20
I
I
I
f
f
I
Ogawa et al.
I
I
AH*/RT
0
kI
1
f
f
I
I
I
I
I
d
2
3
4
5
6
7
10
Number of Prolines Figure 5. Plot of activation parameters AH*/RT (+) and AS*/R(A) versus the number of bridging proline units. nonadiabatic reactions, K ~ =I vel/vn l o A metal-to-metal distance), electron transfer between the donor and acceptor is expected to proceed only through the covalent bonds of the peptide bridge, since electron transfer through space interactions is expected to be extremely slow a t these long distances and thus may be ignored. Our discussion of possible ET mechanisms for the larger bridged systems will thereforeconcentrateon the natureof the intervening peptide bridge. Electronic Interactionsand Peptide Conformations. In general, the electronic interaction between the donor, the bridge, and the acceptor can be divided into several components: the interaction of the donor with the first atom of the bridge HDB,the successive interactions of two different bridging units HB,B~, HB~B,, ...,and finally the interaction between the last bridging proline and the coupling between the two redox sites acceptor H B ~ The . connected by a single covalent bridge18 is given by
-
where Hrpis the electronic coupling matrix element; H Dand ~ H,,A
Electron Transfer across Oligoprolines
The Journal of Physical Chemistry, Vol. 97,No. 44, 1993 11461
SCHEME I1
The quantitative contribution of the secondary structure to the overall electronic factor could not be determined because the nuclear contributions for n = 1-3 could not be separated from 8. However, it is reasonable to assume that 8 is 10.65,the value determined for the [(NH3)s0s11-i(Pro),-Ru~~I(NHs)sl series.1f Future studies with other donors and acceptors should make this determination possible within a single series. Recently, Sneddon and Brooks have published36 an analysis of the conformational energetics and observed electron-transfer rates for the series of proline-linked donor-acceptor complexes [ (NH3)sRUIL~(P~O),-COIII(NH~)S]~+. The minima of their calculated potentials of mean force for n = 1-4 confirm the earlier estimates of internuclear distances that we obtained by molecular modeling. One of the salient points of their calculations is the observation that the barrier for rotation around the angle (the 8-to-a transition) is only 12 kcal/mol, compared to the 22 kcal/mol [(NH3)508-(Pro),Rum(NH3)5];n = 4 barrier for trans-to-cis isomerization about the o angle (both estimates are for “blocked” or amide group terminated dipeptides). are the coupling elements between the orbitals of the donor and Either rotation can result in substantially shorter metal-to-metal acceptor and the atomic orbitals of the adjacent bridge atoms, distances than those of the more stable /?-trans (polyproline 11) 1 and n, respectively; al, and an”are the coefficients of the vth configuration. However, the a-trans configuration is about 5 bridge molecular orbitals at the atoms bonded to D and A; EB” kcal/mol higher in energy than &trans, meaning that less than is the energy of the vth molecular orbital of the bridge; and the 0.1% of the molecules would occupy such configurations at summation is over the molecular orbitals of the bridge. Equation equilibrium. Assuming a preexponential factor of 1013, 8-to-a 6 predicts that, in order for the bridging ligand to be effective in isomerization would occur no faster than lo4s-1, which is slower transferring the charge between the donor and acceptor, it should than most of our observed electron-transfer rates. Moreover, the be made of units that can overlap effectively with the donor or isomerization rates would be expected to further decrease with acceptor orbitals. In the work described here, ET occurs from longer solvent-stabilized oligoprolines. Therefore on the time a state where the electron is a t least partly delocalized over the scales of our experiments, it is unlikely that the above conformethyl carboxy bipyridyl ring (LO) and is thus directly connected mational isomerization would contribute to the observed rates. to the N-terminal of the peptide bridge. This interaction is Further work is required to confirm this. expected to result in mixing of the donor (LO)T* orbital with the Comparison to Electron-TransferStudiesin OtherOligoproline unoccupied 7r* orbital of the first proline peptide unit. This Systems. Several donor-acceptor systems with oligoproline interaction may be a significant contributor to the lower decay bridging groups have been studied over the last The factor 8 observed in this series. The other electronic factors H12, E T rates and their temperature dependencies for a series of oligoprolines, Tyr-(Pro),-Trp, n = 0-5, have been studied by ...,H(,+l),,H , (n 1 2), the interaction between the peptide units, and HBD,the interaction between the a-terminal of the bridge pulse radi~lysis.~ Since the studies were done in aqueous solution, comparison with our results is feasible, especially for n = 4 and and the acceptor, are similar throughout the series studied. 5 prolines, where a similar polyproline I1 secondary structure The Gamow tunneling equation,j7 which describes the electronic exists in both s e r i e ~ . Some ~ ~ ? differences ~~ exist between the Tyrinteraction between two centers in terms of the barrier tunneling (Pro),-Trp, n = 0-5, series and that of the metal-derivatized height, can be applied to the proline studies prolines studied here, especially in the flexibility of the Tyr and Trp donor and acceptor connections to the oligoproline bridge. The connection of the phenol and indole chromophores to the oligoproline bridge in the Tyr-(Pro),,-Trp series is to the flexible amino acid side chains of Tyr and Trp, whereas in the present where Vis the barrier height and m is the electron mass. This studies the donor and acceptor are more rigidly connected to the equation shows that 8 may be a constant within a specific series terminals of the polyproline bridge. For the longer bridges, n = but changes as a function of the barrier height Vor the coupling 4-6, E T results for both series show a weak distance dependence of the donors and acceptors with the bridging ligand. of the electronic factor (@ 0.3 A-’). For the Tyr-(Pro),-Trp A comparison of the bond connectivities for a common member, series, a low 8 is obtained for the entire series, n = 0-5. Recent n = 4 prolines, of the two series [(bpy)~R~11L(Pro),-Co~~~(NH3)~] analysis of these results from temperature-dependence studies of and [(NH~)sOSIL~(P~~),,-R~~*~(NH~)~] is shown in Scheme 11. the rate constant for intramolecular E T concluded that, for n = In both series the metal ion donors are connectectto the proline 1-3, the most likely pathway may not involve the oligoproline bridge through a 4-carboxypyridyl derivative at the N-terminal bridge, while, for the longer bridges, ET involving the proline of the proline peptide and the metal ion acceptors are directly bridge is sugge~ted.~ In the present study the direct attachment connected to the C-terminal of the peptide. of the donor and acceptor to the proline bridge results in a higher A difference between this work and the series studied earlier 8 for n = 1-3 prolines. is the formation of the helical trans-polyproline I1 secondary The different ranges of redox potentials for the Tyr-(Pro),structure adopted by the oligoproline bridge starting with n 1 4 Trp+ and the [(bpy)~Ru1IL‘-(Pro),-Co~~~(NH3)514+ series also suggest that the former might proceed through a “hole-transfer” prolines. This change in the secondary structure of the oligomechanism, while the latter might proceed through an ”electronprolines (n = 4-6) results in a change in the electronic transitions transfer” mechanism.l*b-4eJ4b In Tyr-(Pro),-Trp electron transfer of the polyproline bridge as seen by a red shift in the UV-vis and occurs in radical cations that have a redox potential of approxCD spectra of 0ligoprolines3~-~~ and metal-derivatized oligoproimately 1 V vs N H E and a very small driving force3 In the lines (this work, Figure 2). Thus the secondary structure that present systems electron transfer from the RuIIL‘ center to the oligoprolines adopt may contribute to a more favorable mixing Co(II1) center is the equivalent of electron transfer from a radical between the proline orbitals and the donor T* orbitals. These anion with a potential of -1.2 V vs N H E to the Co(II1) oxidant. new transitions may contribute to the lowering of the electronic This difference in orbital energies of more than 2 V can result factor 8 in the helical trans-polyproline I1 molecules, n = 4-6.
+
-
11462 The Journal of Physical Chemistry, Vol. 97, No. 44, 1993
in different mechanisms where a hole transfer is more favorable in the radical cation case and electron transfer is more favorable in the [(bpy )2RuIlL*-( Pro) ,ColI1( NH3) $14+ case. The contribution of the helical polyproline I1 secondary structure to ET mediation can be further estimated by comparing the ET rate in the polyproline I1 structure to that in the more extended random polyproline conformation. The helix to coil transformation in polyproline I1 is insignificantin the temperature range within which intramolecular ET was studied (25-60 "C), as evidenced by no measurable change in the CD of [(NH3)5Co(Pro)5I3+over this range. However at higher temperatures (ca. 100 "C), Bobrowski et al. observed changes in the CD, consistent with the loss of the polyproline I1 secondary ~ t r u c t u r e . ~ Further work at high temperatures where the polyproline I1 helix changes to a random structure could reveal the effect of rate on helicity in a more direct manner. Although rates of electron transfer could be measured at these long distances of n = 1-6 in the present series of complexes using pulse radiolysis techniques, this series is still not the ideal system for studying ET across prolines. The small absorbance changes between [(bpy)zRu1IL]*and [(bpy)2RuL11] have been used for the determination of the rates and temperature dependence in this series. The ET reaction could only be monitored at the ruthenium donor site because the acceptor [ (NH3)5C0111] does not have a strong chromophore for monitoring the formation of Co", the sole product. Furthermore, the driving force for the electron-transfer reaction cannot be measured directly, because the redox potential at the [(NH3)5C0111]acceptor site cannot be measured but only estimated from self-exchange processes.40 Although the estimated driving force does not affect the accuracy of the rates, it can affect the calculationof the nuclear parameters for the n = 1-3 proline series. Work is in progress to design donor and acceptor systems that have more sensitive chromophores, similar charges and solvation properties, and redox potentials that can be measured dire~tly.~1,42Such complexes should allow the nuclear and electronic effects to be separately evaluated and their contributions from the helical secondary structure of the polyproline I1 bridge to be quantitatively e~aluated.38+~2
Conclusion In this study long-range electron transfer has been observed across a six-proline bridge, a distanceof greater than 30A between the donor and acceptor. This is possible because of the high driving force between the donor and acceptor. Mediation of electron transfer by the polyproline I1 helical structure may also be an important contributor to the small distance dependence observed. Furthermore these results suggest that intramolecular ET across peptides may be observable even beyond 30 A (6 peptide units) in more optimized donor and acceptor polyproline I1 bridged systems. Acknowledgment. The work at Rutgers University was supported by the US.Department of Energy, Division of Chemical Sciences, Office of Basic Energy Sciences under Contract DEFG05-90ER1410. The workcarriedout at BrookhavenNational Laboratory was supported by the US.Department of Energy, Division of Chemical Sciences, Office of Basic Energy Sciences under Contract DE-AC02-76CH00016. M.Y.O. was supported at Rutgers by a fellowship from the N I H (NSRA # 1F32-GM 13223). The workat Argonne National Laboratory was supported by the U S . Department of Energy, Division of Chemical Sciences, Officeof Basic Energy Sciencesunder Contract W-3 1- 109-ENG38. Helpful comments on the manuscript by Dr. Norman Sutin are gratefully acknowledged. Supplementary Material Available: Eyring plots for n = 2-6 ( 5 pages). Ordering information is given on any current masthead page.
Ogawa et al.
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