Long range electron transfer in helical polyproline II oligopeptides

Chemical Physics 176 (1993) 589-600 Nosh-HoUand

Long range electron transfer in helical polyproline II oligopeptides

Michael Y, Ogawa =,I, Icaro Moreira a,‘, James F. Wishart b and Stephan S. Isied *xa a Department of Chemistry, Rutgers, The State University of New Jersey. New Brunswick, NJ 08903. USA b Department of Chemistry, Brookhaven National Laboratory, Upton, NY 11973, USA

Received 23 April 1993

A series of binuclear donor-acceptor complexes with helical polyproline bridges [ (bpy)~Ru’*L-(Pro)=-apy-Ru”x(NH~)~]s*, n=6, 7, 9, where L=4_carboxy-4’-methyl-2,2’-bipyridine, bpy=4,4’-bipyridine, and apy=4_aminopyridine, were synthesized and characterized by absorption spectra, electrochemistry and HPLC. The CD spectra of the complexes confirm that they exist in the helical polyproline II structure. Intramolecular electron transfer within these complexes was studied by generating the [ (bpy),Ru’L’-(Pro),-apy-Ru”‘(NH&] intermediate from the reaction of e., (from pulse radiolysis) with the [ (bpy),Ru%- (Pro),-apy-Ru”‘(NIi&] species in aqueous solution. The driving force for this reaction is estimated to be IAGo = 1.5 V. The rates (k, 25°C) and activation parameters (MT (kcal/mol), AS’ (eu) ) for the intramolecular electron transfer were found to be: 1.08~ lo5 s-l, 5.6, - 17; 6.40~ ~O’S-~, 5.1, - 19; 1.91~ lo4 set, 4.0, and -26 for n=6, 7, 9 respectively. The rate (k, 25“C) and activation parameters (A@ (kcallmol), AS’ (eu)) for the intermolecular reaction between [ (bpy)sRu’L’] and [ (NH,),Ru”‘-apy-Pro] were found to be 2.1 x lo9 M-’ s-l, 3.3 and - 5. This series extends our studies of the distance dependence of rate versus the number of helical prolines bridging a donor and acceptor ruthenium site to a metal-to-metal distance w 40 A. The weak dependence of rate versus the number of prolines observed for n = 6,7, and 9 is very similar to that observed earlier for [ (bpy),Ru”L-(P~~),-CO”‘(NH~)~], n=4-6. The rapid rates observed at these long distances show that long range electron transfer can be observed between an appropriate donor and acceptor directly connected to the proline bridge via peptide bonds at distances similar to the diameter of a small protein.

1. Introduction

Long range intramolecular electron transfer (LRET) with synthetic~y designed peptides has provided new insights into the role of peptides in me- diating electron transfer [ 1 -IO]. Studies using oligoproline peptides as rigid spacers for intramolecular electron transfer across metal complexes have re- sulted in rate variations of more than ten orders of magnitude, depending on the nature of the donor and acceptor metal ions [ I]. The driving force and reorganization energy of the donor-acceptor pair have been used to alter rates of electron transfer to fall into measurable time regimes. Recent results from a study of a series of metal-derivatized oligoproline peptides

* Co~pondi~ author. ’ Present address: Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403, USA. ’ Present address: Departamento de Quimica Organica e Inor- ganica da Universidade Federal do Ceara, Fortaleza-Ce, Brazil.

[ (bpy),Ru11L-(Pro),-Co111(NH3)5] (n=l-6) [ 111 have shown that the rate of intramolecular electron transfer is not a simple function of the length of the peptide joining the donor and the acceptor. At long distances, ra = 4-6 prolines, the distance dependence parameter of the rate of electron transfer, p, was found to be surprisingly small. This study could not be extended beyond n = 6 prolines because of interfering intermolecular reactions [ 111.

In this paper we have investigated intramolecular electron transfer across longer oligoproline peptides, n=6, 7, 9, by studying a series of related metal- derivatized oligoproline peptides of the type

[ (bpy)2Ru11L-(Pro),-apy-Ru”“(NH,)5]5+ , I-III

n=6,7,9,

(L= 4~rboxy-4’-methyl-2,2’-bipyridine and apy = 4-aminopyridine), where the acceptor (NH3)5C~111 in the earlier [ (bpy),Ru”L-(Pro),-Co”‘(NH,),]

0301-0104/93/% 06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved.

590 M.Y. Ogawu et al. / Chemical Physics 176 (1993) 589-600

u(NH&

n-9

Scheme I.

series has been changed to (NH3)SRuu1-apy, a slightly stronger oxidant with substantially lower reorganization energy [ 12 1. The driving force for this reaction is ?5! 1.5 V. This new series of complexes al- lowed us to measure rates of intramolecular electron transfer between a donor and acceptor separated by up to nine proline residues.

Ruthenium bipyridine, [ (bpy)2RunL] (L=4-carboxy-4’-methyl-2,2’-~py~~ne), and ruthenium ammine, [ ( NH3) sRuul-apy] (where apy = 4-ami- nop~~e), complexes are covalently attached to the N and C terminals of the helical oligoproline peptides as shown in scheme I_

This paper describes the synthesis, characteriza- tion, and electron transfer properties of the donor- acceptor complexes with n= 6,7,9 prolines. The weak distance dependence of the rates of intramolecular electron transfer obtained for these oligoproline complexes is discussed in terms of the reorganizational and electronic interactions of the donor and acceptor and the special features of the helical structure of the peptide bridge.

2.1. methods and materials

All the chemicals used were reagent grade unless specified. The solvents CH$&, DMF, CH&N were distilled before use. The [ (NHS)6R~]Cls (Mathey Bishop, Malvem, PA) was used to prepare I (~3hRuC1lcl2, t (~,),RuQl W-)2 (TFA=

trifluoroacetate), and [ (NH,),Ru(OH,)] (PF6)2 according to literature procedures [ 2,13 1. The 1 -proline, Boc- 1 -proline, and Boo l- (proline)* were purchased from Sigma or Peptides International. The 4- aminopyridine was purchased from Aldrich and G.F. Smith. N-methyl morpholine, ~isopropylc~bodi- imide (DPC) and hydroxy~nzot~azole (HOBT )

were purchased from Aldrich. Prep-pack 500-Cls was obtained from the Millipore Corp.

N-(4-pyr~dy~-Boc-proIine (I,’ BocPro, L ’ = IV-(4-pyr- jdy~), 1.0 g BooI-proline (4.7 mmol) and 0.43 g l- hydroxybenzot~~ole (4.7 mmol) were dissolved in approximately 3 ml of 1: 1 CH2C12/DMF. To this solution was added 0.6 g N,N’-diisopropylcarbodiimide (4.7 mmol). A white precipitate appeared within a few minutes and a mixture of 0.49 g of 4- aminopyridine (5.1 mmol) and 0.57 g N-methylmorpholine (5.6 mmol) were then added. The reaction mixture was stirred overnight at room temperature ( 15 h) after which the precipitate was removed by vacuum filtration. The solvent was reduced by rotary evaporation, the residue was dissolved in z 5.0 ml of ethyl acetate, and the unreacted 4-aminopyridine was removed by repeated extraction of the eth- ylacctate solution of the product with water. The ethyl acetate layer was reduced by rotary evaporation, the residual solid dissolved in 1 O”kt aqueous CH,CN, and purified by Cl8 reverse phase chromatography (25 urn particles) gravity column using a step gradient of CH3CN and water, starting from 10% CH&N and stepping the gradient to 25% (in increments of 5%) while monitoring the eluent for unreacted aminopyridine. After the aminopyridine was removed from the column, the product was eluted with 50% aqueous CH&N to remove the Boc-Pro-aminopyridine and monitored by HPLC. Evaporation of the solvent re- sulted in the product N-( 4-pyridyl)-Boc-proline as a white solid. Yield 0.03 g (25% based on 4- aminopy~dine ) .

r~~H~)sRu-N-f-3~Pr~]~PF~~. 0.063 g N- (4-py~dyl)-BocPro (0.22 mmol) was dissolved in 4 ml of degassed acetone in the presence of several pieces of freshly prepared zinc amalgam. Upon addition of [(NH,)sRu(OH2)](PF,)Z (0.135 g, 0.27 mmol), the solution turned dark red. After 1 hour the reaction mixture was poured into 50 ml of degassed diethylether. The crude product was collected by vacuum filtration, washed with diethylether and dried in a desiccator. The Boc-protected complex was purified by Cl8 column chromatography using a 5% methanol-water eluent. The pure material was con- centrated to dryness by rotary evaporation and then deprotected by dissolution in 1 ml of water and 1 ml 48% HBF,. Several drops of Hz02 were then added to this solution and the product was precipitated by

M. Y. Ogawa et al. / Chemical Physics I76 (1993) 589-600 591

the addition of absolute ethanol and diethylether, yield 0.07 g (43%). The complex was characterized by HPLC, absorption spectrum and electrochemistry (cyclic voltammetry and differential pulse polarography ) .

[(NH3),-Co(Pro),,](BFJ3, n= 5, 6, 8. The cobalt protected oligoproline complexes were prepared using procedures developed earlier in this laboratory [ 141 with the following modification. For the complexes described here, [ (tert-butoxyloxy) carbonyl ] tetra- 1 -proline (Sigma Chemicals) was used in place of (tert-butoxyloxy )carbonyl- 1 -proline and coupledto [ (NH,)&o(Pro),] (BF,)3, n= 1,2,4, as described earlier [ 141. The Boc-protecting group was removed by treating the complex with 95% aqueous trifluoroacetic acid (HTFA) for one hour.

[(bpy),Ru”L-(Pro),-Co”‘(NH3)J(BF&, n = 5, 6, 8, where L= 4-carboxy-4’-methyl-2,2’-bipyridine, The binuclear complexes [ (bpy)zRuuL-(Pro).-Co”‘-

W-b),1 W4)4, n=5, 6, 8, were prepared as described previously [ 111.

f(bpy)3RurrL-(Pro),,](BFJI, n = 5, 6, 8 where L= 4- carboxy-4’-methyl-2,2’-bipyridine. The mononuclear ruthenium complexes were prepared by reducing the cobalt center in the corresponding binuclear complexes [ (bpy)zRu*1L-(Pro),-Conr(NH3),] (BF4)4, with zinc amalgam in 0.1 M HTFA solution under argon. The conversion from the binuclear to the mononuclear complex occurred within approximately 30 minutes and was monitored by HPLC. The solution was evaporated to dryness by rotary evaporation, dissolved in a minimal amount of water and purified by Cl8 chromatography. After concentration of the solution of the mononuclear complex, precip- itation was accomplished by addition of ethanol, several drops of HBF4 and diethylether. The solids were collected by filtration, washed with diethylether and dried under vacuum. Yield x60% for the different complexes.

t(bpyl~RurrL-(Pro),-Ru”‘(NH~)~l(BF~)~l(BF~)~, n = 6, 7, 9, where L= 4-carboxy-4’-methyl-2,2’-bipyridine. The 10.0 mg (7.8 umol) of [ (bpy),RuL- ( Pro)5] ( BF4)2 and 2.0 mg of l-hydroxybenzotria- zole ( 15 umol) were dissolved in 200 ul of 1: 1 DMF/ acetonitrile. To this solution was added 2 mg of N,N’- diisopropylcarbodiimide ( 16 umol) . After approximately 5 minutes, 8 mg ( 11 umol) of [ (NH3),RuL- Pro] ( HBF4)3 and 1.3 mg N-methylmorpholine ( 13

umol) were added to the reaction mixture, which was then stirred at room temperature for 24 hours. The orange solid was dissolved in a minimum amount of water and the solution was loaded onto a small (2.5 x 10 cm) cation exchange column (Sephadex C- 25). The unreacted [ (bpy),RuL-(Pro)5]2+ was removed by elution with 0.3 M HTFA and the desired binuclear product was eluted with 1 M HTFA. The product was converted to the BF4 salt by ion-exchange chromatography and final purification was effected by Cls column chromatography using a methanol-water gradient ( 5 to 50%). Solvent was removed by rotary evaporation. The residue was dissolved in methanol and precipitated with diethylether to yield a dark orange solid. Sample purity was checked by HPLC, electrochemical and mass spectral measurements. Yield 5.3 mg (36%).

The heptapeptide was prepared similarly to as described above for the n= 6 species. Yield: 2.7 mg (21%).

The nonapeptide was prepared in a manner similar to that described above. Yield 3.0 mg ( 17%).

2.2. Circular dichroism measurements

Circular dichroism (CD) spectra were measured at room temperature using an Aviv Model 60DS CD spectrometer. The binuclear compounds [ (bpy)zRu*1L-(Pro).-LRu”‘(NH3),] (BF4)4, n= 6, 7, 9, were dissolved in water at x 1 x low5 M.

2.3. Electrochemical measurements

Reduction potentials for the binuclear compounds [ (bpy),Ru”G(Pro),-LRurn(NH,),I (BF,),, n=6, 7, 9, were carried out in 0.1 M NaTFA, pH=3 by differential pulse polarography (DPP) and cyclic voltammetry (CV). A Bioanalytical Systems Inc. Model 1 OOA electrochemical work station was used, fitted with a glassy carbon working electrode, and a platinum wire counter electrode referenced to SCE. Sample solutions were purged with argon prior to the measurements.

2.4. Mass spectral analysis

FAB MS of molecular cations and fragments were obtained at the CAFT facility in Cook College, Rut- gers University.

592 M. Y. Ogawa et al. / Chemical Physics I76 (1993) 589400

The kinetics of intramolecular electron transfer in the complexes [ (bpy)~Ru”~(Pro)~-apy- Ru~(NH~)~] (BF,),, n=6,7,8, were determined by pulse radiolysis techniques as described previously [ 2,111. The kinetics experiments were performed in 0.13 M tert-butanol, 40 mM NaCIO,, argon-satu- rated water at pH 6. Concentrations of the binuclear complexes ranged from 1 O-61 FM, 5-20 pM, and 1 *O-4.0 PM, for n= 6,7,9 prolines, respectively.

The intermolecular electron transfer rate constant was determined using a 20 pM solution of [ ( bpy)zRunL-Pro ] containing 4.4-2 1.8 pM [ ( NH3 ) ,Run’-ap y-Pro ] 3+. The time course of the electron transfer reaction of the mononucle~ and binuclear complexes with aqueous electron was followed spectrophotometrically at 510, 350, and 410 nm. Treatment of the kinetic data is described in refs. 12331.

3. Results

The binuclear complexes (S-III) were synthesized according to the following scheme [ 3 ] :

[ (bpy)~Ru1’L(Pro)~_,-Cox*~(NH3)~] +5H+

(II)

z”,He. [(bpy)zRu”L(Pro),_IOH]

+co2++5NH+ 4 f

I(bpy)2RurxL(Pro),-~OHl

-+ [ (NH3),Ru1n-apy-Pro]3+ x

[ (bpy)2Ru*rL(Pro),-apy-Run1(NHr)~ I’+. (1)

The [ (~3)~Ru~-apy-Pro] was added to the proline bridge in [ (bpy)~RuxiL~Pro)~_~OHl using standard methods of peptide synthesis (with HOBT and DPC) [ 121. The HPLC retention times of all the mononuclear and binuclear complexes used in the synthesis of binuclear ruthenium complexes (I-III ) are shown in table 1. The binuclear complexes were characterized by W-visible spectra, electrochemistry, FAB MS (molecular ion and fragmentation patterns ) .

Electrochemical measurements (DPP and CV) on the mononuclear [ (NH, ) ,Rum-apy-Pro] 3c show a single, reversible reduction at +0.02 V versus SCE, similar to that reported for other [ ( NH3 ) 5Run1py J complexes [ 15 1. The mononuclear [ (bpy)2Ru”L], where L=4-carboxy-4’-methyl-2,2’-bipyridine, also shows a reversible oxidation at + 1.30 V versus NHE [ll~.TheCVandDPPof[(bpy)2Ru~L(P~)~-apy- Ru~~l(NH~)~ls~ in 0.1 M NaTFA, pH 3, show two reversible waves at -t 1.30 V and -t-O.26 V versus NHE (fig. 1). These are assigned to the [ (bpy),Ru”’ II’L] and the [ (NH~)SRun/nf -spy] centers in the binuclear complex, respectively. The observed potentials for the n = 6, 7, 9 binuclear species and the

Table 1 HPLC retention times of mononuclear and binuclear ruthenium proline complexes ‘)

R [ fbpy)zRunL(Pro),Co]5+ [ (bp~)ZRuUL(Pro),lz+ [ (bpy)*Ru’~L(Pro)~-apy-Ru ] ‘+ ‘) (min) (tin) (min)

0 11.00 5 14.15 16.86 6 17.23 21.05 7 21.95 8 14.95 20.16 9 26.00

*) Cl 8 column, 2 i % acetonitrile, 0.2% HTFA. bk bpy=2,2’-bipyridine, L=4-carboxy-4’-methyl-2,2’-bipyridine, apy=4-aminopyridine, CO=CO(NH~)~, Ru=Ru(NH3)5.

Table 2

M. Y. Ogawa et al. / Chemical Physics 176 (1993) 589-600 593

Potentials of the mononuclear and binuclear ruthenium proline complexes ‘)

n Em (V)

[(bpy)2Ru”L(Pro).l*+ [ (bpy)2RunL(Pro),-apy-Ru(NHr)S]5+ b,

6 +1.30 +0.26, + 1.31 I + 1.30 +0.26, + 1.30 8 + 1.30 9 +0.26, +1.31

a) 0.1 M NaTFA, pH 3.0. ‘) [(NH&Ru-aPy(Pro) 1 *+I’+, EL,*= +0.26 V. All potentials versus NHE.

1

E (Volts vs SCE )

A . ..#. _A -- *1 so0 r1.0 l o 5 00.0 -0.500

E (Volts vs SCE ) B

Fig. 1. DPP and CV of [ (bpy),RunL(Pro)e-apy-Ru(NHs)5 in 0.1 M NaTFA, pH 3.0.

corresponding mononuclear ruthenium proline complexes are summarized in table 2. The reduction po-

tential for [ (bpy)zRull/‘L] was determined to be - 1.2 V versus NHE in earlier work [ 111.

The UV-visible spectrum of the [ (bpy)zRunL(Pro),-apy-Rum(NH3)S]5+, n=6, 7, 9, chromophore is seen clearly in the difference spectrum of [ (bpy)2Ru11L(Pro),-apy-Ru11(NHa)5]5+ minus [ (bpy),Ru”L(Pro),-apy-Runl(NH,),l ‘+ obtained by the oxidation of the Ru(II)-Ru(II) complex by & [ Co ( oxalate ) s ] (fig. 2 ) .

The CD spectra of the binuclear complexes, [ (bpy)2Ru11L(Pro),-apy-Rum(NH,),15+, n=6, 7, 9, in aqueous solution (fig. 3 ) show a single, negative CD peak at 207 nm, which is characteristic of the hel-

Fig. 2. Circular dichroism spectra of [ (bpy ),Ru”L (Pro) s-apy- Rum(NH&]. n=6, 7, 9 in water. The concentrations of the compounds were determined from their absorbance at 456 nm (e= 1.4~ 10’ M-’ s-‘) and the resulting CD spectra were nor- malized to a value of 1 uM.

594 M. Y. Ogawu et al. /Chemical Physics I76 (1993) 589-600

‘.c~,,. ...__, _,~~

350 coo 650 so0 550 coo

Fig 3. Visible absorption spectra for [ (bpy)2Ru”L(Pro)9-apy- Rutt(NH,)s] (A) and [ (bpy)2RunL(Pro),-apy-Rum(NHs)S] (R) in aqueous solution, pH 5. The insert is the difference spectrum between the two species A and B.

ical structure of ~an~pol~roline II. The trans-poly- prohne II has a repeat unit of three amino acid residues which increases the length of the helix by 3.1 A/ unit. The magnitude of rotation at 207 nm increases with the number of bridging proline residues (n= 6, 7, and 9) for a constant complex concentration (fig. 3).

3.2. Electron transfer reactions of the(@py),Ru-L- ~Pro)*-ap~-Ru(N~~)~~ complexes, n = 6, 7, 9

Zntramolecular electron transfer in these complexes was investigated using pulse radiolysis techniques. The reduction of [ (bpy),Ru”L’- (Pro),- apy-Run(NH3),] by aqueous electrons (E”= -2.8 V) occurred at each of the two metal sites according to

e, + [ (bpy),Ru”L(Pro),-apy-Rum] II

-+ [ (bpy),Ru’*L’- ~Pro)~-apy-RurxrJ ,

III

4 k

@a)

-+ [ (bpy),Ru”L-(Pro),-apy-Run] ,

Iv (2b)

apy -Ru”‘= 4-aminopyridine-Rur”r ( NH3 ) 5, L= 4- carboxy-4’-methyl-2,2’-bipyridine, n = 6-9; eq. (2a) forms the highly reducing (E*= - 1.2 V) precursor

f (bpy)~Ru”L.-(Pro)~-apy-Ru~‘(NH~~~] and eq. (2b) forms the the~odyn~i~ly stable [ (bpy)~RurlL-(Pro)~-apy-Rul*(NH~)~] complex. The intramolecular electron transfer rate constant was monitored at two wavelengths: I = 5 10 nm for the decay of the reduced bipyridine site and A = 4 10 nm for the formation of the reduced [apy-Ru11(NH3)5 ] species. The same kinetic behavior, corresponding to the electron transfer reaction between the [ (bpy )2Ru11L] * - and the [ ( NH3)5Ru1u-apy] sites, was observed for all the complexes (fig. 4 ). Tables 3 and 4 summarize the in~amole~ul~ electron transfer rates obtained for these oligoproline bridged complexes at different temperatures and concentrations.

The spectrum of the intermediate [ 111 [{(bpy)2Ru11L}‘-(Pro),-apy-Ru11K(NH,),]4+, prior to intramolecular electron transfer, shows an enhanced absorption in the range 3 1 O-400 nm, an

40t I

0 40 80 120

psec

Fig. 4. Pulse radiolysis transient absorption data for n = 7 prolines at A=410 nm showing the increase in the absorption due to the [apy-Ru”(NH&] species and at ,I=510 nm showing the decay of the [ (bpy),Ru”L]‘ species. The initial rapid decay (3 ns ) is from the disappearance of eG.

M. Y. Ogawa et al. f Chemical Physics 176 (1993) 589-600 595

Table 3 Rates and activation parameters for ~~rnol~~r and intermolecufar electron transfer across poiyprol~~ f (bpy )*Ru*~~(Pro f,-apy- Ru*(NH~)~]~+*~=~, 7, Pseries rt (bpy= 2,2’-bipyridine, ~=~c~xy~‘-~et~yl-2,2‘-bipy~~ne, apy =4-aminopyridine)

?l M-M b, knC) AHf *) ASfd’

(A) 25°C (kcal/mol) (caldeg-‘mol-‘) (s-l)

6 31.6 1.08+ 10” 5.650.2 7 34.5 6.40x lo4 5.lkO.2 9 40.8 1.91x104 4.0fO.l

intermolecular reaction (es. (3)) 2.1xlO*M-‘s-’ 3.3rt0.2

*) 0.13 M r-butanol, 0.04 M NaC104. b, Method for cdlculation of distance is described in ref. [ 21. =f Optimal ~n~nt~tions used were 17.8, 10.4, 1.6 pM for n=6,7, and 9, respectively. d, The error limits for the activation parameters represent one standard deviation.

-17fl -19+1 -26&l

-5fl

Table 4 Intramolecular electron transfer rates versus donor-acceptor complex concentration for the [ (bpyf,Ru”L’(Pro).-apy- Ru’~(NH~)~]~+, n=6, 7,9, series *)

n [Cl (FM) k (s-l)

6 61 (1.0~0.3)x105 20 (1.3+0.3)x 105 10 (1.2+0.2)x 105

7 20 (6.3f0.3)x104 10 (6.3f0.3)x104 5 (5.8fOh)x104

9 4 (2.1f0.4)x104 2 (2.1+0.3)x104 1 ~1*9~0.3~x104

a) Conditions: 0.13 M t-BuOH, 0.04 M NaClO,.

isosbestic point at 410 nm, a bleach at 420-460 nm, and a second enhancement at longer wavelengths. The difference spectrum between the reduced [ (bpy),RuuL’-] and oxidized [ (bpy),Ru”L] species is consistent with the formation of a ligand-cen- tered anion radical [ (bpy)zRullL] .-, having a life- time of several milliseconds [ 16,17 1. Direct reduction of the [ (NHJ)SRuln-apy-Pro] site produced an increase in absorption at 410 nm for the formation of the [ (NH,)SRuu-apy-Pro] complex at A=410 nm (ec 104M-* cm-‘).

The ~~~~~olec~ar electron transfer reaction

[ (bpy),Ru”LPro] ’ - + [ (NH3)SRunK-apy-Pro]

+ f (bpy ) t Ru”LPro ] + [ (NH,) s Ruxl-spy-Pro ] ,

(3)

was studied by the reaction of e& with a solution of [ (bpy),Ru”L] (20 PM) containing [ (NH, ) SRulll-apy-Pro ] in concentrations ranging from 4.4 to 2 1.8 PM. The second order rate constant for this reaction was determined to be 2.1 x lo9 M-’ s- ’ at 25°C. The reaction was monitored at the two wavelengths 350 and 410 nm, characteristic of [ (bpy),Ru”LPro] .- and [ (NH,),Ru”*-apy-Pro], respectively. ~~~e~olecular electron transfer rates for [~(bpy)~Ru~1L~‘-(Pro)~-apy-Ru111(NH~)~]4+, n = 6, 7, 9, were also determined empirically by following the intramolecular electron transfer reactions to longer timescales. These results were very similar to the rate obtained for the reaction in eq. (3). No measurable dependence of the intermolecular rate on the number of proline residues was observed as expected.

4. Discussion

4.1. Characteristics of the donor-acceptor series

The series of binuclear complexes studied in this paper [ (bpy)~RullL-(Pro)~-apy-Ruxil(~~)~]s+, n = 6,7,9, span the longest distances studied between a donor and an acceptor bridged by proline peptides.

596 M. Y. Ogawa et al. / Chemical Physics 176 (1993) 589400

The donor-acceptor metal ions are separated by 67, and 9 prolines with metal-to-metal distances ranging from 30 to 40 A in length. The confo~ation for the bridging proline oligomers of this length in aqueous solution is known to be a left handed, solvent stabilized helical polyproline II structure [ l&19 J. This helical polyproline II structure of the bridged peptides is confirmed by the presence of the characteristic negative peak at 207 nm in the CD spectra of the donor-acceptor complexes (fig. 2 ) . The six and nine proline bridged complexes have two and three helical turns, respectively.

(rEI,),]~h [ (bpy),Ru”G(Pro),,-apy-Run’-

, n = 6, 7, 9, series studied here the donor and acceptor metal ions are connected directly to the main chain of the peptide bridge. Such direct connec- tivity minimizes the flexibility at the donor and acceptor connection to the bridging peptide. Both the donor and acceptor ~thenium metal ions in the [ (bpy)*Ru11~(Pro)“-apy-Ruur(NH~)~]5+, n=6, 7,9, series possess reversible redox behavior (fig. 1) . This reversibility and the rich charge transfer spectral characteristics of the donor and acceptor metal ions in the visible region allow the simultaneous monitoring of the electron transfer process at both the donor and the acceptor sites. Also, no other side reactions occur in these complexes on the timescale of the electron transfer reaction.

4.2. Rates of electron transfer

The electron transfer reaction studied (eqs. (2a) and (2b) ) followed unimolecular kinetics for the complexes with n=6, 7, and 9 bridging prolines in the concentration range shown in table 4. The eleo tron transfer process was monitored at both the do- nor and acceptor sites simultaneously. The intramolecular electron transfer rates observed at 25 ‘C were 1.08~ 105, 6.40x IO4 and 1.91 x lo4 s-’ for n=6, 7, and 9 prolines, respectively.

Although the majority of this electron transfer reaction at these low concentrations occurs in the intramolecular mode, a minor component of the electron transfer reaction occurs in the intermolecular mode in most cases. The extent of this intermolecular electron transfer depends on the ~ncentration of the [ (bpy)*Ru~~(Pro~~-apy-Ruru(NH~)~]5+, n=6, 7,9, complex and becomes more pronounced at the

longest distances and slowest rates studied. At the lowest concentration studied for the n=9 proline bridge ( 1 pmol) the cont~bution of the intermolecular reaction was 2: 10% of the overall rate. In order to correct for the ~~~e~olecular efectron transfer contribution to the overall reaction, the intermolecular reaction (es. (3) ) was studied separately, its rate constant was determined at different concentrations and then this was used to correct for the minor intermolecular component in the n = 6, 7, and 9 bridging proline molecules. Alternatively, the overall kinetics observed was fitted to a first and second order rate constant and the rate constant for the second order process was found to be similar to the intermolecular rate constant determined separately (eq. (3) ).

A plot of Ink, the rate constant, versus the through space metal-to-metal distance for n= 6, 7, and 9 is shown in fig. 5. One of the surprising results of this work is the small change in the rate of ~t~rnole~ul~ electron transfer with increasing number of bridging prolines. The electron transfer rate decreased by a factor of six for an increase in distance across the bridge of three additional prolines (9.3 A), i.e. one helical turn. A comparison of the rates of electron transfer in the earlier [ (bpy)2RunL(Pro).Co*n- (NH,),] series for n=4, 5, 6, with those obtained for the present [ (bpy),Ru”L-(Pro).-apy-Rum- (NH&]‘+ series for n = 6, 7, 9 prolines (fig. 5) shows a similar rate of decrease.

The temperature dependence of the rate of the intramolecular electron transfer reaction was determined between 6 and 35°C for n=7 and 9 and between 7 and 25°C for n=6. Table 3 shows the M$ (kcallmol) and A&‘* (eu) values for the intramolecular electron transfer reaction and the corresponding intermolecular reactions in the different molecules studied For a binuclear complex where the donor and acceptor are separated by these long distances ( x 30 A), the change in reorganizational energy with distance is expected to be small [ 20,2 I]. Therefore the changes in rates should be predominantly related to the electronic factor /3 (eq. (5) ). The AH* values for the n = 6,7, and 9 prolines studied here vary between 5.6 and 4.0 kcallmol (table 3), where part of this variation may be due to a temperature effect on the structure of the oligomeric proline peptide bridge. The decrease in AS* between n = 6 and 9 prolines is similar to that observed previously for similar systems

Table 5

M. Y. Ugawa et al, /Chemical Physics I76 (1993) 589-600 597

I%ctron transfer rates versus temperature for [ (bpy)2RunL’ (P~)~-apy-RurKx(NH~)s]‘~

n=6

T (“C)

lo-” k

(s-l)

n=7

T

(“C) IO-‘k

(s-l) 10-4/i W’)

Intermolecular rate

T 10-9k& (“C) (M-’ s-‘)

7.0 5.soIto.03 6.8 3.3OkO.2 5.6 1.08+0.05 8.8 1.38kO.06 16.0 7.49 to.07 IS.7 4.50k0.2 15.1 1.43_+0.06 16.0 1.73rto.10 25.1 10.8rtO.04 25.0 6.40 z!z 0.3 25.1 1.91+0.06 25.4 2.11Ito.05

35.3 8.40 zt 0.2 35.4 2.3840.1 35.0 2.51 kO.08

Fig. 5. Intramolecular electron transfer rates for the [ (bPy)~Ru”L(Pro),-Corn(NH~)~l n= 1-6 (0) and [ (bpy)*RunL(Pm)~-apy-Rum(~~)~] n=6,7, and 9 (0) series versus the distance between the donor and acceptor (i.e. the number of bridging proline units).

where the distance between the donor and acceptor is increased [ 111.

4.3. driving force and the dire&tion of the etectron transfer process

The intramolecular electron transfer reaction studied here, where the direction of electron transfer is from the N-C across the peptide bridge, is shown in eqs. (Za) and (2b). The driving force for this intramolecular electron transfer reaction (eq. (2) ) is = 1.5 V, since the reduction potential is - 1.2 V versus NHE [ 111 for (bpy ) zRunLf (bpy ) 2RunL’ and 0.26 V versus NHE for [apy-RtW” ( NH3)5]. This driving force is higher than the known reorganization energy of the system. The inte~ol~~ar reaction (eq. ( 3 ) ) is therefore expected to be almost diffusion con- trolled and k= 2.1 x 1 O9 s- ’ M - ’ was measured. This

intermolecular reaction (eq. ( 3 ) ) is expected to be a barrierless reaction (or in the “inverted region”)

1231. A study of another intramolecular electron transfer

reaction for the same molecules in different oxidation states is currently under investi~tion by photo- chemical techniques [ 241. The driving force for this reaction is only 1.04 Y:

[ (bpy}~Ru’liL-(P~~~-apy-Rui~(~~)~]s+

-+ [ (bpy)~Ru*~L-(Pro)~-apy-Rurxx(NH~)~]~~ .

(41

The direction of the electron transfer reaction for eq. (4) is from the C-+N terminal of the peptide. This study should provide us with a valuable comparison of an electron transfer reaction in molecules that have the same peptide conformation, but undergo electron transfer in the direction of C-+N rather than N-X across the peptide bridge.

4.4. Peptide co~for~atiun and electronic interaction with the donor and acceptor sites

For these extended oligoproline donor and acceptor complexes with long distances between the donor and acceptor, intramol~~ar electron transfer is expected to involve the intervening bridge. If the eleo tron transfer involves the bonds of the bridging peptide, then a small perturbation in the peptide conformation will not change the “through bond” distance between the donor and acceptor. The rate of int~ol~ular electron transfer, k, for such an extended system can be described using [ 22 ]

598 h4. Y. Ogawa et al. / Chemical Physics I76 (1993) 589-600

H’JA ‘H&~ff * , n= (r--&J/l, t5b)

In cu= --/?I/2 . (5CJ

In these equations HDA is the interaction between the donor and acceptor at distance I and H& is the same interaction at ro, the close contact distance between the donor and acceptor. When r> ro, the reaction is assumed to be non-adiabatic. The bridge is a single chain of n equivalent proline units of length 1, where I is obtained by summing the proline bond lengths of N-C, C-CO, and N-CO. (Y is the product of the decay in the rate (per proline residue) for all the prolines in the bridge, and its relationship to the electronic interaction HDA is given in eq. ( 5b) [ 22 ] . The reor~nizational energy, 11, is the sum of the inner sphere (Ai) and outer sphere (A,) components. The driving force of the electron transfer reaction, AGO, is approximated as the difference in e* between the donor and the acceptor.

From the intramolecular electron transfer rates at 25 “C, a decay coefficient with distance (“through space”), @*0.2 A-’ (or eO.86 per proline unit for “through bond” electron transfer), and a “through space” electronic interaction, HDA z 0.22-0.08 cm- * , are calculated for n = 6-9 prolines, respectively [ 111.

The decay coefficient with distance fi and the electronic interaction HDA calculated in this study are unusually small for donor-acceptor molecules separated by these peptide bridges. In an earlier study using [ (NH~)~Ru(Pro)~Os(NH~)~]‘+, n= l-3, a fi= 0.65 A-’ was calculated, after correction was made for the distance dependence of the reorganizational energy on the rate of electron transfer [ 3 1. Several explanations can account for the small rate decay with distance, /I% 0.2 A-i, observed in the present study. The helical nature of the peptide bridges is one expla- nation for the small decay with distance. The helical polyproline II secondary structure of the bridge, which is known to predominate in oligoprolines with n & 4,

results in a change in the electronic nature as evi- denced by the presence of lower energy electronic transitions in the UV and CD spectra of these helical oligoprolines f 18,191. Also, the interaction of the electron donor [ (bpy),RuL’] with the polyproline bridging ligand may contribute to the lowering of the

rate of decay with distance for these molecules. Since an electron is transferred from the f (bpy ),RuL’ ] intermediate across the o~goproline bridge (eqs. (2a), (2b) ), any additions interaction such as more fa- vorable mixing of the donor L’ x* orbitals with the A* orbitals of the first proline bridging unit may en- hance the interaction between the donor and the bridge in a manner that was not available to the [OS”/ 111(NH3)5] donor in the [ (NH3)SRu(Pro),- OS”‘“’ (NH,),] 5+ series.

In the study of an earlier system, [ (bpy),Ru”L(Pro)$~“*(NH~),], a clear demar- cation is observed in the rate versus distance dependence between n = 1-3 and n & 4 prolines [ 111. While part of the change in slope of the rate constant, Ink, versus distance in that series may arise from the distance dependence of the reorganizational effects of the donor and acceptor, an additional enhancement in electronic coupling introduced by [ (bpy ),Ru”L’ ] may also be involved. The decay coeflicients are similar in the two series, the [ (bpy),Ru11L(Pro),Co111(NH~)5], for n=4, 5, 6 prolines, and the [ (bpy)~Ru”L(Pro),-apy- Ru”‘(NH~),]‘+, for n=6, 7, 9 prolines (fig. 5). However, for the n= 1, 2, and 3 proline bridges, the differences in the rate dependence on distance in the [(NH3)5Ru(Pro),0s(NH3)5]s+ and [ (bpy)2Ru11L(Pro),Con1(NH~)5]5+ series are more pronounced; this difference may be clarified by further evaluation of the reorganizational energy for the [ (bpy)~Rull~(Pro)~~lll(NH~)~]s+ series.

The surprisingly rapid rates observed for the intramolecular electron transfer reaction in this work have very important implications for long range electron transfer in proteins. The distance between the donor and acceptor metal ions in our study varies between 30 and 40 A. Such separation distances are similar to the diameter of a small protein (e.g. cytochrome c). A schematic diagram of cytochrome c and the longest molecule studied in this series n = 9 prolines, drawn on the same scale (fig. 6), shows that the distance across the (Pro )9 bridge is similar to the distance between points on opposite ends of the cytochrome surface.

Long range electron transfer in proteins covalently

M. Y. Ogawa et al. / Chemical Physics 176 (1993) J89-600 599

sults, more work must be done on rigid and flexible peptide bridges related to those described here. Sev- eral experiments are currently being carried out to compare the electron transfer rates determined in the present study with rates for more flexible peptide molecules. An experiment to measure the electron transfer rate in a donor-acceptor complex with a (Gly), bridge and compare it to the rate of the corresponding (Pro)6 molecule will provide an important comparison between electron transfer in two peptide bridges differing only in the flexibility of the (Gly ), versus the rigidity of the (Pro), peptide chain. Other experiments with the [ (bpy ),Ru”G (Pro),- apy-Ruxu(NH3)5]s+, n = 6, 7, 9, complexes will be carried out in a more rigid medium in order to study the effects of a rigid medium on the peptide conformation and the rates of electron transfer. Electron transfer experiments in DzO versus Hz0 may also re- veal any specific hydrogen bonding pathways occur- ring within these solvent stabilized proline helices. These experiments and others on related, more rigid peptide structures should help further the under- standing of the results reported in this paper and bet- ter define facile electron transfer pathways across peptide structures.

Fig. 6. Structure of [ (bpy)~Ru”L-(~o)~-apyRum(NH~)*] versus cytochrome c(tuna) on the same molecular scale. L=4- carboxy-4’-methyl-2,2’-bipyridine; apy=4-aminopyridine.

Acknowledgement

modified with metal ions at specific sites have been analyzed by theoretical models using different pathways provided by the protein matrix. In these calcu- lations pathways involving covalent bonds have been reported to be the most efficient ways for electron transfer between the donor and acceptor sites [ 25 1, The assessment of the contribution of different pathways provided by the protein matrix is still difficult to calculate [ 261. The oligoproline donor-acceptor complexes presented here offer simpler molecules with well defined pathways including the direct con- nectivity between the donor and the acceptor through covalent bonds. Pathway analysis of these oligoproline molecules may allow for simple tests and further re~nements of the analysis of protein long range electron transfer rates [ 25 1.

In order to further understand the oligoproline re-

The work at Rutgers University was supported by the US Department of Energy, Division of Chemical Sciences, Offrice of Basic Energy Sciences under Con- tract DE-FGOS-90ER 14 10. The work carried out at Brookhaven National Laboratory was supported by the US Department of Energy, Division of Chemical Sciences, Office of Basic Energy Sciences under con- tract DE-AC02-76CH00016. MY0 was supported at Rutgers by a fellowship from the NIH NRSA No. lF32-GM 13223. ISM thanks the CNPq (Conselho National de desenvolvimento cientifico e tecnolo- gico, Brazil) for the Post-doctoral fe~owship support (1991-1992). The authors would like to thank Dr. Chang Su for her assistance with the pulse radiolysis experiments.

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Long range electron transfer in helical polyproline II oligopeptides

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