Photochemistry and Radiation Chemistry - American Chemical Society

9

Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/ba-1998-0254.ch009

Long-Range Electron Transfer Between Proline-Bridged Aromatic Amino Acids K. Bobrowski, J. Poznański, J. Holcman, and K. L. Wierzchowski * 1,2

1

3

1

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warszawa, Poland Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Poland RisøNational Laboratory, DK-4000 Roskilde, Denmark 1

2 3

Interpretation of the kinetic pulse radiolysis data for intramolecular Trp --> Tyr radical transformation in aqueous solutions of linear H-Trp-(Pro) -Tyr-OH, n = 0-5, is presented in terms of the Marcus electron transfer theory, taking into account conformational dynamics of the molecules. For this purpose, for each peptide, representative sets of low-energy conformers were selected with the help of experimental methods ( H and C NMR, and circular dichroism) and modeling meth ods (molecular mechanics and dynamics ); and relative electron transfer rates averaged over all the conformers were calculated for two assumed competitive electron transfer pathways: through space (TS) and through the peptide backbone (TB). The TS rates were obtained by taking into account the overlap integrals of aromaticringorbitals calu lated quantum mechanically. By fitting the calculated rates to the experi mentaldata for the rate constants for electron transfer, k with an exponential function appropriate for the two-pathway model, we have demonstrated that in linear short-bridged peptides (n= 0-2), electron transfer predominantly takes the TS pathway, which consists of van der Waals contacts between the aromaticrings,whereas in longer peptides (n = 3-5), it occurs exclusively by the TB pathway, which is made of a -(Pro) -bridge in a helical conformation similar to that of all-trans poly-L-proline II. This pathway is characterized by a low value of the descriptor ofthe exponential distance dependence ofthe electron transfer rate, β = 2.5 ± 0.1 nm , suggesting that helical segments in proteins can function as efficient channels of long-distance electron transfer. n

1

13

et,

n

ΤΒ

-1

* Corresponding author. ©1998 American Chemical Society

In Photochemistry and Radiation Chemistry; Wishart, J., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1998.

131

132

PHOTOCHEMISTRY AND RADIATION CHEMISTRY

Long-range electron transfer (LRET) between various oligoproline-bridged redox pairs has been studied over the past 10 years in several laboratories (1-18) with the aim of elucidating the parameters of L R E T across a single peptide pathway. The choice of oligoprolines for such a study was dictated by the known ability of short H - ( P r o ) - O H peptides to attain, in aqueous solution, a stable helical conformation similar to that of the 3 left-handed helix of alltrans poly-L-proline II (19-22). More recently, similar investigations of L R E T across α-helical peptide bridges were also begun (23-25). These studies com plement present challenging attempts to distinguish molecular pathways in volved in L R E T in proteins (26-30). This chapter briefly summarizes the exper imental results of our earlier pulse radiolysis investigations on intramolecular L R E T accompanying Trp* —• Tyr* radical transformation in linear H-Trp-(Pro)„-Tyr-OH, η = 0-5, peptides (1-3). W e also present our cur rent interpretation of the observed dependence of the rate of L R E T on the separation distance and spatial disposition of the aromatic side chains in terms of the conformational dynamics of the peptides and the theory of L R E T (31 ). n


X

Trp' —> Tyr Radical Transformation in Model Peptides The intramolecular one-electron redox reaction (equation 1) involves electron transfer from the phenol group of the tyrosine side chain to the indolyl radical of the tryptophan side chain (Trp).

Trp*-X-Tyr[OH]

** > H - T r p [ H ] - X - T y r * [ 0 ]

(1)

where X = (Pro)„. This reaction has been studied by pulse radiolysis in aqueous solution at p H 8 (1-3). Reaction 1 was induced by oxidation with the azide radical, N " , of the indole side chain of tryptophan to the neutral Trp' radical (32). The use of small electron doses per pulse for generation of N " radicals, and the low concentration of the peptides, eliminated interference from a slow second-order radical decay and intermolecular radical transformation, respec tively, and thus allowed determination of the first-order rate constants of intra molecular radical transformation with a reasonable accuracy of about 15% (3). Under neutral solution conditions, the electron transfer is accompanied by a net proton transfer due to breakage of the tyrosine O - H bond: Tyr[ O H ] - e - - » Tyr[0]' + H ; and formation of the indole N - H bond: Trp* + e — h H —* Trp[H]. Because O - H and N - H groups in water are involved in very fast proton exchange (k = 1 0 s ) , the protonation-deprotonation equi libria accompanying electron transfer cannot be expected to limit the rate of the radical transformation reaction, which occurs on the microsecond time scale (3, 6). A n additional argument in favor of this conclusion is the recent finding of Mishra et al. (7) that mechanisms of reduction in reaction 1 of T r p [ H ] * and the N-methylated tryptophan radical cation, M e T r p ' , are similar in spite 3

3

+

+

12

-1

+

+


9.

133

BOBROWSKI ET AL. LRET Between Proline-Bridged Aromatic Amino Acids

Table I. Comparison of (k\ , and (k ) Calculated According to the TB + TS Model, and the Experimental k (298 K) Rate Constants of LRET in Linear Peptides TB

TS

et

Peptide

ω Trp-Pro

Trp-Tyr Trp-Pro-Tyr Trp-Pro-Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr Trp-(Pro) -Tyr

cis trans cis trans cis trans cis trans cis trans

2


2

3

3

4 4

5

5

(lo^- )

(10? s- )

(10 s- )

77.0 26.0 26.0 4.9 39.0 1.5 1.5 0.51 0.51 0.30 0.30

48.5 27.6 35.2 9.6 37.2 1.8 1.5 0.69 0.56 0.27 0.22

21.6 14.1 25.4 4.3 33.4 0 0 0 0 0 0

TS

1

—

1

3

1

(10? s- ) 26.9 13.5 9.8 5.3 3.8 1.8 1.5 0.69 0.56 0.27 0.22

TB

1

X

2

1.072 0.017 0.462 2.254 0.011 0.191 0.001 0.470 0.051 0.064 0.510

of the fact that in the latter case electron transfer is not accompanied by a net proton transfer. The first-order rate constants, k , determined by linear least-squares anal ysis of time-dependent absorbance data for Trp* and/or Tyr* (Table I), thus correspond to the one-electron transfer reaction 1. Note that except for the η = 2 linear peptide, where two first-order L R E T processes of similar amplitude were resolved, the kinetic data for all the peptides conformed to a single expo nential. The plot of In fc vs. the number, n, of Pro residues (Figure 1) demonstrates that for longer linear peptides (n = 3-5), the rate of electron transfer decreases exponentially with growing n. However, the k data for shorter (n = 0-2) linear peptides fall off considerably from the plot extrapolated to lower η values. This indicates that the rate of L R E T in short-bridged peptides is faster than would be expected on the assumption of a common mechanism of electron transfer in the whole group of peptides. In order to rationalize these findings in terms of the theory of the distance dependence of L R E T kinetics (33, 34), the separation distances and spatial disposition of the aromatic side chains in the linear peptides studied had to be evaluated from their conformational pref erences and conformational dynamics. et

et

et

Conformational Properties of Trp-(Pro) -Tyr Peptides n

The conformational properties of Trp-(Pro) -Tyr, η = 0-5, peptides were deduced from experimental H and C N M R (35) and circular dichroism (CD) (36) investigations, complemented by molecular mechanics and molecular dyn

l

1 3


134



c

3

4

5

6

7

8

9

number of prolines

Figure 1. Bridge length dependence of the rate of LRET. Plots are of In k^ vs. number, n, of Pro residues for H-Trp-(Pro) -Tyr-OH, η = 0-5 (M); [(bpy) Ru L(Pro) Co (NH ) ] , η = 0-6 (Φ); and [(bpy) Ru jj(Pro) apyRu (NH ) ] , η = 6, 7, 9 (Δ). Solid lines correspond to linear regression for η = 3-5 in the Η-Trp-(Pro ) -Tyr-OH series and for η = 4-6 in the [(bpy) Ru h(Pro) Co ('NE ) ] ' series. Abbreviations: bpy 2,2'-bipyridine; 4-amino-pyridine (apy). n

n

n

3

5

m

3 5

2

4Jh

2

n

4+

n

Xi

m

n

m

3

5

n

2

4n

t

namics modeling (31 ) with the help of A M B E R 3.0 and 4.0 software (37). These properties proved to be governed by a number of interdependent equilibria (cf. Figure 2): (1) trans cis isomerization (ω dihedral angle) about the X - P r c peptide bonds, (2) rotation of Trp and Tyr side chains about C - C and C - C > bonds (χι and χ dihedrals, respectively), (3) extended helix ail-trans polyL-proline (PLP) II type helical conformation within the -(Pro) -bridge in pep tides with η > 3, (4) β (-160°) - » α (-45°) transition at the ψ(Ρτο) angle of the Pro-Tyr fragment, and (5) transition between up and down conformations of the pyrrolidine Pro side chain ( χ ) . Trans *-> cis isomerization occurs most readily at the T r p - P r o bond, so that populations of corresponding major isomers of zwitterionic forms of the peptides are comparable and constitute a 0.85-0.90 molar fraction of the total peptide content. Isomerization about Pro-Pro bonds in peptides with η > 1 results in a small population of at least two additional cis isomers. The rate constant for interchange between trans and cis isomers about the T r p - P r o bond at 298 Κ has been estimated to be close to 10" s (3, 35, 38), that is, 4-6 orders of magnitude slower than that observed for the electron transfer reaction 1. Thus, in this reaction the two isomers should be treated as separate species. Rotations of the Tyr side chain both in cis and trans isomers proved rela tively free, with a marked preference for the Xi(g~) rotamer in longer peptides. a

p

2

n

3

2

_1


p


9. BOBROWSKI ET AL. LRET Between Proline-Bridged Aromatic Amino Acids

135

Rotations of Trp, however, were found to be highly dependent on the configura tion about the T r p - P r o bond. In the trans isomers, the indole ring rotates quite freely, whereas in the cis form of all the peptides its rotation is severely re stricted, which results in a high population, -0.85, of the staggered χ ι ( ί ) re tainer and a χι(ί), X2(-) conformation of the whole side chain. A l l these rotamers, with lifetimes in the time domain of 1 0 " - 1 0 " s, exchange frequently during the hfetime of the Trp* radical. In the short-bridged peptides (n = 0-2), these side chain rotations, combined with oscillations of the backbone ψ angle, lead to the appearance of multiple low-energy conformers characterized by a close edge-to-edge approach of the indole and phenol rings. Conformation of the -(Pro) -fragment varies with the number of adjacent Pro residues and assumes a P L P II—like helical conformation in dll-trans iso mers beginning with η = 3. This conformation also includes the Trp residue. Conformational rigidity of the backbone in the P L P II-like conformation in creases with the growing n. In this conformation the iJi(Pro) dihedral angle generally assumes a value in the β-range ( +160°). Recendy, Sneddon and Brooks (39), on the basis of their C H A R M M simu lations of conformational dynamics of Pro peptides in aqueous solution, have postulated involvement in electron transfer across the -(Pro) -bridge of β —• α transitions at the ψ angle, as the latter occur more rapidly and bring the donor-acceptor distance to a shorter range than the trans cis interconversion 9

12

n

n


136


at the ω dihedral. W e have performed (31 ) similar simulations for T r p - P r o , Pro-Pro, and Pro-Tyr fragments (potential of mean force calculations with explicit H 0 environment in A M B E R 4.0 using umbrella sampling along a reaction coordinate in ψ; 36 molecular dynamics runs consisting of 10-ps equili bration and 30-ps evolution time windows). The calculated energies of, and energy barriers between, the β and α t|/(Pro) conformers of the Pro-Pro frag ment proved similar to those obtained by Sneddon and Brooks (39); it appeared that the ψ(β) conformation is more stable than the ψ(α) one by A G = 3 ± 1 kcal m o l in Pro-Pro and Pro-Tyr, whereas in T r p - P r o , AG amounts to as much as 7 ± 1 kcal m o l , so that the population of T r p - a P r o conformers can be expected to be negligibly small. Using the determined potentials of mean force, we have also evaluated the frequency of β —» α transitions according to the transition state theory: £ _ = 1.5 Χ 10 s" and k ^ = 8 Χ 10 s" for Pro in Pro-Pro and Pro-Tyr fragments, respectively. The mean lifetime of the t|/(a)Pro state in Pro-Pro is thus 6 X 10 ~ s, and the transient population of corresponding conformers of - ( P r o ) - with one Pro residue in this state can be expected to be insignifieandy low on the time scale of the observed electron transfer and was therefore neglected. O n the other hand, β —• a transitions at the Pro residue preceding Tyr, which are much faster on the same scale, led us to include this transition in calculations concerning low-energy conformers. The results of these modeling studies find full support in N M R data, which showed that the location and shape of the C resonances in the C spectra of the T r p - ( P r o ) - T y r peptides (31, 35) were similar to that of P L P II for all the Pro residues studied except that preceding Tyr. The cyclic pyrrolidine side chains of Pro residues undergo very fast, pico second transitions between their up and down equilibrium conformations at the χ angle, as shown by both N M R and molecular mechanics modeling (31 ). To sum up, the H - T r p - ( P r o ) - T y r - O H peptides in solution should be represented by ensembles of fast-exchanging, on the observed L R E T time scale, side chain and ψ( Pro-Tyr) backbone conformers of the major cis and trans isomers about the T r p - P r o bond, and -(Pro) -bridge in a P L P II type confor mation beginning with η = 3. 2

- 1


- 1

β

3

α

1

p

5

a

1

6

n

a

1 3

n

3

n

n

Modeling of LRET Pathways in Trp - (Pro) -Tyr Peptides n

In order to interpret the observed dependence of fc t on the number of Pro residues in the bridge in terms of the theory of the distance dependence of the L R E T rate (34), distances between the two aromatic redox centers along some physically possible molecular pathway(s) had to be evaluated for all repre sentative conformers of the two major isomers, all-trans and cis(Trp-Pro), of each of the peptides. The lowest-energy conformers of zwitterionic forms of the peptides were calculated (31 ) with the use of the A M B E R 3.0 program in e


9.

BOBROWSKI ET AL. LRET Between Proline-Bndged Aromatic Amino Acids

137

united atom parametrization and with a dielectric constant of 81 (to mimic an aqueous environment). For each peptide isomer with a -(Pro) -bridge in the P L P II structure [adopted from X-ray diffraction data (40)], a set of 324 starting conformers, corresponding to all possible combinations of the N-terminal and C-terminal side chain conformations (with values of the χι and χ dihedral angles varied in 30° steps), and α and β Ψ(ΡΓΟ) conformations of the P r o - T y r fragment was generated and subjected to further energy minimization. During the latter step, all dihedral angles, including those of the pyrrohdine ring, were allowed to vary. O f the conformers thus obtained, only those with energies ^42 kj m o l above the lowest-energy one within a given isomeric ensemble were selected for calculation of separation distances and angles between the aromatic rings. Ensembles of the low energy conformers fairly well reproduced the main con formational features of the peptides derived from the experimental N M R (35) and C D data (36), especially populations of conformers in particular rotameric side chain states; and the differences between backbone conformations of the η = 0-2 and η = 3-5 groups of peptides were distinct. We were thus justified in assuming that the distributions of calculated distances between various atoms of the terminal side chains in ensembles of the calculated conformers would satisfactorily resemble those prevailing in solution (3). Similar distributions were also obtained from molecular dynamics simulations (31). The r freeenergy profiles for η = 2 and η = 3 derived therefrom, shown in Figure 3, are representative for the short- and long-bridged systems, respectively. n

2


- 1

e e

Considering the outlined conformational properties of the peptides, two molecular pathways for electron transfer could be reasonably envisaged: (1) through space (TS), viz, directly between the aromatic rings in van der Waals contact and/or mediated by water molecules of the solvation shell, and (2) through the peptide backbone (TB). The corresponding distances for individual conformers (j) within each isomeric ensemble (i), were consequently calcu lated for the TS pathway as ry(C ) between ring carbon, C ^ , (or C and N/O) atoms of the indole and phenol rings, and for the T B pathway, originally (3) as r ^ ( C ) , the shortest distance between C atoms of the terminal Trp and Tyr along the peptide backbone. More recendy (31 ), r^ was calculated, as r y ( C C C ) between the C^ atoms of the terminal amino acids along a path joining the backbone C atoms (cf. Figure 2). The latter parametrization follows a path of highest molecular orbital electronic density and thus better mimics the T B electron transfer pathway. The distance dependence of the rate of L R E T , k, is described (34) by equation 2: a r

ee

pp

i

[3

a

p

p

a

k = k βχρ[-β(Γ-Γ )] 0

0

(2)

where k is the rate constant at r = r , corresponding to the closest edge-toedge approach of redox centers, and the descriptor of the exponential distance 0

0


138


free energy [kcal/mol]


5H

r [nm] ee

Figure 3. Molecular dynamics (10 ns) ^free-energy profiles for cis (solid curves ) and trans (broken curves) isomers of H-Trp-(Pro) -Tyr-OH peptide (top) and H-Trp-(Pro) -Tyr-OH peptide (bottom). 2

3

dependence of k, β = β ι + β , expresses the contribution of electronic (β ι) and nuclear ( β ) factors to the overall distance dependence of the rate. Our approach to distinguish which of the possible molecular pathways is actually involved in reaction 1, and to evaluate the corresponding β descriptor, consisted of the following steps: (1) calculation of relative rate constants, ky of L R E T for individual conformers of a peptide along an assumed pathway, and of average values for each peptide isomer, (k% = k^j (where is the Boltzmann probability of occurrence of a jth conformer of the ith isomer), and then (2) fitting these values to the experimental k data with the use of equation 2, as β

η

β

η

9

et


9.

BOBROWSKI ET AL. LRET Between Proline-Budged Aromatic Amino Acids 139

a fitting function, in an appropriate form for an assumed model of L R E T . Originally, four principal L R E T models were probed, involving either (1) the T B pathway, (2) the TS pathway, (3) the two pathways T B + TS simultaneously, or (4) T B + TS cos Θ, where Θ is the dihedral angle between the planes of the indole and phenol rings, in which the cos Θ function was introduced to roughly account for the dependence of L R E T along the TS pathway on the overlap between the ττ- and σ-orbitals of the aromatic rings (3). The validity of the models was evaluated statistically with the use of the X distribution function, denned as: Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/ba-1998-0254.ch009

2

X

2

=

Σ

ί = 0 >

η

(In ,-ln k^/σ

In

2

fc

(3)

eW

The best agreement between the experimental and calculated rates has been obtained for the last model (4) (documented by the low value of X = 6.23 at the high significance level of 0.513) for the following values of the parameters sought: β = 2.8 ± 0.4 n m " and β = 120 ± 40 n m . The high value of the β parameter indicates that at r ^ C * ) = 3.65 A the contribution to k from the TS pathway vanishes practically to zero, so that T S - L R E T takes place only in conformers with indole and phenol aromatic rings in close van der Waals contact. Because low-energy conformers of this type are dominant in short-bridged (n = 0-2) peptides, the TS pathway proved competitive only in the latter group. In longer peptides (n = 3-5), L R E T thus takes place solely through the T B pathway, characterized by unusually weak decay of electronic coupling between the Trp and Tyr redox centers. We recently calculated (31 ) overlap integrals, I B> between highest occu pied molecular orbitals (HOMOs) of the indolyl radical and phenolringsfor all the low-energy conformers of the peptides. (The calculations were actually performed for H O M O orbitals of 3-methylindole andp-cresol located in space as indolyl and phenol rings in the low-energy conformers.) Using the r ^ C C C ) parametrization for the T B pathway and a fitting function in the form 2

Τ Β

1

Τ δ

τ δ

- 1

8

et

A

8

a

p

k =k

TB

0

βχρ(-β

ΤΒ

Η*« ) + k β

TS

0

(4)

ÎÎB

all the calculated rates (k)i were again simultaneously fitted to the experimental k data. The adjustable parameters thus obtained, (k)i =

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