Electron Transfer Reactions - American Chemical Society

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Long-Range Intramolecular Electron Transfer Reactions Across Simple Organic Bridges, Peptides, and Proteins Stephan S. Isied Department of Chemistry, Rutgers, The State University of New Jersey, P.O. Box 939, Piscataway, NJ 08550 Rates ofintramolecular electron transfer across peptide bridging ligands have been studied by covalently attaching inorganic reagents (Co, Ru, and Os) at their terminals and side chains. In addition to the expected variation in the rates with changes in reorganization energy and driving force as predicted by theory, quantitative information on the participa-tion of the peptide bridging groups in these long-range electron transfer reactions can now be obtained. Our results show that peptides with organized secondary structures such as (proline) (n ≥4) have a low dis-tance decay constant(β~ 0.2-0.3 Å )as compared to β ~ 1 Å for ran-dom coil peptides and saturated hydrocarbons. Thus more rapid rates of intramolecular electron transfer can occur across organized peptides than across rigid hydrocarbons. Experiments on electron transfer across helical, rigid non-proline peptides provide preliminary information that can be compared to the helical proline peptides. This technique has been extended to study intramolecular electron transfer in cytochrome c where the heme protein is an electron donor. These results show that the rate of intramolecular electron transfer between the heme and the metal-modified protein surface site (His 33 or Met 65) cannot be predicted from the distance between the two, even when driving force and reorga-nizationenergy are corrected for. Peptide networks between the heme and the different sites on the surface of the protein play an important role that can account for more than three orders of magnitude change in the rates of electron transfer at similar distances. n

-1

-1

THOSE OF US WHO HAVE BEEN ASSOCIATED with Professor Henry Taube share many memories of watching him analyze and formulate research prob© 1997 American Chemical Society

In Electron Transfer Reactions; Isied, Stephan S.; Advances in Chemistry; American Chemical Society: Washington, DC, 1997.

331

332

E L E C T R O N TRANSFER REACTIONS

lems. H e often communicates his ideas to his associates via common sense arguments (and sometimes even using test tube experiments) full of implica tions and further insights. As graduate students i n his laboratory, we were studying redox reactions of Co(III) and Ru(II) ammine complexes under condi tions in which the time required for substitution is faster than that required for electron transfer. I clearly remember a discussion during which he suggested that i f we could make binuclear complexes of the type [ ( N H ) R u - b r i d g e C o ( N H ) ] , then we could rely on the large disparity in the rates of reduction of Co(III) and Ru(III) ammines (1) to generate the desired precursor com plexes [(NH3) Ru -bridge-Co (NH ) ], which could then be directly used to measure rates of intramolecular electron transfer, independent of the substitu tion rates of the complexes. To accomplish this we carried out a systematic study of the kinetics and thermodynamics of substitution and redox reactions of a variety of substituted Ru(II) tetraammine complexes (2, 3). From the information gained about the substitution reactions of Ru(II) complexes and the redox reactions of coordi nated ligands, we were successful in developing a synthetic procedure to make the [ ( S 0 ) ( N H ) R u - b r i d g e - C o ( N H ) ] complexes (4). Using four closely related pyridine carboxylate bridges (nicotinic, isonicotinic, 3- and 4-pyridine acetic acid), differences in electron transfer rates and mechanism between iso mers were determined and analyzed {4, 5). The first binuclear C o - R u donor-acceptor complex isolated with the isonicotinic acid bridge is shown in the following structure: 3

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 22, 2016 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/ba-1997-0253.ch020

III

3

ni

5

II

5

4

5

3

4

III

III

3

III

5

3

5

n l

m

This approach has now been used for more than two decades, and newer synthetic routes (making use of CF3SO3 chemistry) have succeeded it (6). However, Henry Taube's original ideas on the importance of such bridged bin uclear complexes in the study of the role of bridging ligands in mediating elec tron transfer continues to be a basis for studying intramolecular electron trans fer in donor-acceptor complexes with bridging ligands ranging from single atoms to peptides and proteins. In this chapter I will describe how we have extended this approach to study the electron transfer mediating properties of complex bridges such as peptides and proteins. Interest i n long-range electron transfer in proteins arose from the exami nation of the crystal structures of redox proteins such as cytochromes, copper blue proteins, iron-sulfur proteins, and protein complexes such as cyt c-cyt c peroxidase and, more recently, the cyt c oxidase structure (7-11). Most of these proteins have a redox center (i.e., a heme, a copper ion, or an FeS center) con stituting only a few percent of the total protein, buried within the protein.


20.

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Ekctron Transfer Across Peptide and Protein Bridges

333


ET

METAL ON

PEPTIDE

M6TAO LIN

Scheme I. Donor-acceptor bridged complexes. Crystal structures of multiredox enzyme complexes show that the redox cen ters are separated by peptide residues at distances of > 10-20 Â. The occur rence of rapid electron transfer between redox centers separated by peptide ligands raises questions concerning the role of the polypeptide chain in provid ing electron transfer pathways between these centers. Our approach for investigating the electron transfer properties of peptide and protein fragments is to study them in donor-acceptor complexes similar to that shown in Scheme I. With this strategy, electron transfer pathways in pro teins can be evaluated by designing molecules that emphasize special features such as peptide bonds, hydrogen-bonding networks, polarizable amino acid side chains, and the connectivity of redox centers to the main chain or side chain of the peptide in carefully controlled experiments. In this chapter I will discuss our studies of long-range intramolecular elec tron transfer i n multifunctional biological bridging ligands including amino acids, peptides, and proteins and the more recently developed supramolecular peptide networks with constrained peptides, and electron transfer proteins modified with redox reagents. The goal of these systematic studies is to under stand the distance dependence of long-range electron transfer reactions and its relationship to peptide conformation and specific peptide pathways necessary to achieve rapid rates of electron transfer. As will be seen in the coming sec tions, many interesting and unexpected results have been discovered in these investigations. These results are leading us to a better understanding of the communication between inorganic redox centers and proteins.

Oligoproline Donor-Acceptor Complexes Early studies of energy transfer in biological molecules have made use of oligo proline peptides with organic energy donor and acceptor chromophores placed at the N - and C-terminals of the polypeptide chain. Such studies demonstrated that oligoproline peptides serve as spectroscopic rulers for distance estimation between chromophores. These energy transfer techniques were further used to establish proximity relationships in more complex biological molecules (12).



334


The advantage of the oligoproline peptide bridges as distance spacers over other naturally occurring amino acids and peptides is the very early onset of their solvent-stabilized helical secondary structure [13-16). A left-handed, trans-helical structure of oligoprolines begins to appear with the assembly of only three or four proline peptide units (polyproline II). This same helical sec ondary structure also exists i n solution and i n solid-state structures of tetraand pentaproline peptides, as well as in fiber structure of longer proline poly mers (17-20). Experimental evidence for this helical conformation comes from crystal structure in the solid state (17-20), from C and two-dimensional pro ton N M R of [ ( P r o ^ C o ^ N H ^ (21, 22), and C D of [(bpy) Ru (Pro) apyRu ( N H y j (n = 6, 7, 9) i n solution (16). Redox active transition metal ion chromophores located at the N - and C terminals of oligoproline peptides have been used to study electron transfer across bridging oligoproline peptides. The advantages of using metal ions for these studies is that substantial control over the driving force and reorganiza tion energy of the reaction can be exercised by the choice of different transi tion metal donor-acceptor pairs (13-16, 21, 23, 24). The time scale of the intramolecular electron transfer reaction can be varied to be either slower or faster than peptide conformational changes. A n example of this is seen for the diproline bridge (Table I). The intramolecular electron transfer across the same bridge can vary by more than 11 orders of magnitude, depending on the choice of metal donors and acceptors (13-16, 21, 23, 24). In the C o - ( P r o ) - R u (the first entry in Table I), the rate of electron transfer is slow compared to peptide conformational changes, and hence the equilibration of many peptide confor mations can occur prior to the electron transfer step (24). Experiments with this series, while useful for probing the existence of different peptide confor mations, are not useful for determining the distance dependence of the rate of electron transfer because of complications from the multiple electron transfer 1 3

n

2

n

2

Table I. Intramolecular Rates of Electron Transfer Across the Same (Pro) Bridge with Different Donor-Acceptor Metal Ion Pairs 2

M -M ll

M-M (Â)

m

Co-Ru Os-Co Os-Ru Ru-Co

-AG ~1CH 0.74 3.7 χ 10 1.6 χ 10

14.8 14.8 14.8 14.8

a

fc c d

4 7

0

(-0.5) 0.15 0.25 1.1

°Co-R is [(H 0)(NH ) Ru MPro) -Co (NH ) ]. u

2

3

4

I

III

2

3

*Os-Co is [ ( N H ) O s i - ( P r o ) - C o ( N H 3 ) ] , 3

c

II

5

ni

2

5

5

where

* = isonicotinyl.

Os-Ru is [(NHÔs^-iPro^RuM^NH^J.

Ru-Co is [(bpy) Ru L-(Pro) -Co (NH ) ], and L is 4-carboxy-4'-methyl-2,2'-bipyridine.

d

2

n

2

in

3

5

were bpy is 2,2'-bipyridine,


ni


20.

ISIED

Electron Transfer Across Peptide and Protein Bridges

335

and conformational changes of the bridging peptide that are occurring on simi lar time scales. In order to obtain meaningful distance dependence of rates of electron transfer, the time scale for the electron transfer reaction must be sig nificantly faster than any large conformational changes of the bridging peptide. For oligoproline peptides the major conformational change is a trans-to-cis isomerization with a half-life of 1-2 min at room temperature [and no evidence for any other rapid, large conformational change exists to date (12, 13)]; thus, rates substantially faster than 1-2 min (Tables II and III) (16) occur prior to these conformational changes. In the Os-(Pro) -Ru and (bpy) RuL -(Pro) -Co (last entries in Table I) the intramolecular electron transfer reaction across diproline is faster than trans-cis isomerization by several orders of magnitude (16, 23). 2

Table II. Rates of Intramolecular Electron Transfer Across Polyprolines [(bpy) Ru L--(Pro) -Co (NH ) ] n

2

ii

M-M $

kîs- ) (25 °C)

0 1 2 3 4 5 6

9.0 12.2 14.8 18.1 21.3 24.1 27.3

— >5xl0 1.6 χ 10 2.3 x10 5.1 x l O 1.8 x l O 8.9 x 1ο

#

2

ra

n

3

5

2

3+

ΔΗί ASi (heal mol- ) (cal deg-knoH)

1

1

8 7

s

4 4

3

Intermolecular Reaction 7.2 χ 10 M " s8

1

1

— — 6.0 9.2 9.4 9.0 8.8

— — -6 -3 -5.5 -9 -11

3.63

-5.8

N O T E : bpy is 2,2'-bipyridine; L is 4-carboxy-4'-methyl-2,2'-bipyridine.

Table III. Rates and Activation Parameters for Intramolecular and Intermolecular Electron Transfer Across Polyprolines [(bpy) Ru L-(Pro) âpyRu (NH ) ] (n = 6, 7,9) 2

M-M (λ) 6 7 8 9

31.6 34.5 37.5 40.8

n

ra

n

5

3+

ΔΗί ASi (kcalmot ) [cal (deg-mol)- ]

Κ (25 °C) (s- ) 1.1 χ 10 6.4 χ ΙΟ 3.8 χ ΙΟ 2.0 χ ΙΟ

1

1

5.6 5.1

s

4

1

-17 -19

4

—

—

4

4.0

-26

3.3

-5

Intermolecular Reaction 2.1 χ ΙΟ M - V 9

3

1

N O T E : bpy is 2,2'-bipyridine; L is 4-carboxy-4'-methyl-2,2'-bipyridine; apy is 4-aminopyridine.


336


For two series of oligoproline bridges (Tables II and III), intramolecular electron transfer reactions were studied according to eqs 1 and 2: eâ H-(bpy) Ru L(Pro) -Co q

n

2

n

{(bpy) Ru L}-(Pro) -Co I I

2

n

->

ni

I I I

--^(bpy) Ru L-(Pro) -Co I I

2

n

(i)

1 1

i

(bpy) Ru L-(Pro) + C o n


2

n

2 +

where C o = C o ( N H ) , bpy = 2,2'-bipyridine, L = 4-carboxy-4'-methyl2,2'-bipyridine, and η — 1-6, and 111

m

3

5

eâq + ( b p y ) R u L ( P r o ) - a p y R u -> 2

n

n

m

{(bpy) Ru Lr-(Pro) -apyRu --^(bpy) Ru L-(Pro) -apyRu n

2

n

m

n

2

n

n

where a p y R u = 4 - a m i n o - p y r i d i n e - R u ( N H ) , L = 4-carboxy-4'-methyl2,2'-bipyridine, and η = 6-9. The distance dependence of the rate of intramol ecular electron transfer across the bridge as a function of the number of proline residues was determined using metal donors and acceptors that have a large driving force and/or small reorganization energy. For (Pro) bridges (n = 1-9), the rate of intramolecular electron transfer decreased substantially as the num ber of oligoproline residues increased when {(bpy) Ru L}* donor (bpy = 2,2'bipyridine, L = 4-carboxy-4'-methyl-2,2'-bipyridine) and either [ - C o ( N H ) ] or [ R u ( N H ) a p y ] acceptors (apy = 4-aminopyridine) were used (15, 16). A schematic of one of the donor-acceptor complexes with the (Pro) bridge for each of these series is shown in Figure 1. The rates, activation parameters, and distances for these series are summarized in Tables II and III and Figure 2. 111

m

3

5

n

2

n

m

m

3

3

5

5

6

One of the most surprising results from these studies is the presence of two différent slopes in the plots of the intramolecular electron transfer rate (In fc ) [or (In k + AG /RT) rate corrected for outer sphere reorganizational energy] versus the number of bridging proline residues (i.e., increasing dis tance) (Figure 2). For η = 4-6 prolines, there is a slow decrease in rate with increasing distance, while for η = 1-3 prolines, there is a more rapid decrease in rate with increasing distance (Figure 2). This change in rate with distance coincides with the onset of helical secondary structure adopted by the oligo proline bridge. In comparing the intramolecular electron transfer rates of oligoproline donor-acceptor complexes (where the distance of the proline bridge is the only variable), the distance dependence of the rate depends on the electronic coupling between the metal donor and acceptor after correction is made for the distance dependence of the outer sphere reorganization energy (Figure 2, Tables II and III) (25). At long distances the reorganization energy becomes small and the electronic factor (β) predominates. For these cases the observed rate of intramolecular electron transfer can be expressed in eq 3 et

et

+


20.

ISIED

337


-Com(NH )


3 5

Figure 1. Helical oligoproline complexes with Ru-Co and Ru-Ru donors and acceptors. (Pro) is shown here. 6

k(r) = k ( r

o

(3)

y ^

where k(r ) is approximated to be the rate constant at the contact distance between the donor and acceptor (r ), and fc(r) is the rate as a function of the bridging distance in the oligoproline (n > 4) complexes. A plot of In k versus the distance (r) will yield a slope (-β) that is related to the attenuation of the electron transfer rate with the distance of separation. For η = 4-6 prolines, a low β value (eq 3) ~ 0.3 Â is estimated from the data (compared to a higher β value of 0.7-0.95 Â determined for η =1-3 prolines in different studies) (25). This surprisingly low β value for the helical oligoproline bridges shows that the assembled peptide bridge with a secondary structure is a better mediator of electron transfer than can be predicted by extrapolating the distance depen dence of the rate for η = 1-3 prolines. Such a low β for electron transfer implies that for specific secondary structures long-range electron transfer is not significantly impeded with distance. For even longer helical (Pro) bridges, η = 6-9, a similar low β ~ 0.2 Â is calculated with the {(bpy) Ru L}* donor and the [ R u ( N H ) a p y ] acceptor (Figure 2, D) (16). A l l the helical polyproline bridges (n > 4 prolines) in the different series show similar mediating properties as evidenced by the similar change in rate (corrected and uncorrected for reorganizational energy) with distance observed for the Β and D series and for the A and C series (Figure 2). 0

0

- 1

-1

-1

n

2

n

m

3

5



338


10

15

20

25

30

35

40

45

Distance, Â Figure 2. Intramolecular electron transfer rates for two helical oligoproline series of donor-acceptor complexes versus the bridging distance between the donor and acceptor. The rates (eqs 1 and 2) are plotted as In and aslnk^ + AG /RT, rate corrected for reorganizational energy, versus the number of bridging proline residues. The two series are [(bpy)2Ru U-(Pro) -Co (NH^ (n = 1-6) (A and B) and [^py) Ru U-(Pro) -apyRu (NH^ l (n = 6-9) (C and D) where bpy = 2,2' bipyndine, L = 4-carboxy-4'-methyl-2,2'-bipyridine apy = 4-aminopyridine. +

u

2

II

n

Ili

n

lll

5

i


20.

ISIED

339


Recently in a different system of porphyrin donor-acceptor complexes of heli cal oligoproline bridges (n = 4-8) a similar low β value (0.2 Â ) was estimated from fluorescence quenching studies (26) in ethanol solution (which, like water, is known to stabilize the helical polyproline II structure). The longest peptide bridge across which electron transfer has been mea sured is the helical (Pro) bridge. In this molecule nine amino acid residues separate the metal ion donor from the acceptor (a distance of > 40 Â ) (16). This distance of 40 Â is comparable to the diameter of a small protein such as cytochrome c. Such efficient electron transfer over a long distance (>40 Â) could not be predicted from the results for short proline bridges (n = 1-3). Thus far, unexpectedly fast electron transfer at long distances has been observed i n seven helical oligoproline donor-acceptor complexes. However, the origin of this efficient electron transfer is still not well-understood. The participation of the peptide chain alone and/or the solvation of the oligoproline bridge may be involved in providing this additional channel for electron trans fer at long distances. Experiments in progress in deuterated media with these oligoproline complexes should provide further information on the specific electron-transfer pathway utilized in these peptide bridges. -1


9

Rigid OrHelical Peptide Donor-Acceptor Complexes In the previous section the oligoprolines provided rigid peptide bridges with well-defined distances between metal donors and acceptors. More recently we have begun investigating non-proline α-helical peptide bridges that are cyclic and thus rigid and where the distance between the donor and acceptor is also well-defined (14, 27). These α-helical cyclic peptides are made rigid by crosslinking amino acid side chains with organic and inorganic reagents (see pep tides I, II, and III in Chart I). Similar rigid bicyclic hexapeptides (with organic protecting groups) with two Lys side chains cross-linked to (i + 4) Asp residues (where i refers to the ith amino acid) have been demonstrated to be a helical by C D and N M R analysis (28). Using similar methods, the helical hexapeptide donor-acceptor complex (I), with [(bpy) Ru-L] (L = 4-carboxy-4'methyl-2,2'-bipyridine) at the N-terminal Lys and [(NH ) Ru-apy] (apy = 4-aminopyridine) at the C-terminal Asp, has been recently synthesized and characterized (27). Preliminary fluorescence quenching results suggest that rapid electron transfer (on the microsecond timescale) occurs in this α-helical hexapeptide donor-acceptor complex. Electron transfer studies on this peptide donor-acceptor complex using flash photolysis and pulse radiolysis are cur rently in progress. 2

3

5

The electron transfer properties of the helical peptide donor-acceptor complex (I) will be compared to those of the helical (Pro) peptide bridge in the [(bpy) Ru-apy]-(Pro) -[apy-Ru(NH ) ] donor-acceptor complex studied earlier (Table III). Both complexes have die same donor-acceptor metal ions and are separated by the same number of peptide residues. The main differ6

2

6

3

5


340

ELECTRON TRANSFER REACTIONS

Ν (bpy),Rtf

(CH^4—NHCO


π

CH

2

(UNH iys-His-Ala-Ala-Asp-Hîs-Ala-CONH

2

(bpy),Rir f N

I

I

(CHÎ4-NHCO—CH

2

m M =cis-Ru (NH ) m

3

4

C/wrf /. Rigid helical non-proline peptide donor-acceptor complexes.

ence between these two complexes is the different type of left-handed helical structures in the peptide bridges. In bicyclic peptide complex I the helicity is maintained by crosslinking of the amino acid side chains, while in (Pro) the helicity is maintained by the specific solvation of the oligoproline peptide. The rigid helical peptides II and III were designed to test intramolecular electron transfer pathways between the main polypeptide chain and the pep tide side chain. In the donor-acceptor complexes of both bicyclic peptides II and III, one metal ion is bound at the main chain and the other is bound to the 6


20.

ISIED

His side chain, but with a different number of amino acids, two or four, between the donor and acceptor. Helicity in these two peptides is maintained by a lactam bridge and also by the cross-linking of two His residues to the metal acceptor, [ - R u ( N H ) J . The electron transfer properties of peptides II and III (with the metal-to-metal distance 14 and 17 A, respectively) will pro vide a comparison of the rates of electron transfer between a dipeptide and a tetrapeptide i n a similar rigid environment with peptide main-chain-sidechain connection to the donor and acceptor. Electron transfer in peptides II and III (side-chain-main-chain connectivity) will also be compared to similar diproline and tetraproline bridges (Table II) with main-chain-main-chain con nectivity. Preliminary fluorescence quenching studies with donor-acceptor com plexes of peptides II and III indicate that rapid and measurable rates of elec tron transfer occur between the two metal centers ([(bpy) Ru-L] (L = 4-carboxy-4'-methyl-2,2'-bipyridine) at the Ν terminal and the [Ru(NH ) (His) ] bound to the side chains of peptides II and III) (27). Time-dependent fluores cence and absorption experiments are currently under way on these peptides. The peptides in Chart I represent a new strategy for obtaining rigid small peptides, with amino acids different than proline, where the position of the metal donor and acceptor is well-defined. Further development of this strategy will allow one to study the effect of one or multiple polarizable amino acid side chains on the rates of long-range intramolecular transfer process (by replacing alanine with amino acids with polarizable side chains such as Phe, Tyr, Tip, or Met). The simulation of electron transfer pathways in these small, but conformationally well-defined, peptides should serve as a useful calibration for elec tron transfer in proteins where multiple pathways are difficult to discern. IH


341


3

2

3

4

2

Protein Donor-Acceptor Complexes In the past decade electron transfer proteins have also been modified with a redox reagent (either a donor or an acceptor) that can be bound to different locations on the protein. Horse heart cyt c, modified at surface histidine residues with a variety of ruthenium ammine reagents ([Ru(NH ) L]; L = N H , isn, or py), is one of the best examples of modified protein donor-accep tor complexes (29-36). These modified proteins (analogous to the donoracceptor complex i n Scheme I) have been used to study the dependence of intramolecular electron transfer on distance and the role of the protein net work in facilitating the electron transfer reactions. Rates of intramolecular electron transfer i n proteins have been studied successfully by pulse radiolysis (29-34) and flash photolysis techniques (35, 36). Our studies have focused primarily on pulse radiolysis techniques because of the variety of oxidizing and reducing radicals that can be used to generate the intermediate precursor ruthenium-protein complexes. For example, pulse radiolysis using the C O | (or e ") radical allows one to start with the totally oxi3

3

aq


4

342


dized Ru(III)-heme(III) and generate the precursor intermediate by reduc tion. Using the N radical and the totally reduced protein Ru(II)-heme(II), the precursor complex can be generated by oxidation (29-34). The reactions sum marizing the generation of the precursor complex by oxidation or reduction with pulse radiolysis and the electron transfer measurements i n protein donor-acceptor complexes are shown in eqs 4 and 5. 3


Oxidative Method MXM-L-MÔK)

N- + M^red^L-M^red)

|fc

et

( 4 )

M ^ - L - M a M )

(CO3) Reductive Method

M^re^-L-Môx) CO- + M ^ - L - M ^ o x )

\,k

et

2

(eâ )

( g )

M!(ox)-L-M (red)

q

2

The azide radical N is generated by the reaction of O H * with solutions of sodium azide, and the carbon dioxide radical anion, C 0 , is generated by pulse radiolysis in aqueous solutions by the reaction of O H * with formate ion. Recently, comparative electron transfer studies were carried out i n two different horse heart (hH) cytochrome c (cyt c) donor-acceptor complexes: one modified at Met 65 with [Fe(CN) (H 0)] - (Fe(Met 65)) and one modified at His 33 with [Ru(NH ) isn(H 0)] (isn = isonicotinamide) (Ru(His 33)) (14, 32). The Met 65 and the His 33 are located on opposite sides of the heme in the protein (Figure 3). The through-space distances from Fe(heme) to the S (Met 65) (-15.3 Â) and from Fe(heme) to the N (imidazole)His 33 (16 Â ) are very similar. The direction of electron transfer in both complexes is from the interior of the protein to the surface- bound, transition metal redox reagent, that is, heme(II) to [Fe (CN) ](Met 65) or to [Ru(NH ) isn] (His 33). Fur thermore both metal acceptor complexes have comparable self-exchange rates and redox potentials. Based on these facts, similar rates of intramolecular electron transfer from the heme(II) to F e ( M e t 65) or to R u ( H i s 33) would be expected, i f the protein residues and pathways between the heme and these two metal centers are comparable. Instead, the intramolecular electron transfer rate in cyt c-(Ru(His 33)) was found to be >10 times faster than that in cyt c-(Fe(Met 65)), despite the similar distance of the redox acceptor to the heme, driving force, and reorganizational energy (Table IV). For the heme(II) to R u ( H i s 33), intramolecular electron transfer is the preferred route 3

2

5

3

4

2

2

2

2+

x

m

5

3

m

2+

4

m

3

m



20.

ISIED


343

Figure 3. Two equidistant sites in horse heart cytochrome c: Met 65 and His 33. The Fe(heme) to S and to JVj (imz) distances are 15.3 λ (Met 65) and 16.0 λ (His 33). Horse heart cytochrome c was modified with [Fe(CN)g-J at Met 65 and [Ru(NH^ (isn)-] at His 33 in two separate experiments. 4

(k = 440 s ), whereas for the heme(II) to F e ( M e t 65), intramolecular elec tron transfer is very slow (k < 0.2 s ). Similar results were observed with the tuna cyt c modified with [Fe(CN) ](Met 65) (32). Thus the slow intermolecular electron transfer reaction, heme to Met 65 interaction, has now been observed for two different cyt c species. In both of these reactions the elec tron transfer occurs by an intermolecular pathway where electron transfer occurs from the heme(II) of one cytochrome molecule to the [Fe (CN) ](Met 65) of another cytochrome molecule. Calculation of the tunneling matrix element between the heme and His 33 or Met 65 is in reasonable agreement with these experimental findings (i.e., a lower tunneling matrix element is calculated for the heme —» Met 65 residue than for the heme - » His 33 residue) (37). [For the His 33 a proposed pathway _1

m

_1

5

m


5

344


Table IV. Intra- and Intermolecular Electron Transfer Rates and Driving Forces for Modified Protein Complexes


Hh cyt c -[Ru (NH3) isn(His 33)] 15-4 μΜ Hh cyt c -[Fe (CN) Met 65] 10.3^1.7 μΜ tuna cyt c -[Fe (CN) Met 65] 19.6-1.4 μΜ Ck cyt c -[Ru (NH3) isn(His 33)] Hh cyt c -[Ru (NH ) (His 33)] n

in

(25 °C)

440 ±30

2.8 ± 0.2 χ lO ^

0.18

n

m

Electron Transfer Reactions - American Chemical Society

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