Free-Energy Dependence of Electron Transfer in Cytochrome c

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Downloaded by UNIV OF GUELPH LIBRARY on June 9, 2012 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/ba-1998-0254.ch007

Free-Energy Dependence of Electron Transfer in Cytochrome c Labeled with Ruthenium(II)-Polypyridine Complexes J. L . Wright , K. Wang , L. Geren , A. J. Saunders , G. J. Pielak , B. Durham* , and F. Millett 1

1

,1

1

2

2

1

Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 1

2

The free-energy dependence of the rate constants for intramolecular electron transfer in yeast cytochrome c covalently bound to a series of ruthenium(II)-polypyridine complexes has been determined. The H39C;C102T variant of yeast cytochrome c was attached to a series of ruthenium complexes through a thioether linkage involving the sulfur atom of Cys39 and a methylene carbon of 4,4'-dimethylbipyridine. The free energies of reaction cover a range from 0.75 to 1.26 V with intramo lecularrate constants of 4-9 X 10 s . The observed rate constants are consistent with reactions in which the free energies of reaction are nearly equal to the reorganization energy. In the present case, the reor ganization energy for electron transfer between the ruthenium complex and the heme iron is 1 eV. 5

-1

O v e r the past several years, we have developed a technique that has proven extremely valuable in the study of electron transfer between redox sites in metalloproteins. We have reported kinetic studies of the reaction of cytochrome c with cytochrome c peroxidase (1-3), cytochrome oxidase (4), cytochrome b (5, 6) plastoeyanin (7), and cytochrome C\ (8). In addition, we have been able to show (9,10) that intramolecular electron transfer in cytochromefc covalently 5

5

* Corresponding author. ©1998 American Chemical Society

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

99

100

PHOTOCHEMISTRY AND RADIATION CHEMISTRY

bound to a series of ruthenium complexes follows the dependence on free energy of reaction predicted by Marcus ( I I ) . The technique is based on the photoredox chemistry of derivatives of the well characterized Ru(bipyridine) complex (12). When one of these complexes is covalently attached to a metalloprotein, photoexcitation leads to rapid electron transfer quenching of the ru thenium excited state by the iron center of the protein. The Fe(II)-Ru(III) intermediate thus formed rapidly returns to the original oxidation state through a thermal electron transfer reaction as indicated in Scheme I, where h is Planck's constant, ν is the frequency of the exciting light, and describes the rate of excited state decay by processes other than electron transfer.


3

Ru(II)*-Fe(III)

ι Av \kd 1

Ru(II)-Fe(III)

^ ' Ru(HI)-Fe(H)

Λ.


2+


7. WRIGHT ET AL.

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Free-Energy Dependence of Electron Transfer

These intramolecular electron transfer processes provide an opportunity to examine electron transfer within the protein environment. Addition of a reductant, such as aniline, results in efficient reaction of the Ru(III) with the reductant to form Ru(II), which leaves the heme iron in the reduced state. If a redox active metalloprotein is present in the solution, electron transfer between the reduced heme and the added protein can be observed. Production of reduced heme iron and removal of the Ru(III) intermediate can be accomplished within a few hundred nanoseconds, which allows the study of extremely rapid interprotein electron transfer reactions. In recent publications (5, 6), we described studies of the electron transfer reaction between horse heart cytochrome c and synthetic rat liver cytochrome Z?5. We observed a rate constant for electron transfer of 4 Χ 10 s"" in low ionic strength solutions in which these proteins form a strong 1:1 complex (13). The rate constant presumably describes the rate of electron transfer be tween the two heme centers held together in a specific geometry with a well defined electronic coupling. Alternatively, it may describe electron transfer in a collection of protein complexes in rapid dynamic equilibrium. The binding geometry of the electrostatic complex formed by cytochrome c and cytochrome & has been modeled by Salemme and co-workers (14, 15) and Northrup et al. (16). 5

1

5

If electron transfer between cytochromes c and b% is viewed in the context of the theoretical description outlined originally by Marcus (11 ), then the rate constant for electron transfer, k , is given by equation 1. et

4TT

k

et

-(X

2

exp

= ^-Hl (4IR\k T) B

B

1/2

AGo\2' 4kk T +

(1)

B

This equation describes a model for electron transfer with two fundamental parameters: H , which describes the electronic coupling between the redox centers, and λ, the reorganizational energy, which is a measure of the energy required to alter the solvent and ligands surrounding the redox centers before electron transfer, can take place. The remaining symbols have their usual mean ing; Γ is temperature, h is Planck's constant, k is Boltzmann's constant, and Δ G° is the full energy of reactions. If the rate constant and the reorganizational energy of the reaction are known, then the magnitude of the electronic coupling between the redox centers can be determined. Measurement of the electronic coupling between cytochromes c and h% will provide valuable information about the geometry of the protein-protein interaction. This calculation hinges on an accurate measurement of the reorganiza tional energy. A search of the literature indicates that there is significant uncer tainty in the reorganizational energy of the cytochrome c-b^ system. For exam ple, the self-exchange reactions for both cytochrome c and cytochrome b have been investigated by Dixon and co-workers (17). Intrinsic reorganizational energies of 0.72 and 1.2 eV, respectively, were derived from the self-exchange rate constants. If X(c-bs) = [k(c) + X(b )]/2, then the reorganizational energy AB

B

5

5


102


for electron transfer between cytochrome c and b is 1.0 eV. In contrast, Northrup et al. (16) used a value of 0.7 eV for the intracomplex reorganizational energy. Marcus and Sutin (18) have calculated a value of 1.04 eV for the reorga nizational energy for the cytochrome c, which suggests a value of 1.1 eV or larger for the reorganizational energy for the cytochrome c-b reaction. We have been able to show that the reorganizational energy of cytochrome b is 1.3 eV in the electrostatic complex formed with cytochrome c at low ionic strength. In these experiments, we assumed that the reorganizational energy of cytochrome b was given by the relation X(Ru-fo ) = [X(Ru) + \(b )]/2, where X(Ru) is the reorganizational energy for R u ( b p y ) , where bpy = 2,2 -bipyridine and X(Ru-& ) is the reorganizational energy determined from the intramolecular electron transfer reactions of cytochrome bs covalendy bonded to a series of ruthenium polypyridine complexes. The reorganizational energy, X(Ru-fo ), was obtained from a plot of free energy of reaction versus In (k ) and is equal to Δ G at the maximum point of the curve. The cytochrome b$ used in these studies was genetically engineered to contain a unique reactive site for the ruthenium complex that placed it outside the binding domain for cytochrome c. The fact that the ruthenium complex does not interfere with binding has been verified (5, 6). 5

5

5

5

5

5


3

/

2 + / 3 +

5

5

0 /

et

In the present study we report preliminary data describing the rates of intramolecular electron transfer of cytochrome c covalently linked to several of the same ruthenium complexes used in the cytochrome fc study. Data were obtained with a variant of cytochrome c genetically engineered to place the ruthenium complexes outside of the binding domain. The goal of these experi ments was to determine the reorganizational energy for the intramolecular electron-transfer reactions and calculate the reorganizational energy for cyto chrome c, and for the cytochrome c-b reaction, X(c-b ), as described in the preceding paragraph. 5

5

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Experimental Details Materials. The preparation of the H39C;C102T variant of yeast cyto chrome c has been described by Hilgen and Pielak (19). Reaction of this variant with Ru(bpy)2(BrCH bpyCH3) and subsequent purification and characteriza tion of the labeled protein have been described by Geren et al. (20, 21 ). The other derivatives were prepared by analogous methods. Preparation of the ruth enium complexes has been described by Scott et al. (9) and Ernst and Kaim (22). 2

2+

Kinetic Measurements. Rate measurements were performed using laser flash photolysis techniques. The excitation source consisted of 10-ns pulses of 355-nm light obtained from a Quanta-Ray N d : Y A G laser. The probe beam, at right angles to the excitation pulse, was obtained from a pulsed Xe arc lamp.



7. WRIGHT ET AL.


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The detector was a photomultiplier tube in a five-dynode configuration with associated electronics of local design. Signals were recorded on a LeCroy 7200 series digital oscilloscope as 2000 point records and transferred to a personal computer for storage and analysis. The kinetic equations that describe the reactions given in Scheme I have been reported by Pan et al. (7). Emission at wavelengths greater than 580 nm and transient absorbance changes at 434 and 550 nm were recorded and fitted simultaneously to these equations. The emitted light from the ruthenium com plexes provided a measure of the excited-state decay rate (ki + k^), as did the absorbance changes at 434 nm, which is an isosbestic point for changes in cytochrome c. Absorbance changes at 550 nm reflect changes in the redox state of cytochrome c. Baseline corrections to the 550-nm transient were made with data collected at 556 nm, which is an isosbestic point for cytochrome c. In cases where the oxidation of the heme iron was slow compared to the lifetime of the excited state, £2 was obtained from a simple exponential fit of the transient absorbance changes at 417 or 550 nm. Kinetic measurements were made in solutions containing 1 m M phosphate buffer at p H = 7 at 22 °C. Protein concentrations were 5 to 15 μΜ. The presence of dissolved oxygen had no measurable effects on the transient absor bance changes or the emission measurements.

Results Yeast cytochrome c has been covalently attached to a series of ruthenium-polypyridine complexes at Cys39. A variant of yeast cytochrome c containing cyste ine in place of His39 and threonine in place of Cysl02 was used in this study. The variant was prepared using genetic engineering techniques. The attach ment site was chosen such that binding of cytochrome c to other proteins (i.e., cyt &5, cyt c peroxidase, and cyt c oxidase) would not be affected by the presence of the bulky ruthenium complexes. Cys39 and the attached ruthenium complex point toward the lower part of the back side of cytochrome c, as illustrated in Figure 1. The binding domain, in general, involves the area around the exposed heme edge and the surrounding lysines. Cysteine 102 was changed to a threo nine in this case so that only one surface cysteine residue was available for reaction with the ruthenium complex and to avoid possible disulfide bond for mation between two proteins. The ruthenium complexes were attached to the specified cysteine by for mation of a thioether linkage between the sulfur atom of cysteine and the methylene carbon of one of the bipyridine ligands. The reaction makes use of complexes that contain 4-bromomethyl-4 -methylbipyridine, as indicated. /



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Figure 1. Proposed structure of the H39C;C102T variant of yeast cytochrome c covalently attached to Ru(bpy) (CH bpyCH -) at the sulfur atom of Cys39. The pathway for electron transfer proposed by Wuttke et al (25) extends from the ruthenium complex, through Ser40 and Gly41, and includes a hydrogen bond from the amide nitrogen of Gly41 to the heme propionate group. The structure is based on the X-ray structures of yeast iso-1 -cytochrome c and Ru(bpy) , with modifications made using the molecular modeling program Insight II from Biosym, San Diego, CA. 2

3

2+

2

2+

3

The remaining bipyridine ligands of the ruthenium complexes can be changed to other ligands to give redox potentials that span a range of free energies of reaction comparable to the expected reorganizational energy. In this study, complexes were prepared that contained 4,4 -dimethylbipyridine and 3,3'-bipyridazine. The overall redox potentials for the electron-transfer reactions in vestigated are listed in Table I and cover a range from 0.75 to 1.26 V. /


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Table I. Rate Constants for Intramolecular Electron Transfer in H39C;C102T Variant of Yeast Cytochrome c Covalently Bound to Ruthenium Complexes at Cys39. Rate constants (s ) L

Complex Ru(bpy) (CH bpyCH -) Ru(CH )2bpy) (CH bpyCH -) Ru(bpdz) (CH bpyCH - ) Ru(bpdz) (CH bpyCH -) 2

3

2

2

2

3

3

2

5

2+

5

2+

2

3

6.2 Χ 10 5.2 ts 10 4.0 Χ 10 k [Ru(I)-Fe(III)]

2+

2

3


E°', V

h

2

5

2+

et

a

9.2 X 10 6.0 X 10 6.0 X 10 = 5 X 10 5

5

5

5

1.09, 1.01 1.17, 0.91 0.75, 1.23 1.26

NOTE: Data were obtained with solutions containing 1 mM phosphate buffer, pH = 7, at 22 °C. Estimated standard deviations in rate constants are 15%. "Overall redox potential for reactions of excited-state Ru(II*) and Ru(III) with heme iron of cytochrome c, respectively.

and 434 nm. The rapid decrease in absorbance observed at 434 nm is due to the formation of the ruthenium-complex excited state. The decrease is followed by conversion of the excited state to Ru(III), which subsequently returns to the Ru(II) form of the complex. The absorption of the excited-state form of the complex is nearly identical to that of the Ru(III) form at this wavelength. The increase and decrease in absorbance at 550 nm correspond to the formation and subsequent oxidation of the Fe(II) heme. The rate constants for intramolecular electron transfer in the H39C;C102T variant of cytochrome c are listed in Table I. All the rate constants obtained with the H39C;C102T variant fell within a narrow range of 4 - 9 Χ 10 s" . Rate constants for intramolecular electron transfer from Fe(II) —• Ru(III), Ru(II)* — Fe(III), and Ru(I) — Fe(III) were obtained with the H39C;C102T variant labeled with R u ( b p d z ) ( C H b p y C H 2 - ) , where bpdz = 3,3'-bipyridazine. The Ru(I) form of the complex was generated by quenching of the excited state with 10 m M dimethylaminobenzoate. Formation of the Ru(I) form of the complex was verified by monitoring the absorbance at 504 nm, which is an isosbestic point for cytochrome c. 5

2

3

1

2+

Discussion Rate constants for a series of intramolecular electron transfer reactions involving ruthenium polypyridine complexes attached to Cys39 of the H39QC102T var iant of cytochrome c have been determined. These reactions cover a range of free energies of reaction from 0.7 to 1.26 V. It is of interest that all the rate constants given in Table I, with one marginal exception, fall within a range of two standard deviations. Thus, no statistically significant variation in rate con stants with free energy of reaction was observed. Two explanations for the independence from free energy of reaction are



106 0.0020 0.0000 -0.0020 S

-0.0040

CO


Free-Energy Dependence of Electron Transfer in Cytochrome c

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