1722
J. Am. Chem. SOC.1984, 106, 1722-1726
Ruthenium-Modified Cytochrome c: Temperature Dependence of the Rate of Intramolecular Electron Transfer Stephan S. hied,* Christa Kuehn, and Greg Worosila Contribution f r o m the Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903. Received May 16, 1983
Abstract: The ruthenium-modified horse heart cytochrome c, Ru(III)-cyt c(III), where the ruthenium is bound to the His-33 residue has been synthesized and characterized by ruthenium analysis, UV-vis and CD spectra, and differential pulse polarography and cyclic voltammetry. The intermediate Ru(II)-cyt c(II1) has been. generated by pulse radiolysis with use of four different radicals, COP, (CH3)2COH., (CH,OH),CCHOH, and -02CCH(OH)C(OH)C0,-. The rate of intramolecular electron transfer within the Ru(I1)-cyt c(II1) complex Ru(II)-cyt c(II1)
2Ru(II1)-cyt
c(I1)
and its temperature dependence were determined over a 40 “ C temperature range with the C 0 2 - .radical. A t 25 OC, these values a r e k , = 53 i 2 s-’ (pH 7 , 0.1 M phosphate buffer, 0.1 M N a H C O , ) , AH* = 3.5 i 0.2 kcal mol-’, and AS* = -39 i 1 eu.
There is a considerable body of evidence t h a t electron transfer in biological molecules takes place rapidly over long distances (ca. > 10 A) without direct coupling of *-conjugated molecules between the electron donor and t h e electron acceptor.’ One of t h e important themes in studying t h e mechanisms of electron transfer in biological systems is to understand t h e factors t h a t control this long-range electron-transfer process.2 We have attempted to understand t h e dependence of the rate of electron transfer on distance by measuring the intramolecular r a t e and temperature dependence of electron transfer between a donor and an acceptor separated by a number of peptide residue^.^ In a recent communication3 we have extended this work t o a modified protein donor-acceptor complex, t h e ruthenium-modified horse heart cytochrome c (Figure 1). In this protein the [(NH3)5R~11/111-] ion is covalently bound t o t h e imidazole moiety of His-33. The distance between t h e ruthenium site and t h e iron, estimated from the crystal structure of t h e oxidized tuna cytochrome c, is between 14 and 16 In this paper we report on the temperature dependence of the rate of intramolecular electron transfer over a range of 40 OC and comment on its origin. Different radicals, generated by pulse radiolysis, form t h e intermediate, Ru(II)-cyt c( 111),for which intramolecular electron transfer was measured. Some differences are observed between our results and those of Winker e t al.,5 who studied t h e same electron-transfer reaction with use of flash photolysis and found the r a t e to be temperature independent between 0.5 and 60 OC.
Experimental Horse heart cytochrome c (type VI) (Sigma) was purified by cation exchange chromatography6 on CM-52 cellulose (Whatman). Ruthenium-modified cytochrome c was prepared by using our earlier procedure’ (1) B. Chance, D. DeVault, H. Frauenfelder, R. A. Marcus, J. R. Schrieffer,N. Sutin, Eds., “Tunnelingin Biological Systems”;Academic Press, N.Y., 1979, pp 173, 201, 319, 269, and 417. (2) Stephan S. Isied, ref 1, pp 551 and 552. (3) S. S . Isied, G. Worosila, and S . J. Atherton, J . A m . Chem. Soc., 104, 7659-61 (1982), and references therein. (4) T. Takano and R. E. Dickerson, J . Mol. Biol., 153, 95-115 (1981). Ambiguity in the distance arises from the flexibility of the His-33 side chain. (5) (a) J. Winkler, D. Nocera, K. Yocum, E. Bordignon, and H. B. Gray, J . A m . Chem. Soc., 104, 5798-5800 (1982). (b) K. Yocum, J. R. Winkler, D. G. Nocera, E. Bordignon, and H. B. Gray, Chem. Scr., 21, 29-33 (1983). (c) H. B. Gray, private communication, 1983. (6) (a) Whatman Publication 607A, “Improved Techniques with Ion Exchange Celluloses”,p 3. (b) C. S. Knight, Adu. Chromatogr., 4, 61 (1967). (7) K. Yocum, J. B. Shelton, W. A. Schroeder, G. Worosila, S. S . Isied, E. Bordignon, and H. B. Gray, Proc. Natl. Acad. Sci. W.S.A.,79,7052-55 (1982). 0002-7863/84/ 1506-1722$01.50/0
(the Rutgers preparation). The ruthenium-cytochrome c derivative was further purified by using a second cation exchange resin (Bio Rex 70, column 15 X 1 cm) and eluted with 0.080 M phosphate buffer (pH 7.0) containing 0.25 M NaC1. Colorimetric ruthenium analysis was carried out on the ruthenium-cytochrome c derivative with use of about 3 mg of p r o t e h 8 The heme iron was determined spectrophotometrically by using published extinction coefficient value^.^ Cyclic voltammetry and differential pulse polarography on cytochrome c and ruthenium-cytochrome c were performed by using a gold electrode modified with an adsorbed layer of 4,4’-bipyridyl.’0 The circular dichroism (CD) spectra of the ruthenium-cytochrome c and the cytochrome c were obtained on a Cary 60 spectrometer with a 10 mm path length cell (visible) and a 1 mm path length cell (ultraviolet). High-pressure liquid chromatography of the reduced and oxidized cytochrome c and the ruthenium derivative was done on a Waters Associates H P L C instrument with two M 6000 A pumps and a M 660 solvent programmer and a Perkin-Elmer LC 75 variable-wavelength detector. A weak cation exchange column (Synchrome C M 300,250 X 5 mm) was used. For the HPLC a gradient of 60 80% Buffer B (5 mM NaH2P04,0.5 M NaAc, 1% acetonitrile, pH 6.1) was used in 4 min (1 mL/min) with Buffer A ( 5 mM NaH2P04, 1% acetonitrile, pH 6.1) (Buffers A and B were adjusted with HAC and N a O H , respectively) (A 410 nm). Pulse radiolysis experiments were carried out with a beam of 2-MeV electrons produced by a Van de Graaff accelerator at Brookhaven National Laboratories. Pulse lengths were usually in the range of 0.5-1 FS. Radical concentrations varying between 0.38 and 0.95 fiM were used throughout these experiments. The analyzing light passed through a 2-cm (2 X 1 X 0.5) cell three times for a path length of 6.1 cm. The light was produced by a quartz-iodine lamp with a filter cutoff below 450 nm. Transients were detected spectrophotometrically (A 550 nm) as described in ref 11. Data processing was done with computer programs developed by Dr. H. Scnwartz. In pulse radiolysis, irradiated water forms the following radicalsl2,”
-.
H 2 0 w-
e‘ (aq) (2.8), *OH (2.8), H.(0.6)
(1)
The numbers in parentheses denote G values, the number of radicals formed per 100 eV absorbed. When N 2 0 is present, the hydrated electron is converted to O H radicals, H+
+ e-taq)+ N 2 0
-*
N,
+ .OH
k =8
X
lo9 M-’
s-I
(2)
Reducing radicals COz-., (CH3),C0H, and ( C H 3 0 H ) 3 C C H O Hwere’ produced from the .OH radicals by hydrogen atom abstraction from the (8) F. D. Snell, “Photometricand Fluorometric Methods of Analysis, Part 2, Metals”, J. Wiley, New York, 1978, p I . (9) D. L. Brautigan, S. Ferguson-Miller, and E. Margoliash, Methods Enzymol., 53, 128-164 (1978). (IO) W. Albery, M. J. Eddowes, H. A. C. Hill, and A. R. Hillman, J . A m . Chem. Soc., 103, 3904-3910 (1981). (11) H. A. Schwarz and C. Creutz, Znorg. Chem., 22, 707-13 (1983).
0 1984 American Chemical Society
J. Am. Chem. Soc., Vol. 106, No. 6,1984 1723
Ruthenium-Modvied Cytochrome c
I I C
21@
L
3
iil n X ELENCT
7 0
610
2cc
Its
"r
20
240
I
c
nr 1
n l ELEYC'H
Figure 3. Circular dichroism (CD) spectra of ruthenium-modified horse heart cytochrome c(II1) (-) and native horse heart cytochrome c(II1) (- - -) both at 1.O X M: (A) UV-vis region, 1-cm cell; (B) expanded UV region, 1.1-cm cell. 0 refers to measured ellipticity.
Figure 1. Histidine-33 ruthenium modified horse heart cytochrome c(II1)
(adapted from ref 23).
n
Time [min)
Figure 4. HPLC separation of cytochrome c(II1) and ruthenium-modified cytochrome c(II1) (see Experimental for conditions) I
Scheme I A*
+
-
Ru(II1)-cyt ~ ( 1 1 1 )
k
+
R u ( I I ) - c y t ~(111) A
(3)
I
A* +0.3
tO.l
0
-0.1
+
R u ( I I 1 ) - c y t c(II1)
-k-
R u ( I I I ) - c y t CUI)
+
A
(4)
11 -0.3
E(V)
-
Figure 2. Reduction of [Ru(III)-cyt c(III)] (-) and [cyt c(III)] (- -) by differential pulse polarography: volts vs. SSCE 2 mm gold disk electrode; scan rate = 2 mV/s; pulse amplitude = 25 mV; 0.25 pmol [Ru(III)-cyt c(III)] or [cyt c(III)] in 0.1 M NaC104,0.01 M bipyridine, 0.08 M phosphate buffer pH 7.
corresponding molecules. The .OH radical also decays by dimerization and reaction with H atoms. The rate of reaction of .OH with cytochrome c is k(OH + cyt @I)) = 4 X 1O'O M-I sd1.13 Experiments were carried out at concentrations of 0.1 M sodium formate, 0.1 M isopropanol, and 0.1 M pentaerythritol, and 0.05 M potassium tartrate at pH 7.0 (0.1 M phosphate buffer) and at concentrations of ruthenium-cytochrome c(II1) of 0.8 X lod to 3.2 X loa M.
Results and Discussion (a) Characterization of the Ru(III)-Cytochrome c (III)Species. The ruthenium-modified cytochrome c was characterized as reported earlier by using UV-vis spectra, NMR, cyclic voltammetry, and peptide mapping experiments.' The species used in this study was characterized by using Fe and Ru analysis (Ru/Fe 1.0 f 0.05). Differential pulse polarography gave two overlapping peaks (12) (a) A. B. Ross, "Selected Specific Rates of Reactions of Transients from Water in Aqueous Solution, Hydrated Electron, Supplemental Data"; U S . National Bureau of Standards: Washington, DC, 1975; NSRDSNBS-43 Supplement. (b) L. M. Dorfman and G. E.Adams, "Reactivity of the Hydroxyl Radical in Aqueous Solution"; US. National Bureau of Standards: Washington, DC, 1973; NSRDS-NBS-4 Report. (13) J. Van Leevwen, A. Raap, W. Koppenol, and H. Nauta, Biochim. Biophys. Acta, 503, 1-9 (1978), and references therein.
at +0.26 and +0.13 V (vs. NHE) corresponding to the reduction of the heme and the ruthenium sites, respectively (Figure 2). CD spectra of the ruthenium(II1)-cytochrome c(II1) were identical with those of the native cytochrome c(II1) in the UV and visible region. Figure 3 shows both C D spectra for a solution of 1.O X lov5M . High-pressure liquid chromatography of the native cytochrome c(II1) and the ruthenium(II1)-cytochrome c(II1) derivative on a weak cation exchange column is shown in Figure 4. Figure 4 shows the resolution obtained thus far on HPLC. Further work on the N M R and the interaction of the ruthenium-cytochrome c derivative with cytochrome c oxidase is in progress. (b) Kinetics and Temperature Dependence of the Intramolecular Electron-Transfer Process. In an earlier communication3 we reported our preliminary results on the rate of reduction of Ru(111)-cyt c(II1) with COz-. to generate the Ru(I1)-cyt c(II1) intermediate with use o f pulse radiolysis. The rate of intramolecular electron transfer from Ru(I1) to the heme was measured to be k, = 82 f 20 s-l (pH 6.7, 2 mmol phosphate buffer, 0.1 M sodium formate buffer). In Scheme I the sequence of reactions leading to Ru(I1)-cyt c(II1) intermediate is shown where A- is the reducing radical produced at a concentration lower than that of the Ru(II1)-cyt c(II1) by more than one order of magnitude. At that radical concentration, A- selects between the [(NH3),Ru"'-] site and the heme site. The Ru(I1)-cyt c(II1) intermediate was generated with use of four different reducing radicals, A = COz-., (CH3)2C--OH, (CH20H)&CHOH., and (-02CCH(OH) C(OH) COz-). Figure
1124 J . Am. Chem. Sac., Vol. 106, No. 6, 1984
Isied, Kuehn. and Worosila Table I. Temperature Dependence of the Intramolecular Electron-Transfer Reaction from the Ru(l1) hemc(ll1) Site in Ru(I1)-cyt ~(111)'
cn 3 CHa- F; - OH
d
-f
co;
T , "C
k,, s-'
T. "c
43.8 43.5 43.5 43.8 43.7 43.9 43.9
77.0 14.5 84.6 85.6 81.8 82.9 79.8
24.8 24.9 24.8
52.0 53.8 52.3
33.9 34.1 34.2 34.2
63.5 67.0 60.2 12.3
10.8 10.6 10.4 10.4 10.7 10.4
41.1 38.7 35.3 36.7 37.0 37.7
4.2 3.9 2.9 2.8
32.3 33.4 31.2 29.8
k,,
s-1
a CO;, radical as reductant. [ Ru(II1)-cyt c(III)] = 1.6 x in 0.1 M NaHCO,, 0.1 M phosphate buffer. pH 7.0.
M?
Table 11. Effect of [Ru(III)-cyt c(III)] Concentration (M)on the Rate of Intramolecular Electron Transfera [Ru(III)-cyt c(III)]
25
50
0.80 X
54.8 54.4 55.3 54.9 54.8
1.6 X
53.8 52.0 52.3 52.0 52.3 51.7 52.0
Time (m s e d
Reduction of ruthenium(II1)-cytochrome c(II1) by (CH,),COH, COP, and (CH20H),CcHOHradicals (see Experimental for conditions). Figure .5.
k , s-]
3.2 X
T = 25 "C, 0.1 M NaHCO,, 0.1 M phosphate buffer, pH 7.0.
Table 111. Effect of Radical Type on the Rate of Intramolecular Electron Transfer' -1.50
-
radical
F d -2.00
-
53
T
(CHJ,COH - O,CCH(OH )C(OH)CO;
57
t
(CH,OH),CCHOH a T = 25 "C. [Ru(III)-cyt c(III)] = 1.6 x 0.1 kl phosphate buffer, p H 7.0.
i
I
3.20
I
3.40
I
I
3.60
lo3 (IIT)
Figure 6. Temperature dependence of the intramolecular electrontransfer rate constant in histidine-33 ruthenium modified cytochrome c.
5 shows the absorbance vs. time profile for three of these radicals. The radicals derived from sodium formate (COP),isopropanol ((CH,),COH), potassium tartrate. (-02CCH(OH)C(OH)C02-) and pentaerythritol (C(CH,OH),CHOH) reduce cyt c(II1) with second-order rate constants of 1.3 X lo9, 3.8 X lo*, 1.7 X lo8, and 95% (pentaerythritol), depending on the choice of the radical. The selectivity of the different radicals for the heme and the ruthenium sites is a function of the charge and solvation properties. The hydrophobic 2-propanol radical has the highest preference for the hydrophobic heme site, while the negatively charged formate and tartrate radicals prefer the hydrophilic ruthenium site. The polyhydroxylic pentaerythritol radical selects the ruthenium site almost exclusively. Earlier work by Simic et al.I4 reported an upper limit on the rate of reduction of cyt c(II1) with (CH,OH),CCHOH of
