Symposium: Applications of Inorganic Photochemistry
Symposium: Applications of Inorganic Photochemistry
Ruthenium(II) Polypyridine Complexes and the Electron-Transfer Reactions of Metalloproteins Bill Durham* and Frank Millett Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701
Reactions involving the transfer of a single electron are very common in many biological processes. Respiration and photosynthesis, for example, require several one-electron transfer reactions. Respiration in eukaryotic cells takes place in small compartments called mitochondria. A simple schematic diagram of some of the electron-transfer reactions that take place in mitochondria is shown in Figure 1. The diagram highlights cytochrome c, which functions as an electron-transfer shuttle and is probably the most intensely studied metalloprotein involved in these reactions. One of its primary roles is to shuttle electrons from the cytochrome bc1 complex to cytochrome c oxidase, where the electrons are used to reduce molecular oxygen to water. The reduction is accompanied by the formation of a proton gradient across the membrane in which cytochrome c oxidase is embedded. The proton gradient is used in the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). ATP is used by the cell as an energy source for numerous other reactions. Cytochrome c is a relatively small protein (ca. 100 amino acids) containing a single iron atom held by four nitrogens of a porphyrin ring, the nitrogen of histidine, and the sulfur atom of methionine (1). The iron atom is the center of the redox activity and cycles between Fe(II) and Fe(III) forms. The X-ray crystal structures of cytochrome c from numerous different organisms show that only a very small portion of the porphyrin ring is exposed to the surface of the protein. Such structural studies prompted an intense study of the electron-transfer reactions of cytochrome c. The initial questions invariably focused on how electrons could be transferred effectively between two proteins over the long distances (20 to 40 Å) that separated the redox centers. Other questions were pursued with equal intensity. For example, how is the flow of electrons regulated in cells? It is clear from Figure 1 that cytochrome c can accept or donate electrons from several different sources. Are there structural features that provide a means for recognition between the proper partners? Are there features that aid in regulating priorities among the various donors and acceptors? How is electron transfer coupled to the formation of proton gradients and energy production? Measurement of the rates of electron transfer is a key element in investigation of these questions, yet this remains a very difficult experimental problem. The difficulty stems from the extremely fast nature of the reactions. In fact, many of these reactions are too fast to measure by even the fastest mixing techniques. Over the past two decades only a few techniques have been developed to study these reactions. All of these rely on some *Corresponding author. Phone: 501/575-5178; fax: 501/5754049; email:
[email protected].
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Figure 1. Schematic diagram of mitochondrial electron-transfer reactions involving cytochrome c.
rapid means of generating a reactive state of one of the proteins without the need for mixing two separate solutions. For example, with a technique known as pulse radiolysis, a very short burst of high-energy electrons can be used to rapidly reduce some of the components of the solution (2). Tollin and co-workers (3) have made extensive use of laser light–induced reduction of flavin derivatives to generate reactive states of metalloproteins. Both of the these techniques produce nonselective reductants, which can potentially react with more than one protein in a reaction mixture. Several investigators (4) have utilized cytochrome c with the iron replaced by zinc or other metals. These metal-substituted analogues have longlived excited states that can transfer electrons. The excited states can be rapidly produced with laser light sources and are easily detected through emitted light and large changes in absorbance. Unfortunately, although the overall structural integrity of the proteins substituted with different metals appears to be retained, the reactions of these proteins lack some of the interesting features of the native iron-containing proteins. Pioneering studies by Gray and co-workers (5) and Isied and co-workers (6) demonstrated that Ru(NH3)52+ could be covalently linked to the nitrogen atom of histidine residues on the surface of cytochrome c. In subsequent studies with cytochrome c and many other proteins, the rate constants for electron transfer between the metal center of the protein and the ruthenium complex were reported. The early work made use of simple ammonia complexes of ruthenium, which do not have excited-state properties that can be used to initiate electron transfer. Instead, a variety of techniques such as pulse radiolysis and zinc-substituted metalloproteins were employed to measure the rates of intramolecular electron transfer. These studies focused on the question
Journal of Chemical Education • Vol. 74 No. 6 June 1997
Symposium: Applications of Inorganic Photochemistry of how electrons were transferred over long distances through a protein. Several theoretical developments describing electron transfer in such an environment paralleled the experimental work (7, 8). Later developments demonstrated the utility of ruthenium complexes containing bipyridine ligands [e.g., Ru(bipyridine)32+ ] in the study of electron transfer. Photoexcitation of a ruthenium bipyridyine complex bonded to a metalloprotein produces a long-lived excited state that can act as a strong oxidant or reductant. In many cases, rapid electron transfer to the iron center of the protein follows photoexcitation. The electron transfer reactions can be monitored spectrophotometrically over an extensive time range (nanoseconds–seconds) after excitation by a short laser pulse. Over the past 8 years the ruthenium-labeled proteins have been used extensively to study intramolecular electron transfer (Ru to Fe) as well as intermolecular electron transfer between two metalloproteins. A detailed description of the chemistry involved in the use of ruthenium bipyridine complexes for the study of electron transfer reactions in metalloproteins is presented in the following sections. For a review of the chemistry of ruthenium polypyridine complexes and their excited state properties see references 9–11.
ligands is replaced by 4-bromomethyl-4′-methylbipyridine, which will react selectively with the sulfur atom of cysteine at pH 10.
CH2Br N
(bpy)2Ru
CH2-S-Protein N
Protein-SH
(bpy)2Ru
N
N CH3
CH3
Again, genetic engineering of the proteins has greatly strengthened this methodology and has been used to place cysteine at optimal locations for electron transfer in rat liver cytochrome b5 and yeast cytochrome c (14, 15). Photoinitiated Electron Transfer Photoexcitation of an appropriate ruthenium bipyridine complex covalently bound to cytochrome c or b5 initiates the reaction sequence shown in Scheme I.
Ru(II)*-Fe(III)
Covalently Binding Ruthenium Complexes to Cytochromes
k1 hν
A number of different chemical means have been developed by which ruthenium complexes can be covalently bound to metalloproteins. Attention will be focused primarily on cytochrome c and cytochrome b5, since these proteins (together with myoglobin) represent a significant portion of the work published in this field. Initial success in this area relied heavily on the fact that ruthenium(II) shows a strong tendency to bind to nitrogen-containing heterocyclic bases. The amino acid histidine contains an imidazole ring, and ruthenium complexes, such as Ru(bpy)2(H2O)22+ , selectively bind to the nitrogen atom in the imidazole ring. 1 The resulting complex Ru(bpy) 2(H2O)(His-cytochrome c) has a very short excited-state lifetime. However, further reaction with excess imidazole yields the complex Ru(bpy)2(imidazole)(His-cytochrome c), which has an excited state lifetime of 80 ns (12) and has been used extensively in the study of electron-transfer reactions (13). The above reaction is specific for histidine residues found on the surface of the protein. Histidine residues are not very common, and often reactions are limited to one or two specific sites on the protein. Progress in this area has been aided greatly by the rapidly developing area of genetic engineering. Through this technology, histidine residues can be selectively placed at specific locations on the surface of the protein. In addition, specific histidine residues that are potential interfering sites can be removed so that only a single product is formed. Gray and co-workers (13) have used this strategy to explore electron transfer between the metal center of various proteins and the covalently bound ruthenium complex. The sulfur atom of cysteine residues will react with a number of reagents containing reactive bromo or iodoalkyl functional groups to form a thioether link. This chemistry provides a means of covalently binding ruthenium complexes containing three bipyridine ligands. Many complexes of the type Ru(bpy)32+ have excitedstate lifetimes of several hundred nanoseconds in aqueous solution. In this binding scheme one of the bipyridine
Ru(III)-Fe(II)
kd
Scheme I k2 Ru(II)-Fe(III)
Ru(II)-Fe(III), in this and subsequent schemes, represents the covalently bound ruthenium complex and the iron center of cytochrome c or b5. Absorption of light leads to the formation of the excited state, Ru(II)*, which rapidly transfers an electron to the iron center of the protein. Since the resultant Ru(III) is now in its ground state, it readily accepts an electron from the iron center to return the system to the initial oxidation states. The rate constants k1 and k2 describe the forward and reverse electron transfer, and kd describes all other processes (such as light emission) that return the excited state to the ground state. The progress of the reaction can be followed by monitoring changes in the absorbance at specific wavelengths after excitation by a short laser pulse. An example of transient absorbance changes showing the formation and subsequent decline of the reduced form of the protein is illustrated in Figure 2a. In this example, the absorbance changes were monitored at 424 nm because the protein shows a very large increase in absorption upon reduction at this wavelength. The rate constants can be obtained by fitting the time-dependence of the transient absorbance changes for the protein and the ruthenium complex to the appropriate rate equations (16). Successful measurement of electron-transfer rate constants using Scheme I relies on adequate production of the Ru(III)-Fe(II) intermediate. The efficiency of production of the intermediate depends on the relative magnitudes of kd and k1 (i.e., ideally k1 > kd). Simply stated, success requires very rapid intramolecular electrontransfer quenching or a long excited-state lifetime. Alternative schemes can be derived from Scheme I which can be employed for cases where the excited state is short-lived or intramolecular electron transfer is slow.
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Symposium: Applications of Inorganic Photochemistry For example, Scheme II shows the use of an external quencher for the formation of Ru(III). Q Ru(II)*-Fe(II) Q-
Scheme II
hν kd
Ru(III)-Fe(II)
k2
Ru(II)-Fe(III)
Ru(II)-Fe(II)
In this scheme, known as the “flash-quench” method, the protein is in the reduced state initially and the excited complex reacts with an external quencher. The quenching reaction produces Ru(III), which can then react with the reduced iron center of the protein. High concentrations of external quencher can be used to improve the
a
quenching efficiency. The scheme also allows for very slow reactions between the iron and the ruthenium complex. Note that the rate constants k1, k2 , and kd describe the same reactions in all schemes. Use of Schemes I and II has provided a large body of experimental data from which a detailed picture of electron transfer in these model systems has emerged (13, 17). For example, the electron-transfer reactions of cytochrome b5 , labeled at position 65, with a variety of ruthenium complexes have been shown to follow the freeenergy relation suggested several years ago by Marcus (11, 18): ket = (4π3/h2λkBT)1/2(HAB )2 exp[{ (∆G°′ + λ)2/4λk BT] (1) where λ is the reorganizational energy, HAB the electron tunneling matrix element, and ∆G°′ is the free energy for the reaction (obtained from electrochemical measurements). The reorganization energy is a measure of the energy required to alter the ligand and solvent orientation and polarization before electron transfer can take place. H AB is a measure of the electronic coupling between the redox centers and indicates the probability that electron transfer will occur once the transition state is reached. Figure 3 shows a plot of ln(k et) vs. ∆G°′. As predicted by eq 1, the rate constants increase to a maximum with increasing free energy of reaction, and then decrease with further increases in free energy. The maximum in the plot corresponds to the point where λ = {∆G°′. In the present example, the reorganizational energy is 1.0 eV. Since there is very little change in the geometries of the ruthenium complex and the iron center of the protein in these reactions, it has been concluded that the reorganization energy is dominated by reorientation and repolarization of the solvent and protein.
ln k et
b
c
{∆G° ′ (eV)
Figure 2. Transient absorbance changes following laser photoexcitation of (a) 10 mM solution of cytochrome b5 labeled at Cys-65 with Ru(bpy)2 (CH3bpyCH2–) 2+ in phosphate buffer pH = 7; (b) solution “a” with 10 mM N-phenylglycine added; and (c) solution “b” with 10 µM cytochrome c added. Increasing absorbance indicates formation of Fe(II) cytochrome b5 at all wavelengths indicated.
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Figure 3. Plot of ln(k et) vs. {∆G ° ′ for the intramolecular electrontransfer reaction of cytochrome b5 labeled at position 65 with a series of ruthenium(II) bipyridine complexes. The specific complexes are numbered as follows: 1: Ru(bpy)2(CH3bpyCH 2–)2+ 2: Ru[(CH3)2bpy]2(CH3bpyCH 2–)2+ 3: Ru(CH3bpyCH 2OH)bpdz(CH3bpyCH2–) 2+ 4: Ru(bpy)bpym(CH3bpyCH 2–)2+ 5: Ru(CH3bpyCH 2OH)bpyz(CH3bpyCH 2–)2+ The rate constants k 1 are indicated by m and the rate constants k2 are indicated by d.
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Symposium: Applications of Inorganic Photochemistry The free energy plot also provides a measure of HAB , which depends on the distance between the ruthenium complex and the iron center as well as on the intervening medium. Gray and co-workers have explored this relation extensively (13). Several investigators, including Beratan and co-workers (7, 8, 19), Marcus and co-workers (20), and Kuki (21), have focused on the theoretical aspects of the electronic coupling and have tried to develop models describing how the electron travels through or is coupled through the protein. One of the most popular models is that of Beratan and co-workers, which develops the idea of dominant coupling pathways. The dominant pathways are derived by searching for combinations of covalent bonds, hydrogen bonds, and through-space jumps that maximize the coupling between the redox centers. In this treatment, covalent bonds offer the best coupling and through-space jumps the poorest. In the present example, the metal centers are coupled through 12 covalent bonds and the probability of electron transfer in the transition state is on the order of 10{7. Protein-to-Protein Electron Transfer The electron-transfer reactions depicted in Scheme I are extremely fast and in many cases are complete within 500 ns. If, however, an electron donor “D” that will react with the transient Ru(III) is present, the reduced form of the metalloprotein can be produced permanently as indicated in Scheme III. Ru(II)*-Fe(III)
k1 hν
D Ru(III)-Fe(II)
kd
D+ Ru(II )-Fe(II )
Scheme III k2 Ru(II)-Fe(III)
Several electron donors, including aniline, EDTA, Nphenylglycine, and dimethylaminobenzoate have been used successfully. An example of transient absorbance changes under these conditions is shown in Figure 2b. Since the reduced form of the protein can be produced extremely rapidly, the scheme provides a means of investigating electron-transfer reactions between the initially reduced protein and some other protein that may be present in solution. If, for example, Fe(III)cytochrome c is added to a solution containing cytochrome b 5 labeled with ruthenium in the presence of 5–10 mM aniline, the transiently reduced cytochrome b5 will transfer an electron to cytochrome c. This reaction mixture has been used to explore the reactions of these two proteins, and transient absorbance changes that accompany the electron transfer are shown in Figure 2c. The course of the reaction can easily be established by monitoring the redox changes of both proteins. The rate of disappearance of one reduced protein should mirror the formation of the reduced form of the other. The location of the ruthenium complex is critically important if the measured rate constants are to be representative of the rate constants that would be obtained if the reaction could be measured without the ruthenium present. Ideally, the ruthenium complex should be placed far from the surface domains involved in the interaction of the two proteins under investigation. In the case shown above, the ruthenium complex was placed on Cys-65, which, according to modeling studies, is outside the binding domain occupied by cytochrome c. Figure 4
Figure 4. Model of electrostatically stabilized complex formed between cytochrome c (left side) and cytochrome b5 labeled with a ruthenium(II) bipyridine complex. The polypeptide backbone is represented by the ribbon. Geometry based on the calculated structure reported by Salamme (27).
shows the placement of the ruthenium complex and the binding interaction between cytochromes b5 and c. Identical rate constants were also obtained when the ruthenium complex was linked to Cys-73, which is even further removed from the binding domain. Several other experimental measurements (22) have clearly shown that cytochrome b5 labeled with ruthenium complexes at Cys-65 very closely resembles the unlabeled protein in nearly all respects. Placement of the ruthenium complex is also critical to efficient production of reduced cytochrome b 5. The ruthenium label must be sufficiently close to the iron center to undergo electron transfer with a high yield. Otherwise, the concentration of reduced protein formed by laser excitation will be too small to detect. Thus the best location for the ruthenium complex is a compromise between very close to the iron center and very far away from the iron center. Striking the best compromise, however, is not always absolutely necessary, since valuable information can be obtained about binding geometry in those cases where the ruthenium complex interferes with protein–protein interactions. Formation of the Protein–Protein Complexes Until now we have focused only on electron transfer. In reality, the overall electron-transfer reaction between two proteins also involves formation of a protein– protein complex followed by its dissociation after electron transfer. The fact that these reactions involve more than simply electron transfer is evident from the dramatic effects caused by changing the ionic strength of the reaction. In the case of cytochromes c and b5 , transient absorbance changes at 1 mM ionic strength show fast and slow components. The fast component has a rate constant of 400,000 s{1. Addition of 300 mM salt reduces the reaction to a single slow phase with a rate constant of 500 s {1. Many of the intricacies of the reaction can be understood in terms of association, dissociation, and changes in the concentrations of the protein–protein complex. Thus, by examining the reaction over a wide variety of conditions, information about these aspects of the reaction can be obtained. In the reaction between cytochromes c and b5 described above, the ionic strength
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Symposium: Applications of Inorganic Photochemistry was made very low to ensure that essentially all the proteins in solution were in the form of a 1:1 complex. Under these conditions the observed rate of reaction is actually a measure of the rate of electron transfer in the protein complex. The slow phase appears to be due to a small fraction of protein that is not bound at a reactive site. If the ionic strength is increased to 100 mM or higher, only a small fraction of the proteins are associated in protein–protein complexes and the overall reaction is limited by formation of the complex rather than by electron transfer. If a 1:1 mixture of cytochrome c and labeled cytochrome b5 is reacted in the presence of excess unlabeled cytochrome b5, the reaction will become limited by dissociation of the protein–protein complex. In this case, reaction can only occur with the labeled cytochrome b5. Only a small fraction of this will be able to bind to cytochrome c when in competition with the excess unlabeled cytochrome b5 in solution. Therefore, reaction is limited by the rate at which reduced labeled cytochrome b5 can find free cytochrome c, which is readily related to the rate of dissociation of the protein complex. Studies of the kinetics of the reactions over a range of ionic strengths provide a detailed picture of the overall electron-transfer reaction that is readily related to physiological conditions. Further information concerning specific interactions can be obtained by combining the kinetic measurements with genetic engineering. Specifically, key residues thought to be important in protein– protein binding or electronic coupling can be changed and the resulting impact on electron transfer measured. Common examples of this strategy involve substitution of neutral amino acids for charged amino acids assumed to be important in the electrostatic interactions of the protein–protein complex. Conclusion The use of ruthenium polypyridyl complexes to photoinitiate electron transfer represents a powerful means of obtaining kinetic measurements of intraprotein and interprotein electron-transfer reactions. The technique provides for very fast measurements (nanoseconds to seconds) with minimum perturbation of the proteins. It has been successfully applied to the reactions of cytochrome c with cytochrome c oxidase (23), cytochrome c peroxidase (24), cytochrome b5 (22), cytochrome c1 (25) and plastocyanin (26).
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Note 1. Abbreviations: bpy = 2,2′-bipyridine; bpym = bipyrimidine; bpdz = bipyridazine; bpyz = bipyrazine; NPG = N-phenylglycine, Cys = cysteine; His = histidine.
Literature Cited 1. Moore, G. R.; Pettigrew, G. W. Cytochrome c. Evolutionary Structure and Physiochemical Aspects; Springer: Heidelberg, 1990. 2. Isied, S. S.; Kuehn, C.; Worosila, G. J. Am. Chem. Soc. 1984, 106, 1722–1726. 3. Tollin, G.; Hazzard, J. T. Arch. Biochem. Biophys. 1991, 287, 1–7. 4. Peterson-Kennedy, S. E.; McGourty, J. L.; Ho, P. S.; Sutoris, C. J.; Liang, N.; Zemel, H.; Margoliash, E.; Hoffmann, B. M. Coord. Chem. Rev. 1985, 64, 125–133. 5. Nocera, D. G.; Winkler, J. R.; Yocum, K. M.; Bordignon, E.; Gray, H. B. J. Am. Chem. Soc. 1984, 106, 5145–5150. 6. Isied, S. S.; Worosila, G.; Atherton, S. J. J. Am. Chem. Soc. 1982, 104, 7659–7661. 7. Beratan, D. N.; Betts, J. N.; Onuchic, J. N. Science 1991, 252, 1285. 8. Regan, J. J.; Risser, S. M.; Beratan, D. N. J. Phys. Chem. 1993, 97, 13083. 9. Kalyanasundaram, K. Photochemistry of Polypyridine and Porphyrin Complexes; Academic: New York, 1992. 10. Watts, R. J. J. Chem. Educ. 1983, 60, 824–828. 11. Sutin, N.; Creutz, C. J. Chem. Educ. 1983, 60, 809–813. 12. Durham, B.; Pan, L. P.; Hahm, S.; Long, J.; Millett, F. In Electron Transfer in Biology and the Solid State; Johnson, M. K.; King, R. B.; Kurtz, D. M.; Kutal, C.; Norton, M. L.; Scott, R. B., Eds.; Advances in Chemistry 226: American Chemical Society: Washington, DC, 1990; pp 181–193. 13. Winkler, J. R.; Gray, H. B. Chem. Rev. 1992, 92, 369–379. 14. Willie, A.; Stayton, P.; Sligar, S. G.; Durham, B.; Millett, F. Biochemistry 1992, 31, 7237–7241. 15. Geren, L.; Hahm, S.; Durham, B.; Millett, F. Biochemistry 1991, 30, 9450–9457. 16. Durham, B.; Pan, L. P.; Hall, J.; Millett, F. Biochemistry 1989, 28, 8659–8665. 17. Scott, J. R.; Willie, A.; McLean, M.; Stayton, P.; Sligar, S. G.; Durham, B.; Millett, F. J. Am. Chem. Soc. 1993, 115, 6820–6824. 18. Marcus, R. A. J. Phys. Chem. 1956, 24, 966–978. 19. Beratan, D. N.; Betts, J. N.; Onuchic, J. N. J. Phys. Chem. 1992, 96, 2852–2855. 20. Siddarth, P.; Marcus, R. A. J. Phys. Chem. 1990, 94, 2985–2989. 21. Kuki, A. In Structure and Bonding; Palmer, G. A., Ed.; Springer: Berlin, 1991; pp 49–83. 22. Durham, B.; Fairris, J. L.; McLean, M.; Millett, F.; Scott, J. R.; Sligar, S. G.; Willie, A. J. Bioenerg. Biomemb. 1995, 27, 331–339. 23. Pan, L. P.; Hibdon, S.; Liu, R.; Durham, B.; Millett, F. Biochemistry 1993, 32, 8492–8498. 24. Millett, F.; Miller, M.; Geren, L.; Durham, B. J. Bioenerg. Biomemb. 1995, 27, 341–351. 25. Heacock, D. H.; Liu, R. C.; Yu, C. A.; Yu, L.; Durham, B.; Millett, F. J. Biol. Chem. 1993, 268, 27171–27175. 26. Pan, L. P.; Frame, M.; Durham, B.; Davis, D. J.; Millett, F. Biochemistry 1990, 29, 3231–3296. 27. Salemme, F. R. J. Mol. Biol. 1976, 102, 563–566.
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