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Recognition and Modulation of Cytochrome c’s Redox Properties using an Amphiphilic Homopolymer Britto S. Sandanaraj, Halil Bayraktar, Kothandam Krishnamoorthy, Michael J. Knapp,* and S. Thayumanavan* Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003 ReceiVed October 18, 2006. In Final Form: January 10, 2007 An amphiphilic homopolymer scaffold has been used to bind to the protein, cytochrome c. This interaction is analyzed using cyclic voltammetry, native gel electrophoresis, UV-visible absorption, and circular dichroism spectroscopy. The polymer binds to cytochrome c with micromolar affinity and the association of polymer with cytochrome c leads to a structural change of the protein. This conformational change exposes the heme unit of the protein, which affords an opportunity to reversibly modulate its electron-transfer properties. We have also shown that the electrostatic binding of polymer to cytochrome c can be used to disrupt its interaction with its natural partner, cytochrome c peroxidase.
Introduction Complementary electrostatics is a key feature in many biomolecular interactions, such as protein surface recognition.1,2 The electrostatic aspects of these interactions contribute substantially to complex stabilization.3 Although designing ligands for a concave site of a protein is relatively easily conceived, design of molecules to recognize the solvent-exposed surface area of proteins is more challenging due to their large surface area (>600 Å2).4 Earlier reports in this area focused on the development of aspartate-rich cyclopeptides on calixarene scaffolds or glutamate-rich peptides on porphyrins for surface recognition of cationic proteins.5 A general strategy to increase the binding affinity of a receptor for its target protein is to attach multiple copies of a single weak binding motif on the receptor.6 For example, a new way to develop high affinity inhibitor for cholera toxin was demonstrated by attaching five copies of an R-D-galactoside to a pentacyclen core unit.7 Inhibition of human leukocyte elastase by linear anionic oligomers works on a similar principle.8 More recently, nonspecific recognition of a cationic * To whom correspondence should be addressed. E-mail: thai@ chem.umass.edu. (1) (a) Berg, T. Angew. Chem., Int. Ed. 2003, 42, 2462-2481. (b) Arkin, M. R.; Wells, J. A. Nat. ReV. Drug DiscoVery 2004, 3, 301-307. (c) Guo, Z.; Zhou, D.; Schultz, P. G. Science 2000, 288, 2042-2045. (d) Ghosh, I.; Chiemielewski, J. Chem. Biol. 1999, 5, 439-445. (e) Schramm, H. J.; de Rosny, E.; ReboudRevaux, M.; Butttner, J.; Dick, A.; Schramm, W. Biol. Chem. 1999, 380, 593596. (f) Wakeling, A. E.; Guy, S. P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Barker, A. J.; Gibson, K. H. Cancer Res. 2002, 62, 5749-5754. (2) (a) DeLano, L. W.; Ultsch, H. M.; De Vos, M. A.; Wells, J. A. Science 2000, 278, 1279-1283. (b) Bogan, A. A.; Thorn, K. S. J. Mol. Biol. 1998, 280, 1-9. (c) Capasso, C.; Rizzi, M.; Menegatti, E.; Ascenzi, P.; Bolognesi, M. J. Mol. Recognit. 1997, 10, 26-35. (3) Sheinerman, F. B.; Norel, R.; Honig, B. Curr. Opin. Struct. Biol. 2000, 10, 153-159. (4) (a) Stites, W. E. Chem. ReV. 1997, 97, 1233-1250. (b) Lijnzaad, P.; Argos, P. Proteins 1997, 28, 333-343. (c) Golumbfskie, A. J.; Pande, V. S.; Chakraborty, A. K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11707-11712. (5) (a) Hamuro, Y.; Calama, M. V.; Park, H. S.; Hamilton, A. D. Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683. (b) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999, 121, 2479-2493. (c) Park, H. S.; Lin, Q.; Hamilton, A. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5105-5109. (d) Jain, R. K.; Hamilton, A. D. Org. Lett. 2000, 2, 1721-1723. (6) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem, Int. Ed. 1998, 37, 2754-2794. (b) Choi, S.-K. Synthetic MultiValent Molecules; WileyVCH: New York, 2004. (7) Merritt, E. A.; Zhang, Z.; Pickens, J. C.; Ahn, M.; Hol, W. G. J.; Fan, E. J. Am. Chem. Soc. 2002, 124, 8818-8824.
protein, R-chymotrypsin, using surface functionalized anionic gold nanoparticles was reported.9 Similarly, inhibition of hemagglutination was achieved by recognition using dendrimers containing multiple copies of ligands.10 Nanoscale objects containing multiple copies of complementary ligands are interesting for surface recognition of proteins,11 since these objects are commensurate in size. One of our groups have recently reported that functionalized gold nanoparticles could disrupt the interaction between cytochrome c (Cc) and cytochrome c peroxidase (CcP).12 Polymer-based nanoparticles are interesting for recognizing protein surfaces, since polymers are softer in nature and are therefore likely to be able to better adapt to the surface of the proteins. One of our groups has recently reported on an amphiphilic homopolymer that forms assemblies of about 40 nm in size in aqueous solutions.13 In those studies, we showed that the hydrophilic carboxylate groups of the amphiphilic polymer are buried in the interior of an inverted micelle-type assembly in apolar organic solvents, whereas they are presented on the exterior of a micelle-type assembly in aqueous solution. The structure and morphology of these assemblies were thoroughly characterized using transmission electron microscopy.13a The nanocontainer properties of these assemblies were studied by encapsulating both hydrophilic and hydrophobic dyes in both organic and aqueous solvents, respectively. 13b,c (8) Regan, J.; McGarry, D.; Bruno, J.; Green, D.; Newman, J.; Hsu, C.-Y.; Kline, J.; Barton, J.; Travis, J.; Choi, Y. M.; Volz, F.; Pauls, H.; Harrison, R.; Zilberstein, A.; Ben-Sasson, S. A.; Chang, M. J. Med. Chem. 1997, 40, 34083422. (9) (a) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023. (b) Fischer, N. O.; Verma, A.; Goodman, C. M.; Simard, J. M.; Rotello, V. M. J. Am. Chem. Soc. 2003, 125, 13387-13391. (c) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 739-743. (10) (a) Woller, E. K.; Walter, E. D.; Morgan, J. R.; Singel, D. J.; Cloninger, M. J. J. Am. Chem. Soc. 2003, 125, 8820-8826. (b) Schlick, K. H.; Udelhoven, R. A.; Strohmeyer, G. A.; Cloninger, M. J. Mol. Pharm. 2005, 2, 295-301. (c) Wolfenden, M. L.; Cloninger, M. J. J. Am. Chem. Soc. 2005, 127, 12168-12169. (d) Roy, R.; Zanini, D.; Meunier, S. J.; Romanowska, A. Chem. Commun. 1993, 1869-1872. (11) Strong, L. E.; Kiessling, L. L. J. Am. Chem. Soc. 1998, 121, 6193-6196. (12) Bayraktar, H.; Ghosh, P. S.; Rotello, V. M.; Knapp, M. J. Chem. Commun. 2006, 1390-1392. (13) (a) Basu, S.; Vutukuri, D. R.; Shyamroy, S.; Sandanaraj, S. B.; Thayumanavan, S. J. Am. Chem. Soc. 2004, 126, 9890-9891. (b) Basu, S.; Vutukuri, D. R.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127, 16794-16795. (c) Savariar, E. N.; Aathimanikandan, S. V.; Thayumanavan, S. J. Am. Chem. Soc. 2006, 128, 16224-16230.
10.1021/la063063p CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007
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Chart 1. Structure of Polymer 1 (DP: Degree of Polymerization, PDI: Polydispersity Index)
In this paper, we report on our studies on the ability of the amphiphilic homopolymer to interrupt the interaction between Cc and CcP. The Cc:CcP complex is an excellent model system to probe the ability of polymers to interrupt protein-protein interactions, since these proteins and the complex are well characterized both from structural and thermodynamic viewpoints.14 A tangible goal of this work is to modulate electrontransfer characteristics of proteins through interactions with an artificial scaffold. Although electron-transfer modulation has been achieved by specific mutations on proteins,15 accomplishing this with a noncovalent composite material containing polymers and proteins could find use in a variety of applications. An example of such applications is hydrogen production.16 It is important however that we understand the most basic effects that these amphiphilic polymers have on the properties of the protein upon binding, and this is the focus of the work presented here. We show in this paper that: (i) amphiphilic homopolymer 1 binds to Cc and this binding event disrupts the Cc:CcP interaction; (ii) electrostatic interaction is the major driving force for the formation of Cc-polymer 1 complex; (iii) binding of polymer 1 results in the exposure of heme group of Cc accompanied by change in global conformation of Cc; (iv) the binding event and therefore the conformational change is reversible; (v) the polymer-protein interaction changes the electron-transfer characteristics of Cc.
Results and Discussion Cc is a cationic protein with a pI of 10.3; the heme edge of Cc is surrounded by an array of lysine and arginine residues.17 CcP is an anionic protein with a pI of 5.25, with 45 glutamate and aspartate residues. The interaction between Cc and CcP is primarily driven by electrostatics, and the binding affinity understandably decreases with increasing ionic strength.18 Since polymer 1 (Chart 1) is negatively charged at 10 mM sodium phosphate buffer pH 6.0, we anticipated that it would bind to the cationic protein, Cc.19 The interaction of Cc with polymer 1 was characterized by native gel electrophoresis (Figure 1). The positive control, 28 µM Cc, completely moved toward cathode, and there was no protein left in the well (lane 1). This is understandable, considering the cationic nature of Cc. Similarly, 5 µM of the anionic polymer 1 completely moved toward the anode as anticipated based upon charge (lane 8). The migration of Cc and polymer 1 toward cathodic and anodic sides of the gel show the charge complementarity of these two macromolecules. At a ratio (14) (a) Pelletier, H.; Kraut, J. Science 1992, 258, 1748-1755. (b) McLendon, G. L.; Zhang, Q.; Wallin, S. A.; Miller, R. M.; Billstone, V.; Spears, K. G.; Hoffman, B. M. J. Am. Chem. Soc. 1993, 115, 3665-3669. (15) (a) Raphael, A. L.; Gray, H. B. Proteins 1989, 6, 338-340. (b) Raphael, A. L.; Gray, H. B. J. Am. Chem. Soc. 1991, 113, 1038-1040. (c) Bren, K. L.; Gray, H. B. J. Am. Chem. Soc. 1993, 115, 10382-10383. (d) Fumo, G.; Spitzer, J. S.; Fetrow, J. S. Gene 1995, 164, 33-39. (e) Wallace, C. J.; Clark-Lewis, I. J. Biol. Chem. 1992, 267, 3852-3861. (16) Elgren, T. E.; Zadvorny, O. A.; Brecht, E.; Douglas, T.; Zorin, N. A.; Maroney, M. J.; Peters, J. W. Nano Lett. 2005, 5, 2085-2087. (17) Scott, R. A.; Mauk, A. G. Cytochrome c : A Multidisciplinary Approach; University Science Books: Sausalito, CA, 1996. (18) Kresheck, G. C.; Vitello, L. B.; Erman, J. E. Biochemistry 1995, 34, 8398-8405. (19) Liang, L.; Yao, P.; Gong, J.; Jiang, M. Langmuir 2004, 20, 3333-3338.
Figure 1. Native gel electrophoresis of Fe(III) Cc (28 µM) and polymer 1 (0-28 µM) at 1% agarose, 10 mM phosphate, pH 6.0. The ratios corresponding to each lane are listed at the top of the figure.
of 1:8 and 1:6 (polymer 1:Cc) in lanes 2 and 3, respectively, the intensity of protein band in the cathodic side decreased and two new bands corresponding to the neutral and negative complex appeared in the middle and anodic side. The above results imply that even at 1:8 ratio of polymer 1:Cc, most of the protein was bound to the polymer. At a ratio of 1:5 (lane 4), there was no more uncomplexed protein left in the cathodic side. At ratios of 1:4, 1:2, and 1:1 in lanes 5, 6, and 7, respectively, all of the polymer-protein complex migrated toward the anode. The anionic complexes arise from the uncompensated negative charges of polymer due to low ratio of Cc present. We took these results to suggest that the binding ratio of polymer to Cc is approximately 1:8. We realize that this is only a semiquantitative estimate of the binding ratio. However, this is a useful supporting evidence for a more quantitative analysis of the binding ratios below. Spectroscopic Studies. In order to further understand the nature of interaction of Cc with polymer 1, we carried out systematic spectroscopic studies to investigate the complexation of polymer 1 with Cc. First, the interaction of Cc with polymer 1 was probed using linear absorption spectroscopy (Figure 2). Horse heart Cc is a well-characterized small protein, which contains 24 basic residues and is a positively charged protein at neutral pH.17 The absorbance of the heme group in the Soret region reports on the identity of the ligands and the spin state of the iron. Heme iron in Cc exists in a low-spin state with two axial ligands provided from His18 and Met80.20 A Soret band with a maximum intensity at 410 nm (λmax) is typical of the native state of Cc,21 indicative of the presence of these ligands. Native Cc also exhibits a peak at 695 nm indicative of Met80 coordination. In microperoxidases derived from proteolytic digestion of Cc and in the acid denatured form of Cc, Met80 is replaced with a water ligand17 which leads to a blue shift of the Soret band to 402 nm and loss of the peak at 695 nm. Binding of polymer 1 to Cc causes spectral shifts similar to those seen in acid denatured Cc, indicating that the heme of polymer-bound Cc has lost the Met80 ligand and has likely acquired a water molecule as the ligand. Circular dichroism was also used to investigate the structural transformations of Cc upon interaction with polymer 1. The farUV CD spectrum of Cc shows a characteristic peak at 222 nm, due to the high R-helix content of the protein23a (Figure 3A). Addition of polymer 1 to Cc resulted in a presumably decreased helicity of Cc. Similarly, the near-UV CD region (250-330 nm) (20) Imdiani, C.; Sanctis, G. C.; Neri, F.; Santos, H.; Smulevich, G.; Coleta, M. Biochemistry 2000, 39, 8234-8242. (21) (a) Babul, J.; Stellwagen, E. Biochemistry 1972, 11, 1195-1200. (b) Muga, A.; Mantsch, H. H.; Surewicz, W. K. Biochemistry 1991, 30, 7219-7224. (c) Elove, G. A.; Bhuyan, A. K.; Roder, H. Biochemistry 1994, 33, 6925-6935.
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Figure 2. Absorption spectra of native Fe(III) Cc (10 or 50 µM) in the presence of polymer 1 (1-30 µM), 10 mM phosphate, pH 6.0. (A) Soret region. (B) methionine-Fe absorption region.
provides information on the packing of aromatic side chains (Figure 3B). The near-UV CD spectrum of Cc displays distinct minimum at 288 nm, which has been assigned to the tertiary structural packing of Trp59.22 Binding of polymer 1 to Cc diminishes the intensity of this band, which indicates the loss of a tightly packed core in Cc upon interaction with polymer 1. The CD spectrum in the Soret region can provide further insight into the integrity of the heme unit (Figure 3C). The optical signature in this region is due to the coupling of heme π-π* transition dipole moments with those of the nearby aromatic residues in the protein.23 The spectrum for Cc in its native conformation exhibits a strong negative band at about 418 nm and a positive band at 405 nm due to the Soret-Cotton effect.23 Addition of polymer 1 to Cc resulted in a gradual loss of negative features at 418 nm, along with an increase in intensity of the peak at 405 nm due to the removal of the heme from the protein core. The CD results are consistent with the UV-visible absorption spectroscopic results in that both indicate that the interaction of polymer 1 with Cc perturbs the heme environment.24 Reactivity of Cc. The changes in the spectroscopic properties of Cc upon polymer binding indicate that the heme is solvent exposed and suggest that water has replaced the Met80 ligand, conditions conducive for peroxidase activity in Cc. To investigate this possibility, we tested the reactivity of Fe(II) Cc toward hydrogen peroxide in the presence of polymer 1 (Figure 4). As the heme group of native Cc is deeply buried inside the protein, (22) Davies, A. M.; Guillemette, J. G.; Smith, M.; Greenwood, C.; Thurgood, A. G. P.; Mauk, A. G.; Moore, G. R. Biochemistry 1993, 32, 5431-5435. (23) (a) Myer, Y. P. Biochemistry 1968, 7, 765-776. (b) Sanghera, N.; Pinheiro, T. J. Protein Sci. 2000, 9, 1194-1202. (24) Pinheiro, T. J. T.; Elove, G. A.; Watts, A.; Roder, H. Biochemistry 1997, 36, 13122-13132.
Figure 3. CD spectra of native Fe(III) Cc (50 µM) in the presence of polymer 1 (0-50 µM), 10 mM phosphate, pH 6.0. (A) Far-UV CD region. (B) Near-UV CD region. (C) Soret region.
and Met80 prevents the binding of H2O2 to iron, the rate of reaction with peroxide is very low.25 When the Met80 ligand is replaced by the labile H2O and the heme group is solvent exposed, the rate of reaction with H2O2 increases significantly as seen with unfolded and chemically modified Cc.25 Fe(II) Cc (5.4 µM) was preincubated with polymer 1 (0-1.2 µM) and then exposed to H2O2 (100 µM). The observed rate of oxidation of Fe(II) Cc (kobs) was obtained from the single-exponential decrease in absorbance at 550 nm. In the absence of polymer 1, Fe(II) Cc is slowly oxidized by H2O2 with kobs ) 0.00184 s-1 (Figure 4A). Binding Fe(II) Cc to polymer 1 increases the rate of oxidation to a maximal value of kobs ) 0.0137 s-1. These peroxidation rates correspond to second-order rate constants of ca. 100 M-1 s-1, comparable to other peroxidase reactions with Cc.26 The above trend in the reaction rate coincides with the CD results, suggesting that exposure of the heme group of Cc to bulk solvent could be achieved by increasing the concentration of polymer 1. (25) Turrens, J. F.; McCord, J. M. FEBS Lett. 1988, 227, 43-46. (26) (a) Belikova, N. A.; Vladimirov, Y. A.; Ospivo, A. N.; Kapralov, A. A.; Tyurin, V. A.; Potapovich, M. V.; Basova, L. V.; Peterson, J.; Kurnikov, I. V.; Kagan, V. E. Biochemistry 2006, 45, 4998-5009. (b) Prasad, S.; Maiti, N. C.; Mazumdar, S.; Mitra, S. Biochim. Biophys. Acta 2002, 1596, 63-75. (c) Chen, Y.-R.; Deterding, L. J.; Sturgeon, B. D.; Tomer, K. B.; Mason, R. P. J. Biol. Chem. 2002, 277, 29781-29791. (d) Diederix, R. E.; Ubbink, M.; Canters, G. W. Biochemistry 2002, 41, 13067-13077.
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Figure 5. (A) Kinetics of reduction of Fe (III) Cc (14 µM) by ascorbic acid (2.8 mM) in presence of polymer 1 (0-5.0 µM), 10 mM phosphate, pH 6.0 (B) Concentration dependent reduction of Fe(III) Cc by sodium ascorbate in the presence of different concentrations of polymer 1.
Figure 4. (A) Kinetics of oxidation of Fe(II) Cc (5.4 µM) by hydrogen peroxide (100 µM) in the presence of polymer 1 (0-1.2 µM), 10 mM phosphate, pH 6.0 (B) Concentration dependent oxidation of Fe(II) Cc by hydrogen peroxide in the presence of different concentrations of polymer 1.
Modulation of protein function using a synthetic macromolecular scaffold is an exciting area of research. We have shown in our previous studies that substrate selectivity of an enzyme, R-chymotrypsin, can be achieved by using amphiphilic homopolymer.27 In that case, we observed that ChT exhibited significant inhibition toward negatively charged substrates, compared to the neutral and positively charged ones. The observed selectivity was attributed to the anionic nature of the “chymotrypsin-polymer” complex. Assay studies of Fe(II) Cc with hydrogen peroxide clearly demonstrated that this neutral reagent has better access to the heme group of Cc in the presence of polymer 1. This observation was attributed to the exposure of the heme group of Cc upon interaction with polymer 1. It is interesting to ask whether solvent exposure of the heme or charge-charge repulsion would dominate the reactivity of Cc toward a negatively charged substrate. Thus, we have carried out kinetic studies of reduction of Fe(III) Cc to Fe(II) Cc using anionic ascorbic acid in the presence of polymer 1.28 Fe(III) Cc (14 µM) was preincubated with polymer 1 (0-5 µM), and then, ascorbate (2.8 mM) was added to initiate reaction. The reduction of Fe(III) Cc was observed as a single-exponential increase at 550 nm. In the absence of polymer 1, Fe(III) Cc is rapidly reduced by ascorbic acid with kobs ) 7 × 10-2 s-1 (Figure (27) Sandanaraj, B. S.; Vutukuri, D. R.; Simard, J. M.; Klaikherd, A.; Hong, R.; Rotello, V. M.; Thayumanvan, S. J. Am. Chem. Soc. 2005, 127, 1069310698. (28) (a) Mochan, E.; Nicholls, P. Biochem. Biophys. Acta 1972, 267, 309319. (b) Petersen, L. C.; Cox, R. P. Biochem. J. 1980, 192, 687-693. (c) Lin, Q.; Park, H. S.; Hamuro, Y.; Lee, C. S.; Hamilton. A. D. Biopolymers 1998, 47, 285-297.
5A), which is consistent with previously observed values.29a Addition of polymer 1 to Fe(III) Cc decreased both the rate of reduction and the total amplitude. These observations indicate that the polymer bound form of Fe(III) Cc is unreactive toward ascorbate. A binding model in which free Cc binds to n noninteracting sites of polymer 1 characterized by a dissociation constant KD was used to relate the ascorbate reactivity to polymer binding.29b The binding constant and the ratio of the polymer 1:Fe(III) Cc were obtained by plotting the amplitude of reduction of Fe(III) Cc against polymer concentrations (Figure 5B). The dissociation constant was determined to be KD ) 6 ( 3 × 10-7 M with a binding ratio of n ) 7.5 ( 0.8. The binding ratio estimated here is consistent with the results obtained from gel electrophoresis study above. We have been assuming that the nature of the interaction between the protein and the polymer is based on electrostatics. If this were indeed the case, then we should be able to reverse the binding by increasing the ionic strength of the solution.30 In order to demonstrate that the complexation between Cc and polymer is reversible, we carried out UV-visible spectroscopic studies of a Cc-polymer mixture at higher ionic strength of the medium. In a 1.0 M NaCl solution, the Cc-polymer mixture restores the heme signature of native Cc, which suggests that Cc dissociates itself from the polymer scaffold due to screening of electrostatic attraction between Cc and polymer at high ionic strength (Figure 6). However, the molar extinction coefficient of heme group is higher than that of native Cc, which is attributed to the shift of baseline due to the presence of polymer. These studies show that the interaction between the polymer and Cc is based on electrostatics and that the binding is reversible. Probing Protein-Protein Interactions through Cyclic Voltammetry. Cyclic voltammetry is a useful probe to report (29) (a) Cannon, J. B.; Erman, J. E. Biochem. Biophys. Res. Commun. 1978, 84, 254-260. (b) Detailed procedures for the estimation of binding constants and ratios from assay studies are outlined in the Supporting Information. (30) Goldkorn, T.; Schejter, A. J. Biol. Chem. 1979, 254, 12562-12566.
Redox Modulation of Cc with an Amphiphilic Polymer
Figure 6. Effect of ionic strength on Cc (5.0 µM) binding to polymer (0-4 µM), 10 mM phosphate, pH 6.0.
Figure 7. Cyclic voltammogram of Cc (10 or 50 µM) in the absence (A) and presence (B) of polymer 1 (10 µM), 10 mM phosphate, pH 6.0. Electrode area ) 0.31 cm2, Scan rate ) 1-10 mV s-1.
on the microenvironment of the heme moiety within Cc.31 The redox potential of Cc depends on several factors, including the polarity of the heme environment,32 the identity of ligands coordinated to Fe, and the accessibility of heme to the solvent.33 As the spectroscopic and reactivity studies indicate that the heme (31) Moore, G. R.; Pettigrew, G. W. Cytochrome c: EVolution, Structural and Physiochemical Aspects; Springer: New York, 1990; pp 115-159. (32) Kassner, R. J. J. Am. Chem. Soc. 1973, 95, 2674-2677. (33) Stellwagen, E. Nature 1978, 275, 73-74.
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is solvent exposed when Cc binds polymer 1, we anticipated corresponding changes in the redox behavior of Cc. The redox behavior of Cc was studied by cycling the potential between 300 and -100 mV vs Ag/AgCl. The reduction peak (Ec) was observed at -7 mV and the separation between Ec and the reoxidation peak was 141 mV, from which the heterogeneous electron-transfer rate constant (k0) was calculated to be 2.3 × 10-4 cm/s (Table 1). The peak observed at -7 mV arises from the reduction of His/Met coordinated Fe(III) heme34 and the slow heterogeneous rate constant for Cc indicates that the Cc heme is deeply buried inside the protein. In order to define the effect of polymer 1 on redox kinetics of Cc, the heterogeneous electron transfer of Fe(III) Cc in the presence of 10 µM of polymer 1 was measured. Addition of polymer 1 to Cc decreased the peak separation (∆Ep ) 61 mV) and the rate constant for electron transfer (2.3 × 10-2 cm/s). The increase in electron-transfer kinetics and decrease in ∆Ep for Cc in the presence of polymer 1 is due to the fact that heme moiety of Cc is more solvent exposed upon binding. It has been previously shown that the current due to reduction of His/ Met coordinated heme decreases with the concurrent appearance of His/His reduction peak at higher reduction potentials.34 By the addition of polymer 1, we observed that the peak current due to His/Met coordinated heme diminished but no peak due to His/His was observed. It is likely that oxygen interferes with the observation of the latter redox process; however, removal of oxygen from Cc solution was not possible in the presence of the amphiphilic polymer 1,34 due to frothing. The surfactant properties of amphiphilic polymer 1 simply precluded the evacuation of oxygen from the Cc-polymer 1 solution. Results from these electrochemical studies of the Cc-polymer 1 complex encouraged us to test whether the disruption of CcCcP interactions by polymer 1 can be probed by electrochemistry (Table 1). In order to do that, we first studied the Cc-CcP complex. Fe(III) Cc (10 µM) was incubated with 10 µM CcP reconstituted with Zn-PPIX (Zn-CcP, 10 µM) for sufficient time at room temperature, and then a cyclic voltammogram was recorded. Zn-CcP was used in the place of Fe-CcP to eliminate potential interference from redox chemistry of CcP. Note that the association constant of FeCc-ZnCcP pair is same as that of FeCc-FeCcP pair for a given condition35 as the association of Cc with CcP is driven by charged residues present on the exterior surface of two proteins and not by the metal ion in the heme group, which is present in the interior of the protein. As CcP binds near the solvent-exposed face of the heme in Cc, such binding is anticipated to reduce the ability of the heme moiety to interact with the electrode surface, thereby decreasing the rate of electron transfer. The electron-transfer rate (k0) of Cc in the presence of Zn-CcP was smaller (6.4 × 10-5 cm/s) than in the absence of Zn-CcP. Note that the diffusion coefficient of the Cc:CcP complex (3.0 × 10-7 cm2/s) is an order of magnitude smaller than that of Cc (6.6 × 10-6 cm2/s) which is also consistent with the formation of the Cc:CcP complex, a species with a higher molar mass. We anticipated that polymer 1 would be able to outcompete CcP for Cc, based upon gel electrophoresis, CD, and assay studies. Polymer 1 was added to preformed Cc:CcP complex, and the electron-transfer reactivity of Cc was measured. (34) (a) Bixler, J.; Bakker, G.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6938-6939. (b) Ferri, T.; Poscia, A.; Ascoli, F.; Santucci, R. Biochim. Biophys. Acta 1996, 1298, 102-108. (c) Zhu, Y.; Dong, S. Bioelectrochem. Bioenerg. 1996, 41, 107-113. (d) Pineda, T.; Sevilla, J. M.; Roman, A. J.; Blazquez, M. Biochim. Biophys. Acta 1997, 1343, 227-234. (e) Fedurco, M.; Augustynski, J.; Indiani, C.; Smulevich, G.; Antalik, M.; Bano, M.; Sedlak, E.; Glascock, M. C.; Dawson, J. H. Biochim. Biophys. Acta 2004, 1703, 31-41. (e) Fedurco, M.; Augustynski, J.; Indiani, C.; Smulevich, G.; Antalik, M.; Bano, M.; Sedlak, E.; Glascock, M. C.; Dawson, J. H. J. Am. Chem. Soc. 2005, 127, 7638-7646. (35) Wei, Y.; McLendon, G. L.; Hamilton, A. D.; Case, M. A.; Purring, C. B.; Lin, Q.; Park, H. S.; Lee, C.; Yu. T. Chem. Commun. 2001, 1580.
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Table 1. Electrochemical Studies of Protein-Polymer Interactionsa
Cc Cc-polymer 1 Cc-CcP Cc-CcP-polymer 1
n
∆Ep (mV)
Ec vs Ag/AgCl (mV)
E1/2 (mV)
Ea (mV)
D0 (cm2/s)
k0 (cm/s)
1 1 1 1
141 61 181 61
-8 -2 -60 -2
63 29 31 29
133 59 121 59
6.6 × 10-6 2.3 × 10-7 3.0 × 10-7 4.7 × 10-7
2.3 × 10-4 2.3 × 10-2 6.4 × 10-5 1.5 × 10-2
a n ) number of electrons; ∆E ) peak separation between anodic and cathodic processes; E ) anodic peak potential; E ) cathodic peak; E p a c 1/2 ) midpoint potential; D0 ) diffusion coefficient; k0 ) heterogeneous electron-transfer rate constant.
The k0 of Cc:CcP complex (6.4 × 10-5 cm/s) increased by nearly 3 orders of magnitude in the presence of polymer 1 (1.5 × 10-2 cm/s) which is very close to the value of the Cc:polymer 1 complex (2.25 × 10-2 cm/s). Note that the diffusion coefficient of the Cc:polymer 1 complex is also smaller than for Cc, consistent with the increased molar mass of the complex compared to the native protein. The observed results further confirm our model that polymer 1 has the ability to disrupt the interaction of Cc with CcP, by selectively binding to Cc.
Summary We have used an amphiphilic polymer scaffold to disrupt the interaction of Cc with its natural partner, CcP. This was achieved through a reversible, noncovalent, electrostatic binding of polymer 1 with Cc. Binding of polymer 1 to Cc alters the secondary structure of Cc, as evident from the absorption and CD spectroscopic studies. The binding event exposes the heme group of Cc to solvent, which results in an enhanced activity of Cc toward a neutral oxidizing agent, hydrogen peroxide. However, the Cc-polymer complex is less reactive to ascorbate than free Cc. This could either be due to the electrostatic repulsion between the polymer and ascorbate or the change in the redox potential of the heme. We were unable to test the latter due to oxygen interference. We have also quantified the effect of polymer 1 binding upon the heterogeneous electron-transfer rates for Cc using electrochemistry. Diffusion coefficients and the electrontransfer rate constants demonstrate that the polymer interrupts the Cc:CcP interaction. Using an artificial, polymeric scaffold to noncovalently and reversibly modulate the electron-transfer kinetics of metalloproteins offers new prospects for the use of polymer-protein composites in areas such as hydrogen production, which is a topic of current investigation in one of our laboratories. Experimental Section Materials. Recombinant yeast cytochrome c peroxidase MKT (CcP) was expressed and purified as described in a reported procedure.36 Preparation of zinc-substituted cytochrome c peroxidase was accomplished using Yonetani’s method.37 The concentrations of Zn-CcP were determined from a Soret extinction coefficient of 196 mM-1 cm-1.38 Horse heart cytochrome c and all other chemicals were purchased from commercial sources and used as received unless mentioned otherwise. Synthesis of polymer 1 was achieved using reported procedure.13 Reduction of Fe(III) Cc to Fe(II) Cc. Fe(III) Cc was dissolved in 10 mM phosphate buffer, pH 6.0. Ascorbic acid was dissolved in water, and the pH of the solution was adjusted to 6.0 by addition of dibasic sodium phosphate buffer. Cc (14 µM) was mixed with varied concentrations of polymer 1 (0.05-5 µM). The temperature was adjusted to 25 °C using a thermo controller. Finally, ascorbic acid was added to reach a final concentration of 2.8 mM. The reduction of Cc was monitored at 550 nm37 with a UV-visible spectrophotometer and the data was fitted to a first-order rate equation. (36) Goodin, D. B.; Davidson, M. G.; Roe, J. A.; Mauk, A. G.; Smith, M. Biochemistry 1991, 30, 4953-4962. (37) Yonetani, T. J. Biol. Chem. 1967, 242, 5008-5013. (38) Stemp, E. D. A.; Hoffman, B. M. Biochemistry 1993, 32, 10848-10865.
Oxidation of Fe(II) Cc to Fe(III) Cc. Fe(II) Cc was prepared by adding excess dithionite and purified on a G-75 column. The final Fe(II) Cc product was more than 95% reduced. The concentration of hydrogen peroxide stock solutions was determined from the absorbance at 240 nm using an extinction coefficient of 39.4 M-1 cm-1;39 5.4 µM of Cc was mixed with polymer 1 (0.1 to 1.2 µM), then, hydrogen peroxide was added (100 µM) to initiate oxidation. The time dependent change of absorbance at 550 nm was used to monitor the oxidation. UV-Visible Spectroscopic Studies. Absorbance spectra were obtained for samples containing 10 or 50 µM Fe(III) Cc in 10 mM sodium phosphate buffer pH 6.0 in the absence and presence of polymer 1, for polymer concentrations ranging from 5 to 50 µM. Spectra were recorded at room temperature on a Varian UV-visible spectrophotometer. For salt-based release, 4 µM of polymer 1 was mixed with 5 µM of Cc. Then the salt concentration was increased to 1 M by adding sodium chloride. Absorption spectra of the solution were then recorded. Circular Dichroism Studies. CD spectra of far-UV (200-250 nm), near-UV (250-350 nm), as well as the Soret region (350-450 nm) were recorded on a JASCO spectropolarimeter. Spectra were obtained for samples containing 50 µM Fe(III) Cc in 10 mM sodium phosphate buffer pH 6.0, in the absence and presence of polymer 1 (5-50 µM). CD spectra were measured using quartz cells of 1 mm path length. All spectra were background corrected. Three scans were taken for each sample with a bandwidth of 1.0 nm with a scanning rate of 100 nm/min. All experiments were performed at a constant temperature of 20 °C with a 5 min equilibration before the scans. Gel Electrophoresis Studies. For gel electrophoresis, agarose gels were prepared in 10 mM phosphate, pH 6.0 buffer at 1% final agarose concentration. Appropriately sized wells (40 µL) were formed by placing a comb in the center of the gel. A stock solution of 28 µM of Cc and 10 µM of polymer 1 in 10 mM sodium phosphate buffer (pH 6.0) was used to prepare 30 µL samples at varied Cc: polymer 1 ratios. After a 60-min incubation period at room temperature, 3 µL of 80% glycerol was added to ensure proper loading in the well (30 µL) and a constant voltage (100 V) was applied for 70 min for sufficient separation. Gels were placed in staining solution (0.5% Coomassie blue, 40% methanol, 10% acetic acid aqueous solution) for 1 h, followed by extensive destaining (40% methanol, 10% acetic acid aqueous solution) until protein bands were clear. Gels were scanned on a flatbed scanner after staining to visualize the bands. Cyclic Voltammetry. ITO coated glass was used as working and counter electrodes, and Ag/AgCl acted as the reference electrode. The working electrode area was fixed by cello tape that had a 2 mm diameter hole on it. The cyclic voltammograms were recorded immediately after inserting the reference electrode into the analyte solution (10 mM phosphate, pH 6.0). We found that Ar purging produced lather in experiments when polymer 1 was present. Therefore, Ar purging was avoided in all of the experiments. The potential cycling was started at 300 mV and allowed to proceed till -100 mV, and the cycle was completed by stopping the scan at 300 mV (scan rate 1-10 mV/s). The diffusion coefficient (D0) was calculated using Randle-Sevcik equation (eq 1).40 (39) Nelson, D. P.; Kiesow, L. A. Anal. Biochem. 1972, 49, 474-478. (40) Nicholson, R. S. Anal. Chem. 1965, 37, 1351-1355.
Redox Modulation of Cc with an Amphiphilic Polymer ip ) (2.69 × 10-5)n3/2AD01/2Cν1/2
Langmuir, Vol. 23, No. 7, 2007 3897 (1)
ip is the peak current, n is the number of electrons transferred, A is the area of the electrode, D0 is the diffusion coefficient, C is the concentration of the analyte, and ν is the scan rate. The peak current is measured from the cyclic votammogram and using the known electrode area, concentration, and scan rate, the diffusion coefficient was obtained. The anodic and cathodic peak separation (∆Ep) obtained from the CV was used to obtain a dimensionless parameter ψ. The k0 was calculated from ψ, scan rate, and diffusion coefficient as was reported by Nicholson.40
Acknowledgment. Support from NIGMS of the National Institutes of Health (GM-65255 to ST) is gratefully acknowledged. M.J.K. acknowledges University of Massachusetts and Grant # IRG 93-033 from the American Cancer Society. We thank Dr. D. B. Goodin (Scripps) for providing the plasmid encoding CcP. Supporting Information Available: Detailed procedures for the estimation of binding constants and ratios from assay studies. This material is available free of charge via the Internet at http://pubs.acs.org. LA063063P
