The Journal of
Physical Chemistry
0 Copyright 1995 by the American Chemical Society
VOLUME 99, NUMBER 15, APRIL 13, 1995
LETTERS Structural Changes and Internal Fields in Proteins: A Hole-Burning Stark Effect Study of Horseradish Peroxidase Jiirgen Gafert and Josef Friedrich" Physikalisches Institut und Bayreuther Institut f i r Makromolekiilforschung, Universitat Bayreuth, 0-95440 Bayreuth, Germany
Jane M. Vanderkooi Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 191 04
Judit Fidy Institute of Biophysics, Semmelweis Medical University, P.O. Box 263, H-I444 Budapest, Hungary Received: January 4, 1 9 9 9
W e compare the Stark spectra of photochemical holes bumt into a globular chromoprotein, namely mesoporphyrin-substituted horseradish peroxidase, with the respective behavior in a glass sample. From the distinct pattems between glass and protein as well as between various bum frequencies in the protein, we can clearly demonstrate that the chromophore in the protein is decoupled from the host glass and, hence, definitely probes the protein. Moreover, the electric fields in the protein pocket are quite different for the various tautomer states. These characteristic features are linked to allosteric-like behavior of proteins.
The extraordinary specificity of enzymatic reactions arises from the influence of the polypeptide chain in controlling the reactions of the prosthetic group. It has long been recognized that the protein may hold the prosthetic molecule in a particular torsional or twisted active form.' More recently, the role of protein dynamics in determining the reactivity of the prosthetic group has also been recognized, especially for heme proteins which bind small diatomic These two effects stress the importance that the positions of the neighboring atoms have in deciding reactivity and specificity. A third control feature of the protein polypeptide chain is to directly influence the orbitals of the electrons of the prosthetic group by exerting
* To whom correspondence @
should be addressed. Abstract published in Advance ACS Abstrucrs, April 1, 1995.
an electric field. The amino acids contain charged, dipolar, and polarizable groups that can produce large local electric fields. Although some work has been carried out to simulate the electric field effects in proteins: there is very little direct experimental data that would indicate the magnitude and directionality of electric fields induced by the protein upon the active prosthetic group.6 Electric fields can be expected to be particularly important in the function of heme proteins that contain the large electronrich porphyrin as the prosthetic group and which are involved in electron transfer reactions. In this work we use spectral hole buming7.*to study the effect of the polypeptide chain in altering electric field-induced changes on the prosthetic group of horseradish peroxidase (HRP), a protein of -34 000 MW in which the heme is nearly completely surrounded by the peptide.
0022-365419512099-5223$09.00/0 0 1995 American Chemical Society
5224 J. Phys. Chem., Vol. 99, No. 15, 1995
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a / cm-' Figure 1. Conventional spectrum of MP at 4.2 K (a) MP-IX (obtained from Porphyrin Products. Logan, UT) was dissolved in dimethylformamide/glycerol/aqueous buffer matrix (1:2:1 v/v/v). (b) HRP isoenzyme C2 was prepared from horseradish root2' and donated to us by K.-G. Paul. The heme was replaced by MP as described previously.** The buffer was 50 mM ammonium acetate adjusted to pH 8.0 with K2HP04. Glycerol was added (1 : 1 v/v) to ensure transparency at low temperature. Arrows indicate the frequencies where holes were burnt, and letters denote the tautomers. MP concentration was 20-40 pM.
The protein is modified to emphasize proteidporphyrin interactions by substituting free-base mesoporphyrin (MP) for the native iron-containing heme. Previous work demonstrated that the modified protein retains its substrate binding ability, showing that the protein folding is nearly identical to the native proteins9 Unlike the iron-containing heme, MP has a long excited singlet lifetime (-20 ns),'O and hence, its intrinsic 0,O optical line width is narrow. In addition, the porphyrin undergoes photochemical tautomerization reactions involving the central hydrogens on pyrrole nitrogens that make it an ideal system for persistent spectral hole-burning studies.' ' - I 3 The interaction of the polypeptide chain with the chromophore is already evident in the conventional absorption spectra taken at 4.2 K in a glass and in the protein (Figure la,b). In the glass only one 0,O band is seen; this band does not change upon irradiation. Probably, the chromophore acquires various tautomeric forms, but the amorphous matrix does not produce distinguishable 0,O bands.I3 In contrast, three well-separated origin absorption bands are seen in the region of MPhorseradish peroxidase (Figure lb). These bands were earlier identified as 0,O bands of the various pyrrole tautomeric forms of MP in the heme pocket of the p e r o ~ i d a s e .In ~ previous work we have carried out spectral hole-burning studies on these different tautomeric forms. The barriers separating the tautomeric forms were found to be distributed over a considerable range, I and additionally, temperature-dependent spectral diffusion o c c ~ r r e d . ' ~ .Both ' ~ features are characteristic of a disordered protein matrix. From pressure broadening experiments on spectral holes it could be shown that the scale of disorder compares well with the glassy state.I6 Pressure-tuning experimentsI6-l8 in the various tautomeric bands gave rise to remarkably different compressibility values of the HRP protein. These results gave strong evidence that chromophore and matrix structures are interrelated in In other words, if
local structural changes are induced in the chromophore, e.g. by photochemistry, the apoprotein adapts its structure. This structural adaption may involve larger parts of the protein. It may even have global character. This behavior is very similar to the allosteric effect. Structural rearrangement is inevitably connected with a change of the internal electric fields. If the conclusions from the pressure experiments, namely a correlation between tautomer configuration and protein structure, were right, the internal protein fields must change as one goes from one tautomer to another. In order to test this conjecture, we performed holeburning Stark effect experiments. The narrow photochemical holes are very sensitive to perturbations. The reason is that spectral shifts, broadening, or changes in the line shape can be measured on the scale of the natural line width which is in the megahertz range. This work represents a comparative Stark effect study between a protein, namely MP-HRP, and MP in a glass. It is from this comparison that we can learn a lot on the details of the physics involved: A glass is homogeneous and macroscopically isotropic. A protein solution (as seen from the chromophore) is nonhomogeneous and no longer isotropic. A Stark experiment is sensitive to such a symmetry breaking. The details of the Stark effect in a hole-burning experiment have been worked out by various groups (for reviews, see refs 20 and 21). However, applications in protein physics are quite scarce.22 Before discussing the results, we briefly go through the characteristic features of a hole-burning Stark experiment focusing on the qualitative aspects: First of all, whenever a Stark experiment is carried out within the inhomogeneous band, the effect is linear, irrespective of the molecular symmetry. The reason is that the random internal fields are much larger than the external field, and the symmetry of the chromophore will always be broken. However, the spectral pattern observed depends in quite a characteristic fashion on whether the chromophore has inversion symmetry or not. Let us first consider a chromophore without inversion symmetry in a disordered matrix. An external field induces a frequency shift Av of the transition under consideration:
EO is modified through the matrix. Figure 2 represents model calculations of a hole in an electric field which are based on a theoretical treatment by Schatz and Maier.23 We will discuss the specific features of a Stark experiment 0 2the basis of these calculations: The difference vector 40between the permanent dipole moment in the ground and electronically excited state has a molecular frame fixed orientation with respect to the transition dipole moment. This orientatiocis described by the angle y . In case the exciting laser field EL is parallel to the external field EOand y is zero, 40 is also oriented parallel to EO, and the respective spectral line will split (Figure 2a). Whether this splitting is pronounced or not depends on the simultaneous field-induced broadening of the line. This broadening originates from the random matrix fields and is characterized by the paramegr uLL.In the case where the exciting field is perpendicular to EO,there is broadening only. Although the actual pattern observed depends on the angle y , the basic features, namely splitting and broadening for the two polarization directions, are independent of it. Three cases, namely y = O", 45",and 90°, are modeled in Figure 2 . What is the spectral patternif the chromophore has inversion symmetry? In this case, Ap0 = 0, and hence, the Stark experiment is only sensitive to the dipole moments which are
f accounts for the fact that the external field
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Figure 2. Model calculations for the Stark effect of a low-symme%hromophore
in a random matrix. EL is the laser field, EO the external field,
y the angle between transition dipole moment and difference vector A ~ ofo the permanent dipole moments, and upthe width of the distribution of
induced dipole moments. Note that the hole spectra are inverted.
induced in the chromophore by the matrix. If the matrix is random, like a glass, the induced dipole moments have any size and directions. As a consequence, the holes will be broadened only, irrespective of the polarization of the laser field. The experimental results are shown in Figures 3 and 4. The inserts show the broadening and splitting as a function of EO.
These data confirm that the effect is linear in EO,in all cases, as stressed above. In the glass (Figure 3), we observe a broadening only, but no splitting. In the protein the situation is quite different, depending on the tautomer origin where the experiment is performed (see also Figure 1). In the BIband (Figure 4a), the situation is similar to the glass. There is a rather
5226 J. Phys. Chem., Vol. 99, No. 15, 1995
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Figure 3. Hole bumt into MP-IX in a glass. Conditions are given in Figure 1. The polarization of the bum light was parallel or perpen-
dicular to the applied field, as indicated, with the field strength EObeing 0 and 10 kV/cm. The solid line indicates the fit based on Ap0 = 0. Inset: field-induced hole broadening as a function of applied field. ,
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Figure 4. (a) Hole bumt into MP-HRP, B 1. Conditions are as given in Figures 1 and 3. The solid line indicates the fit again based on Ap0 = 0. Inset: field-induced broadening as a function of applied field. (b) Hole bumt into MP-HRP, B2. Conditions given in Figures 1 and 3. The solid line indicates the fit to the hole using the parameters h-lf A ~ =o 0.133 GHzkV cm-l, h-lfo, = 0.046 GHzkV cm-l, and y = 63". Inset: field-induced broadening and splitting as a function of applied field.
weak broadening, but no splitting. However, when the experiment is performed in the B2 tautomer band (Figure 4b), there is, in addition to the broadening, also a splitting whose
magnitude depends on the tautomer state. The same pattern is observed in the B3 band. From these results several important conclusions can be made: First, the field-induced broadening is of the same order of magnitude in the protein and in the glass. As mentioned above, this broadening is a result of structural randomness of the environemnt of the chromophore. Although the broadening scales with the local field factorf (eq l), a quantity which we do not know, we can say that, on a qualitative level, the scale of randomness is the same for the protein and the glass. This conclusion is in agreement with pressure experiments.I6 Second, since in the glass there is broadening only but no splitting, irrespective of polarization, we conclude that the n-electron system of the chromophore must have an effective inversion symmetry, despite the low molecular symmetry. Similar broadening pattems have been found in random matrices for molecules with true inversion symmetry like octaethylporphyIn the protein, we do observe a splitting in the B2 and B3 band. This implies that the inversion symmetry must be broken by the protein. It is broken because the protein is structurally organized. It induces a dipole moment in the chromophore, but this induced moment is not random. Instead, it is fixed in a molecular frame due to the organized structure of the protein. Hence, this induced moment acts much like a permanent moment. In the B1 band, on the other hand, there is no splitting. Obviously, the chromophore retains its inversion symmetry. The most obvious conclusion is that, for the B1 tautomer, the protein pocket must be almost free of electric fields. In any case one result is quite clear: The various tautomer states experience quite different internal fields. Clearly, the different internal fields arise from different protein structures. Hence, the Stark experiments in the various tautomer origins of mesoporphyrin-IX in HRP support in a quite convincing way what has been concluded from pressure-tuning experiments: There seems to be a strong correlation between the structure of the prosthetic group and the structure of the protein. Similar conclusions were drawn by Mathies and Stryer from Stark experiments on retinal in solutions.25 Again, we stress that this is a generalization of allosteric behavior. In summary, Stark spectroscopy can be used to monitor electric field effects imposed on the chromophore by the protein. The comparison of the Stark results leads to the conclusion that the absence or presence of inversion symmetry for MP is achieved by the interaction of the chromophore and the matrix. Since the diameter of the heme protein is about 30 8, and the porphyrin diameter is around 10 A, the average distance of the porphyrin to the surface is 10 A or less. Therefore, results showing differences between MP in the protein and in the amorphous matrix indicate that the electric field effects arise from close range forces within the protein itself. We consider this as an important outcome. It means that the spectral features of the chromophore are not influenced by the host matrix. Instead, they reflect properties of the protein. When we switch the MP configuration photochemically into different tautomeric structures at low temperature, we may achieve various internal field distributions that are dramatically different from the starting configuration.
Acknowledgment. We gratefully acknowledge grants from the DFG (Graduiertenkolleg "Nichtlineare Dynamik') (J.Fr.), the Fonds der chemischen Industrie (J.Fr.), the WTZ program between Hungary and Germany (J.Fi. and J.Fr.), and the NIH GM 48130 PO1 (J.M.V.).
Letters References and Notes (1) Perutz, M. F. Nature 1970,228, 736. (2) Frauenfelder, H.; Par&, F.; Young, R. D. Annu. Rev. Biophys. Biophys. Chem. 1988,17, 451. (3) Henry, E. R.Biophys. J . 1993,64, 869. (4) Levitt, M.; Sharon, R. Proc. Natl. Acad. Sci. U.SA.1988,85,7557. (5) Sharp, K. A.; Honig, B. Annu. Rev. Biophys. Biophys. Chem. 1990, 19,303. Holst, M.; et al. Proteins 1994,18,231. (6)Anni, H.; Vanderkooi, J. M.; Sharp, K. A,; Yonetani, T.; Hopkins, S. C.; Herenyi, L.; Fidy, J. Biochemistry 1994,33,3475. (7) Reddy, N.R. S.; Lyle, P. A.; Small, G. J. Photosynth. Res. 1992,
31,167. (8) Friedrich, J. In Methods in Enzymology; Sauer, K., Ed.; Academic Press: San Diego, 1995;Vol. 246,pp 226-259. (9)Fidy, J.; Paul, K.-G.; Vanderkooi, J. M. Biochemistry 1989,28, 7531. (10)Hone, T.;Vanderkooi, J. M.; Paul, K.-G. Biochemistry 1985,24, 7935. (1 1) Gorokhovskii, A. A.; Kaarli, R. K.; Rebane, L. A. JETP Lett. (Engl. Transl.) 1974,20, 216. (12) Volker, S. In Relaxation Processes in Molecular Excited States; Fiinfschilling, J.,Ed.; Kluwer: Dordrecht, The Netherlands 1989;pp 113-
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J. Phys. Chem., Vol. 99,No. 15, 1995 5227 (13) Fidy, J.; Vanderkooi, J. M.; Zollfrank, J.; Friedrich, J. Biophys. J . 1992,61,381. (14) Zollfrank, J.: Friedrich, J.; Vanderkooi, J. M.; Fidy, J. J . Chem. Phys. 1991,95, 3134. (15) Zollfrank, J.; Friedrich, J.; Fidy, J.; Vanderkooi, J. M. Biophys. J . 1991,59, 305. (16)Gafert, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. J. Phys. Chem. 1994,98, 2210. (17)Zollfrank, J.; Friedrich, J.; Fidy, J.; Vanderkooi, J. M.J . Chem. Phys. 1991,94,8600. (18) Friedrich, J.; Gafert, J.; Zollfrank, J.; Vanderkooi, J. M.; Fidy, J. Proc. Natl. Acad. Sci. U.S.A. 1994,91,1029. (19)Gafert, J.;Friedrich, J.; Parak, F. J . Chem. Phys. 1993,99,2478. (20) Maier, M. Appl. Phys. 1986,B41,73. (21) Kohler, B.E.; Personov, R. I.; Woehl, J. C. In Laser Techniques in Chemistry; Rizzo, T., Myers, A. B., Eds.; Wiley: New York, 1994. (22) Gafert, J.; Friedrich, J.; Parak, F. Proc. Natl. Acad. Sci. U.S.A., in press.
(23) Schatz, P.; Maier, M. J . Chem. Phys. 1987,87, 801. (24)Meixner, J.; Renn, A,; Bucher, S. E., Wild, U. P. J . Phys. Chem. 1986,90,6777. (25) Mathies, R.; Stryer, L. Proc. Natl. Acad. Sci. USA. 1976,73,2169.
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