2253
J. Phys. Chem. 1989, 93, 2253-2261
The Mechanism of Phototautomerization in Mesoporphyrin Horseradish Peroxidase. Studies by Fluorescence Line-Narrowing Spectroscopyt Judit Fidy,*vl K.-G.Paul,$ and J. M. Vanderkooi*,* Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 191 04, and Department of Physiological Chemistry, University of Umea, Umea, Sweden (Received: April 4 , 1988; In Final Form: July 28, 1988) High-resolutionemission spectra of mesoporphyrin IX (MP) were determined under site-selectiveconditions for MP horseradish peroxidase (HRP) at pH 5.1 and 8.0, and for MP in diethyl ether (DEE) and polycrystalline n-octane in the 5-40 K temperature range. Spectra corresponding to MP tautomeric forms were identified, and the range of 0 0 emissions was used for determining the site distribution functions. The distribution of 0 0 energies was found to be narrow, of 60-70 cm-l in HRP and n-octane, and resolvable for the tautomeric components. A downward shift in 0 0 energies was found parallel with the strength of crystal field effect as HRP > octane > DEE. The photoconversion kinetics of MP-HRP at pH 5.1 was found to be non-single-exponential. The transition probability was temperature independent below 15-20 K; the temperature dependence found at higher temperatures was mainly accounted for by a ground-state, phonon-assisted phenomenon. Deactivation from the triplet state was studied by phosphorescencelifetime measurements. The decay was non-single-exponential,and the slight temperature dependence of the lifetime components was in accordance with the kinetic results for the tautomerization reaction. MP in HRP was found to be photochemically stable at pH 8.0; only one of the tautomeric forms is populated.
-
+-
+ -
1. Introduction How the polypeptide chain and its motions interact with the prosthetic group of heme proteins to determine function is of great biological significance. Among the optical spectroscopic methods relevant to this question, site selection spectroscopy offers high selectivity and resolution. However, in the literature, in only a few cases was it successfully applied for the study of native proteins.'v2 The capability of this method in the case of photosynthetic systems seemed to be hindered by energy-transfer3 and electron-transfer4 processes, and by the strong interaction with phonon vibrationsh6 leading to the strong broadening of emission lines. In some of our previous works, however, we have successfully applied the technique for fluorescent derivatives of cytochrome c, horseradish peroxidases, leghemoglobin, and and for the study of phototransformation processes in In this study we applied fluorescence line-narrowing techniques to study mesoporphyrin horseradish peroxidase C2 (MP-HRP). Horseradish peroxidase C2 is a heme glycoprotein, one of at least seven isoenzymes that catalyzes the oxidation of indoleacetic acid and other aromatic compounds in plant roots by hydrogen peroxide.'* The prosthetic group of the native resting enzyme is ferric protoporphyrin IX, which we have replaced by mesoporphyrin IX (MP). MP-HRP is photochemically stable and binds substrate with about the same affinity as the native enzyme.*^'^,'^ We have previously reported" that the site distribution function for M P in H R P is surprisingly narrow: The width of the Soo SIo(0 0) transition energy is 50-75 cm-', suggesting that the inhomogeneous broadening is small; i.e., the position of the M P molecules in the heme pocket is well defined. For these reasons, MP-HRP appears to be a good model system for the native protein, having strong resemblance in its heme pocket structure. Our goal in these spectroscopic studies is to describe the role of the protein in the electronic-vibronic transitions of the heme, which form a basis for its important photochemical/photophysical processes. In our previous, fluorescence line-narrowing studies of Zn-cytochrome c,Io the detected line-broadening effects and phototransformation reactions were found producible by the presence of a proper organic glass around the chromophore as well as by the protein. In the present study, the use of a metal-free porphyrin has the advantage, through its well-known ability for
-
+
'A preliminary report was presented at the 16th Annual Meeting of the American Society for Photobiology, March 13-17, 1988; Abstr.: Photochem. Photobiol. Suppl. 1988, 47, 10s. *University of Pennsylvania. 'University of Umea. On leave from Institute of Biophysics, Semmelweis Medical University, Budapest, H-1444, POB 263, Hungary.
0022-3654/89/2093-2253$01.50/0
tautomerization transition within the pyrrole hydrogens,I5J6that it can produce a system where the structural symmetry and vibrations of the surrounding protein will be sensitively represented in the emission spectra. In this paper we interpreted the high-resolution emission spectrum and the site distribution function of M P in H R P by comparison with M P in crystalline octane and in amorphous diethyl ether (DEE) and by comparing MP-HRP at different pH values. The existence of two tautomeric forms of M P in H R P was verified on the basis of the spectra and the site distribution function. These forms were shown to photochemically interconvert, and the nature of the reaction was examined from the temperature dependence of the tautomerization reaction, the phosphorescence lifetime, and the ground-state reequilibration kinetics. The tautomerization reaction was proved to be strongly sensitive to the environment of the chromophore, showing a strongly asymmetric crystal field potential within the protein and the presence of vibronic interactions. The temperature dependence of the tautomeric transition in MP-HRP indicated the presence of the interaction with low-frequency vibrational modes of the protein.
2. Experimental Section 2.1. Materials. Horseradish peroxidase C2 was isolated and purified as d e ~ c r i b e d .The ~ protein was split to heme and apoprotein with the butanone extraction method." After exhaustive (1) Friedrich, J.; Scheer, H.; Ziokendracht-Wendelstadt, B.; Haarer, D. J . A m . Chem. SOC.1981, 103, 1030. (2) Avarmaa, R.; Renge, I.; Mauring K. FEBS Lett. 1984, 167, 186. (3) Avarmaa, R.; Jaamiso, R.; Mauring, K.; Renge, I.; Tamkivi, R. Mol. Phys. 1986, 57, 605. (4) Won, Y . ;Friesner, R. A. Proc. Natl. Acad. Sci. U S A 1987,84, 551 1. (5) Platenkamp, R. J.; den Blanken, H. J.; Hoff, A. J. Chem. Phys. Lett. 1980, 76, 35. (6) Vink, K. J.; de Boer, S.; Plijter, J. J.; Hoff, A. J.; Wiersma, D. A. Chem. Phvs. Lett. 1987. 142. 433. (7) Angiolillo, P. J.;'Leigh, J. S., Jr.; Vanderkooi, J. M. Photochem. Photobiol. 1982, 36, 133. (8) Horie, T.; Vanderkooi, J. M.; Paul, K.-G. Biochemistry 1985, 24,7935. (9) Vanderkooi, J. M.; Moy. V. T.; Maniara, G.; Koloczek, H.; Paul, K . 4 . Biochemistry 1985, 24, 7931: (10) Koloczek, H.; Fidy, J.; Vanderkooi, J. M. J . Chem. Phys. 1987,87, 4388. (1 1) Fidy, J.; Koloczek, H.; Paul, K.-G.; Vanderkooi, J. M. Chem. Phys. Lett. 1987, 142, 562. (12) Paul, K.-G. Enzymes (3rd Ed.); 1963, 227, 8. (13) Paul, K.-G.; Ohlsson, P.-I. Acta Chem. Scand. Ser. B. 1978, 32, 395. (14) Ugarova, N. N.; Savitski, A. P.; Berzin, I. V. Biochim. Biophys. Acta 1981, 662, 210. (15) Abram, R. J.; Hawkes, G. E.; Smith, K. M. Tetrahedron Let?. 1974, 16, 1483. (16) Zalesski, I. E.; Kotlo, V. N.; Sevchenko, A. N.; Solovyev, K. N.; Shkirman, S . E. Sou. Phys. Dokl. (Engl. Transl.) 1973, 17, 1183. (17) Teale, F. W. J. Biochim. Biophys. Acta 1959, 35, 543.
0 1989 American Chemical Society
2254
The Journal of Physical Chemistry, Vol. 93, No. 6, 1989
dialysis of the protein against water to remove 2-butanone, M P was stoichiometricaly added and incubated in 10 mM Tris HCI at pH 8.0 for 3 h in the cold. This was followed by column chromatography on a DEAE-Sephadex A-50 using 0.1 M sodium acetate, pH 5.0. To assure the transparency of the samples at low temperatures, 50% glycerol was added; the final concentration was about 30 pM for MP-HRP. In the kinetic studies for the protein, the samples were pretreated by first irradiating them at 5 K for 20 min and then equilibrating at 70 K in the dark for 5 min. Model systems with M P were prepared by dissolving mesoporphyrin IX from Aldrich Chemical Co. (Milwaukee, WI) in diethyl ether (DEE) from Sigma Chemical Co. (St. Louis, MO), or n-octane from Fisher Scientific (Malvern, PA). The stock solution obtained this way was diluted in the actual matrix used to a concentration of 30-60 pM. The samples after preparation were stored in liquid nitrogen. 2 . 2 . Instrumentation. Conventional fluorescence and phosphorescence spectra at room temperature and 77 K were measured by a Perkin-Elmer LS5 luminescence spectrophotometer equipped with a Xenon flash lamp excitation system. For measuring high-resolution emission spectra, the excitation source was a single-frequency Coherent 599 dye laser with Rhodamine 6G, pumped by a continuous wave Coherent argon ion laser.* The intensity of the laser beam was -50 mW, focused in a spot of 1.7-mm radius to minimize local heating effects. The emission spectrum was measured at 90' from the excitation direction through a 1-m JY Ramanor HG2S double monochromator equipped with holographic gratings. The instrumental spectral resolution was 1 cm-l. The detector was composed of a cooled GaAs photomultiplier (RCA 3C1034-RF) and photon counter (Princeton Applied Research Model 1105). The sample was cooled by a liquid helium EPR dewar (Air Products, Allentown, PA). Phosphorescence lifetime measurements were performed by an instrument described elsewhere.'8 An EG&G MCP 1/FY900 xenon flash lamp of 2-ps pulse width, powered by an H P 6263 direct current power supply, was used for excitation. A combination of filters allowed for excitation energies in the Soret band at wavelengths 700 nm by a Hammamatsu R926 photomultiplier (PM) tube. After preamplification and current/voltage transformation, the analog signal of the PM tube was converted by a Scientific Solutions Lab Master 40-kHz 12-bit resolution analog to digital converter. Data acquisition and synchronous triggering of the flash lamp was regulated by an AT&T 6300 computer. The adjusting of data acquisition parameters and the evaluation of decay curves were preformed by the Asystant+ software package. The lifetime values were determined from the average of 50-100 decay curves by an iterative nonlinear regression method. 3. Results 3.1. Fluorescence Site Selection Spectra and Site Distribution Functions for M P in Different Systems. The fluorescence site selection spectra of M P in HRP, 50% octane/DEE, and DEE, excited by an energy of 17 300 cm-I, are shown in Figure 1. We also studied the 50% methanol/DEE system, but no difference compared to the case of DEE was found (not shown). The insert shows the Q bands of MP as they appear in the room-temperature absorption spectrum of MP-HRP, with the arrow indicating the location of excitation energies used in the experiments; the absorption spectra are basically the same in each of the model systems. All of the emission spectra presented in the figure have the same features; within the range of 15900-16200 cm-l intense 0 0 emission lines can be observed, while below this energy range the emissions from the SI lowest vibronic state to the ground-state vibrational levels appear. The emission energies for these latter transitions, as expected, are shifted in comparison to the main 0 0 transition energy by about the same energy for + -
+-
(18) Green, T. J.; Wilson, Biochem. 1988, 174, 13.
D. F.; Vanderkooi, J. M.; DeFeo, S. P. Anal.
Fidy et al. C
>
I-
v)
z
w
z
W
0
z
W
n
0
tn W
0:
0 3 -I Y
I
15°K
1
311°K
L ;
!
~
:
:
~
I5IlOO
I4511(1
I
~!
:
I
:
:
I/
~:
i5500
i : IWW
I
CU,d
Figure 1. Fluorescence site selection spectra of different M P systems excited at 17 300 cm-I: (A) MP-HRP, pH 5.1 at 30 K; (B) MP-50% octane/DEE at 15 K; (C) MP-DEE at 10 K; (insert) absorption spectrum for MP-HRP, pH 6 at room temperature.
- -
each case. The intensities of these SIo So' lines are very small compared to those of the 0 O's, having the highest intensity in the case of DEE and the smallest in HRP. This difference between the three porphyrin systems shows that the surrounding matrix affects the transition probabilities. The low probability found for the SIo So" emissions in the case of MP-HRP compelled us to use the range of 0 0 emissions for further studies. A shift toward lower energies in the 0 0 emission ranges can be found from Figure 1C to Figure lA, H R P showing more resemblance to the octane/DEE system than to DEE. The difference in 0 0 energies indicates that the excitation energy hits the three types of samples at different vibronic levels; however, without knowledge of the site distribution functions characteristic for the systems, it is hard to tell whether the inhomogeneous distribution is different or the whole set of S I vlevels is shifted under the influence of a different matrix. In Figure 2 the site distribution f~nctions'~ for the three systems are shown determined and plotted as the intensity of the emission found at Au = 1210 cm-l below the excitation energy in the function of the actual energy of excitation. The figure thus shows
-
-
- -
(19) Fuenfschilling, J.; Zschokke-Granacher, I.; Williams, D. F. J . Chem. Phys. 1981, 75, 3669.
The Journal of Physical Chemistry, Vol. 93, No. 6, 1989 2255
Fluorescence Site Selection Spectroscopy of H R P
TABLE I: Parameters of Gauss Approximations to tbe Site Distribution of 0 + 0 Energy for M P in Different Systems'
loo-- A
DEE
DEE maximum position of subpopulations energy separation of subpopulations width (2a) of the Gauss function
50 --
16200
0 ( 750 nm and a rather strong delayed fluorescence (dashed line in Figure 6C). As delayed fluorescence in this case is expected to arise from thermally activated population of the S, level from T I , at lower temperatures delayed fluorescence is expected to cease, while (26) Gouterman, M.; Khalil, G.-E. J . Mol. Specfrosc. 1974, 53, 88. (27) Gradyusko, A. T.; Tsvirko, M. P. Opt. Specrrosc. (Engl. Transl.) 1971, 31, 291. ( 2 8 ) Burgner, R. P.; Ponte Cancalves, A. M . J . Chem. Phys. 1974, 60, 2942.
4 0 -4
Figure 7. (A) Phosphorescence decay of MP/DEE at 32 K, excited in
the Soret band and registered at X > 700 nm. (B) Residuals plotted on the same time scale fbr one-component fit. (C) Residuals in the case of two-component fitting. phosphorescence gains intensity. A typical phosphorescence decay curve measured at 32 K can be seen in Figure 7. Generally the decay curves could not be fitted well by supposing a single-exponential phenomenon. The residuals for one-exponential fit shown in the figure also indicate the presence of a second component. The lifetime results coming from two-exponential fittings are presented in Table 11. To characterize the experimental reproducibility of the results, we presented two data sets (I and 11) for the MetOH/DEE sample at 5.6 K, which were determined in two different series of experiments. The close similarity of the data shows the reliability of the techniques. In a comparison of MP-HRP, pH 5.1, with the model system, we see that in the protein, T, is not affected by the temperature; however, the lifetime for component 2 is decreasing (decay rate is increasing) somewhat with the temperature increase. The proportion for T , ( A , ) is decreasing toward higher temperatures. In the model system, the T , component is somewhat longer. In this case, during temperature increase, both components show a lifetime decrease, while the percentage of component 1 is maintained at a high value. 3.6. Effect of p H in MP-HRP. Prior to this section, all the data presented for MP-HRP have referred to pH 5.1. Equilibrium spectra of MP-HRP achieved by prolonged irradiation are shown for pH 8.0 at different temperatures in Figure 8, using the same excitation energy as for pH 5.1 in Figure 5. The spectra for the two pH values, though similar, are not identical at any of the temperatures indicated. At pH 8.0, the molecules emitting at 16 100 cm-I are represented by a high intensity, but the emission is very weak at 16 000 cm-', and the spectrum is not affected by the temperature. When comparing the spectra with those shown in Figure 3A at different stages of the irradiation process for pH 5.1, we can see that the predominant spectral characteristics of the sample at pH 8.0 correspond to those of the sample at 5.1 at the start of the irradiation experiment, when all the molecules
The Journal of Physical Chemistry, Vol. 93, No. 6, 1989 2259
Fluorescence Site Selection Spectroscopy of H R P
- -
32%
‘1. I
5.5%
4
3 (cm-l)
16ooo 16300 Figure 8. Equilibrium spectra of MP-HRP at pH 8.0, excited by 173 IO-cm-’ energy at different temperatures, as indicated. Insert shows site distribution function for the same sample measured by Av = 1210 cm-I vibronic excitation at 5.5 K as a function of the excitation energy (upper abscissa) or the emission energy (lower abscissa).
were in tautomer 1 state. The difference between the two cases can be demonstrated better on the basis of their site distribution functions, shown in the insert of Figure 8 and in Figure 2C. The subpopulation characterized by 16000-cm-’ 0 0 energy for pH 5.1 is practically missing from the sample at pH 8.0, and the distribution is narrower at pH 8.0: 50 cm-’. The phosphorescence lifetime results shown in Table I1 for MP-HRP at pH 8.0 are very similar to the pH 5.1 case; the difference in the phototautomerization is not sensitively represented in this parameter.
-
4. Discussion 4.1. Spectroscopic Results. A schematic of the electronic
energy levels and considered transitions for M P is shown in Figure 9A. In the spectroscopic measurements we excited the samples into a higher vibronic energy level, S I v in SI. After internal conversion to SIo,two types of fluorescent transitions were registered: Soo SIo(0 0) and Sov SIo(v 0). As previously demonstrated, in the case of HRP, the effects leading to line broadening do not hinder the resolution of the site selection technique; thus not only for the model systems but also for the protein, well resolved spectral lines could be d e t e ~ t e d . ~The .~ intensity relations for 0 0 and v 0 emissions were in accordance with literature data concerning photosynthetic model systems6 Both the emission energies represented by 0 0 emission lines and their intensity relations were found dependent on the system used. The registration of the spectra within the energy range of 0 0 emissions at various excitation energies and the resolution of the technique made it possible to determine the site distribution functions for the three different systems of MP. These distribution functions revealed that the matrix around the chromophore may
- -
- -
- -
-
-
- -
affect both its characteristic (mean) 0 0 transition energy, and the energy separation (in 0 0 energies) between the tautomeric forms. The magnitude of the effect for the various systems is as follows: 1, energy separation, HRP, pH 5.1 > octane > DEE; 2,O 0 transition energy, DEE > octane > HRP, pH 5.1. The data for the 0 0 energy of M P in DEE is in agreement with that published by Romanovskii et aLzo To interpret the effect of the protein as a matrix on the spectral characteristics of MP, we turn to the model suggested and used by Voelker and van der Waalsz4for interpreting the phototautomerization of porphin in n-octane. A schematic corresponding to this model is shown in Figure 9B,C. In Figure 9B the tautomeric forms of unsubstituted free base porphyrin (porphin) are represented by two harmonic potential functions having minimal positions at -Qoand +Qoalong a vibrational coordinate Q, which is considered Jahn-Teller (JT) The authors considered the tautomers originating from a common structure (schematized as a cube for the position of the methylene groups and corresponding doubly degenerate potential function shown by dashed line) in which, in imagination, the two inner-porphyrin hydrogens have a smeared position, symmetric among the four inner nitrogens of the ring. From this state of high symmetry, the tautomers are produced by JT-type deformations along the vibrational coordinate involved in vibronic interactions resolving deformationally the electronic d e g e n e r a ~ y . ~The ~ - ~surrounding ~ matrix (octane) was handled as a source of a perturbing electrostatic potential, the “crystal field”, which in the case where its direction corresponds to that of the vibronically active mode, will lead to an energy splitting (a) between the deformed forms (see Figure 9C). EJT in the figure indicates the stabilization energy achieved by vibronic interactions. The significanceof the JT effect compared to crystal field effects in the case of 4-fold symmetric metal-porphyrins (of point group D4h) has been the subject of theoretical and experimental studies, which have produced experimental evidence for the existence of the JT coupling and also showed that the significance of the effect is dependent on the nature of the actual chromophore-matrix The involvement of the JT effect in ligation problems connected to the out-of-plane displacement of the metal atom was theoretically studied by Bersuker and S t a ~ r o v .An ~~ important feature of the model based on JT interactions in our case is that it allows for interconversion between the vibronically coupled forms: here, in-plane tautomers. Upon applying the above-described model to mesoporphyrin, we have used the term “crystal field” in a general sense.32 There has been experimental evidence indicating that asymmetric substitution may act as a crystal field on the electronic energies of the p ~ r p h y r i n . Thus ~ ~ * ~we~ have considered M P as a molecule undergoing an additional crystal field “per sen because of its slight asymmetry compared to that of porphin, and we have followed the concept proposed by Voelker and van der Waals. (29) Jahn, H. A.; Teller, E. Proc. R. Soc. London Ser. A . 1937,161,220. (30) Englman, R. The Jahn-Teller Effect in Molecules and Crystals; Seitz, F., Turnbull, D., Eds.; Wiley-Interscience: New York, 1960. (31) Bersuker, I. B. The Jahn-Teller Ejject and Vibronic Interactions in Modern Chemistry; Plenum Press: New York, 1984. (32) Hoffman, B. M.; Ratner, M. A. Mol. Phys. 1978, 35, 901. (33) Child, M.S. Mol. Phys. 1960, 3, 601. (34) Hougen, J. T.J . Mol. Spectrosc. 1964, 13, 149. (35) Personov, R. I.; Korotaev, 0. N. Sou. Phys. Dokl. (Engl. Transl.) 1969, 13, 1033. (36) Chan, I. Y.; van Dorp, W. G.; Schaafsma, T. J.; van der Waals, J. H.Mol. Phys. 1971, 22, 741; Ibid. 1971, 22, 753. (37) Shulman, R. G.; Glarum, S. H.; Karplus, M. J. Mol. Eiol. 1971,57, 93.
(38) Gouterman, M.;Yamanashi, B. S.; Kwiram, A. L. J. Chem. Phys. M.Ann. N . Y. Acad. Sci. 1973, 206, 70. (39) Canters, G. W.; Egmond, J.; Schaafsma, T. J.; Chan, I. Y.; van Dorp, W. G.; van der Waals, J. H. Ann. N . Y. Acad. Sci. 1973, 206, I1 1 . (40) Hoffman, B. M.;J. A m . Chem. SOC.1975, 97, 1688. (41) Shelnutt, J. A,; Cheung, L. D.; Chang, R. C. C.; Yu, N.-T.; Felton, R. H. J . Chem. Phys. 1977, 66, 3387. (42) van der Poel, W. A. J. A,; Nuijs, A. M.; van der Waals, J. H. J. Phys. Chem. 1986, 90, 1537 and citations therein. (43) Bersuker, J.; Stavrov, S . S.; Chem. Phys. 1981.54, 331; Ibid. 1982, 69, 165. 1972, 56, 4073. Gouterman,
A
B
C
-
Returning to our spectroscopic results, the data gave energy separations and 0 0 energies for the three studied systems. The energy separation values now will be considered as crystal field splitting data. The fact that the protein at pH 5.1 achieves the largest crystal field splitting may originate from two circumstances: (1) there is a strong potential present; (2) the direction of the crystal field is in accordance with the JT-active deformations. It is interesting that the splitting is smaller in crystalline octane: The protein seems to achieve a larger asymmetry in the potential around the heme in its plane than an ordered crystalline matrix of hydrocarbons. That DEE produces an even smaller crystal field effect shows that polarity and possible orientation effects of polar molecules around the chromophore alone cannot lead to a large splitting. Concerning DEE, we have to mention that according to literature data amorphous matrix systems can be well described by supposing the presence of a distribution of crystal field eff e c t ~ . ~If ~this , ~ is~ the case, then DEE cannot be described by one splitting value; however, the mean value of the distribution cannot be larger than the estimation used by us. Polarity differences also may affect the 0 0 energies. The fact, however, that the nonpolar octane has a middle position in terms of 0 0 energies indicates that polarity alone cannot be responsible for the large shift observed in MP-HRP (Figure 2). The 0 0 energy data characteristic for the systems may be indicative for different EJTstabilization energies as well, Le., for different amounts of distortions through vibronic interactions. Unfortunately, no structural data are available for the protein, and it is not possible to tell whether some differences in possible deformations of M P in the given systems or differences in the interactions with some close-lying atomic groups cause the observed differences in transition energies. The downward shift in this parameter shows the same tendency as the crystal field splitting in accordance with the statement by Ham, that the strength of the crystal field splitting effect is proportional to the strength of the JT coupling.46 Both effects are strongest in the case of the protein at pH 5.1. 4.2. p H Effect on Tautomerization in MP-HRP. The pH effect on the site distribution function is similar to the effect reported by Jansen and Noort4' for Mg-porphine in crystalline hydrocarbons when, in case of some ligated forms, only one of the JT-distorted structures was found to be populated in the system. This effect, however, is not yet understood. Literature data for Fe-HRP4* indicate that changing the pH from 5 to 8 +
-
+
(44) Sutherland, J. C . ; Axelrcd, D.; Klein, M. P. J . Chem. Phys. 1971, 54,
ma.
(45) Canters, G. W.; Jansen, G.; Noort, M.; van der Waals, J. H. J . Phys. Chem. 1976, 80, 2253. (46) Ham, F. S. In Electric Paramagnetic Resonance; Geschwind, S . , Ed.; Plenum Press: New York, 1972. (47) Jansen, G.; Noort, M. Spectrochim. Acta 1976, 32A, 747.
D
may lead to conformational changes in the protein, detected through the Fe-histidine stretching mode. Our data, indicating that these changes do not affect the symmetry of the pocket potential in the plane of the porphyrin, are complementary to the literature data. We think that the lack of population of the lower energy tautomeric form in the case of pH 8.0 may be connected to the presence of the two histidines, which in the native form are ligated to the Fe but now are unligated and able to form hydrogen bonds with the pyrrole hydrogens. This interaction may result in a pH-dependent stabilization of one tautomeric form over the other. 4.3. Nature of Phototautomerization Kinetics. The kinetics of the photochemical processes could be detected only in the case of the protein, as discussed in 3.2. The kinetic curves for the decrease in the intensity attributed to tautomer 1 and the increase in that for tautomer 2 shown in Figure 3B were parallel up to the last 5% of the change found in the signal for tautomer 1. After an irradiation time of 10 min was reached, the intensity for tautomer 2 seemed to be stabilized while the line intensity for tautomer 1 was still decreasing within 5% of the total change. This compatibility was found to be dependent on slight changes in the excitation energy, indicating that the phenomenon is site dependent. We based our conclusions on the kinetics followed by the decrease in the emission for tautomer 1 at 16 100 cm-' when excited at 17 310 cm-' (Figure 3B-1). Semilogarithmic plotting of the decay data for tautomer 1 seen in Figure 4 showed that the decay of the signal is not single-exponential. The most evident interpretation for this is based on the fact that the term "single site" is only an approximate terminology in this technique. By the decay curves, in fact, we have detected the signal from all of the sites that met the excitation and emission energy conditions; Le., because of line-broadening processes (for zero phonon lines) and phonon wing contributi0ns,4~ this means an array of sites in reality. A log-log representation shown in the insert of Figure 4 gives a quasi-linear function (neglecting the first two points of high experimental errors). A similar time course was found for the binding of CO to myoglobin49 and for other monomeric heme proteins.50 This type of decay could be interpreted on the basis of a distribution for the reaction barriers because of the inhomogeneity of the molecules.50 4.4. Temperature Effect on the Kinetics of Tautomerization. One result from the study of the kinetics was that the kinetic (48) Teraoka, J.; Kitagawa, T. J . Biol. Chem. 1981, 256, 3969. (49) Personov, R. I . In Spectroscopy and Excitation Dynamics of Condensed Molecular Sysrems; Agranovich, V. M., Hochstrasser, R. M., Eds.; North-Holland: Amsterdam, 1983; Chapter 10. (50) Austin, R. H.; Beeson, K.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I . C.; Marshall, V . P. Science 1973, 181, 541. ( 5 1 ) Frauenfelder, H.; Young, R. D. Comments Mol. Cell. Biophys. 1986, 3, 347 and citations therein.
The Journal of Physical Chemistry, Vol. 93, No. 6, 1989 2261
Fluorescence Site Selection Spectroscopy of H R P constant(s) K do not show significant temperature dependence in agreement with what was reported for porphin in n-octane within 4.2-77 K.24 This means, on the basis of the approximation for K of formula 1, that the sum of the probabilities p,, is only slightly dependent on the temperature. Consequently, the temperature dependence of the data points presented in Figure 5B is coming mainly from the transition probability p Z lalone. Thus our results showed that the probability for an excited type 2 tautomer of M P in H R P at pH 5.1 to be transformed into the tautomer form 1 is increasing with the temperature within 5-40 K. Our dark reaction studies showed that this probability increase can almost be fully accounted for by a transformation from 2 to 1 in the ground state. The slight difference between the photochemical and dark transformation is significant, however, in one respect. Dark recombination absolutely does not change below 15-20 K, while the overall probability shows a continuous increase through the whole temperature range. We discuss the meaning of p21 by considering excited- and ground-state pathways on the basis of the schematics in Figure 9D. In the phototransformation reaction the contribution from singlet-state pathways is estimated to be low on the basis of the high yield of triplet formation for porphin, reported as -90% by Gradyusko and T ~ v i r k o .Our ~ ~ singlet decay measurementsll showing single-exponential decays for both tautomers did not indicate additional pathways within the singlet lifetime. Our present spectroscopic data showed that in the protein, the SI energy level for tautomer 2 is below that of tautomer 1 by about 100 cm-'. 1 Voelker and van der Waals found24that a singlet-state 2 transformation could not be achieved in the case of porphin at 4.2 K even though it has a much smaller splitting value. On the basis of these, we neglected this pathway. Thus we interpret pZl in the form of p21= p21T + p z l G ,accounting for triplet- (T) and ground-state (G) processes. The temperature effect on p Z I Gwas independently measured by dark recombination. The data show that this probability has a very low, temperature-independent value below 15-20 K, and then the probability increases with the temperature. We performed the studies only up to 40 K, but our experiences showing that the site distribution is changed by reaching 100 K, and photochemical hole-burning experiments of others detecting significant linebroadening effects at 77 K,24 indicate that the temperature dependence could not have been studied in a much wider temperature range without affecting the reaction itself. Having thus a small range and data loaded with experimental error, it is impossible to perform a detailed functional analysis for the temperature effect. In NMR studies on tetraphenylp~rphyrin,'~ a large (-3500 cm-I) energy barrier was found between the ground-state tautomeric forms. This seems contradictory to a classical thermal activation of the transformation at low temperatures, although the activation energy coming from a thermal activation model, 30 cm-', agrees with our estimate for the ground-state energy difference. It is more probable that the temperature effect obeys a power function relation, which would account for interaction with phonons both From in tunnelingS2or in JT-type deformational intercon~ersion.~~ such a fit, a power of 1.5-2 follows, allowing for one- and twophonon processes. We have to note, furthermore, that the cited N M R results, formally contradictory to our data, did not refer to temperatures below -80 "C. On the basis of our results and those discussed above, we believe that in terms of interactions with the low-frequency vibrations of the protein matrix, the behavior of the chromophore is very different below 40 K from that at around
-
( 5 2 ) Frauenfelder, H. In Tunneling in Biological Systems; Chance, B., DeVault, D., Frauenfelder, H., Marcus, R. A., Schrieffer, R. A., Sutin, N., Eds.; Academic Press: New York, 1979; p 627.
200 K. Thus the N M R measurements and ours may not have characterized the same phenomenon. Concerning pzlT,literature data from Hoffmanm and van Dorp et aLs3 showed direct evidence for interconversion in the triplet state between JT-distorted forms of metal-porphyrins. We also believe that the leading 1 2 phototransformation process in MP-HRP at low temperature is via triplet interconversion. In Figure 5B, the transition 2 1 in the triplet state is almost hidden by pzlG;however, if we consider that we neglected a 25% increase in the denominator of the plotted values, one can feel that this probability is not evidently negligible. Triplet lifetime data showing two components indicate that this method through formula 4 is an appropriate technique to measure the triplet transition rate for tautomerization separately for the two tautomers. Thus p21Tcould be experimentally determined. In our measurements the transition rate corresponding to 7 2 would be characteristic for 2 1 transition, and its observed slight temperature dependence is compatible with the data in Figure 5B. In this sense, T I being much shorter indicates a higher transition rate independent from the temperature. In such a measurement the low-temperature long-lifetime component would be characteristic for triplet ground state interconversion alone; thus the contribution due to photochemical depopulation can be estimated from the comparison of the lifetime components. In our experiments the technique of data collection and averaging used in the lifetime measurements increased the capability of the method significantly. However, because of the low phosphorescence quantum yield, more capable low-temperature laser excitation techniques would be necessary for a fully reliable two-component analysis. The absolute value for the phosphorescence lifetime of M P is in agreement with literature data, the short component is shorter, and the mean value based on two components is longer than the 16 ms r e p ~ r t e d ~ ~ , ~ ' on the basis of single-exponential decay. 4.5. Relevance to the Structure of HRP. Concerning the data for temperature effects, it is interesting to find supporting evidence for the model elaborated by Frauenfelder and c o - ~ o r k e r for s~~ protein fluctuations related to different temperature ranges. Our experience showed that below 80-100 K, motions of the protein did interact with the electronic states of the chromophore but did not affect the 0 0 energy pattern. Above about 100 K, however, the motions reach a significantly different amplitude, where the site distribution can be rearranged. This stepwise change in the fluctuational amplitudes within a characteristic temperature range corresponds to the model described by the cited authors for myoglobin. Mesoporphyrin in horseradish peroxidase proved to be a useful spectroscopicmonitor for the structure of the heme crevice through its capability for inner porphyrin tautomerization. The comparison of the protein with M P in other matrices invoking the JT effect showed the existence of a strongly asymmetric potential in the plane of the porphyrin within the heme pocket, more significant than in crystalline octane. Out-of-plane deformations of the porphyrin play a major role in the function of heme proteins. The tautomerization transition being an in-plane phenomenon yields information concerning the in-plane symmetry relations in the crevice, thus completing those based on out-of-plane phenomena.
-
-
-
-
Acknowledgment. This work was supported by National Science Foundation Grant PCM84-00844 and by the Swedish Research Council. Registry No. MP, 493-90-3; HRP, 9003-99-0. (53) van Dorp, W. G.;Schoemaker, W. H.; Soma, M.; van der Waals, J. M. Mol. Phys. 1975, 30, 1701. (54) Ansari, A,; Berendzen, J.; Bowne, S.F.; Frauenfelder, H.; Iben, I. E. T.; Sauke, B.; Shyamsunder, E.; Young, R. D. Proc. Natl. Acad. Sci. USA 1985, 82, 5000 and citations therein.
