J. Phys. Chem. 1995,99, 16527-16529
16527
Electric Field and Structure in the Myoglobin Heme Pocket Peter Geissinger, Bryan E. Kohler,* and Jdrg C. Woehl Chemistry Department, University of California, Riverside, California 92521 Received: August 2, 1995; In Final Form: September 25, 1995@
Recently we showed that intemal electric fields for linear polyenes in n-alkane crystals could be determined by high-resolution optical hole burning spectroscopy in combination with a novel quantum mechanical approach to data analysis. Here we demonstrate that the same ideas can be applied to protoporphyrin IX substituted horse myoglobin to determine the two intemal electric field components in the porphyrin plane. The orientation of the intemal field shows that the dominant contribution is from the deprotonated propionic acid side chains of the porphyrin ring; the magnitude gives a measure of the contribution of microscopic heme pocket structure to the local field factor.
The distribution of charges (electrons and nuclei) in a molecule generate an electric field. Although the field from a neutral molecule vanishes at distances large compared to molecular dimensions or when averaged over all possible orientations, its strength at typical condensed phase intermolecular distances can be orders of magnitude larger than experimentally attainable fields. Thus, at the microscopic level the intemal electric field can be expected to be a key determinant in processes that generate or separate charge in a variety of substances ranging from electronic microstructures to biomolecules. Recently, it has been suggested that electrostatic steering is instrumental in guiding the neurotransmitter acetylcholine to the active site in the enzyme In this light the proposal that it is the electrostatic field of the asymmetric protein environment of the photosynthetic reaction center that forces unidirectional electron transfer rather than any subtle property of the center itself3 seems quite reasonable: recent experiments by Steffen et aL4 demonstrate asymmetry between the right and left hand protein environments. Until recently, it was not possible to experimentallydetermine the magnitude and orientation of intemal electric fields. We have shown that this can be achieved for linear polyenes in n-alkane crystals by high resolution optical hole-burning spectroscopy in combination with a novel quantum mechanical approach for data a n a l y s i ~ . ~Here .~ we demonstrate that these same ideas can be applied to protoporphyrin IX substituted horse myoglobin to determine the two intemal electric field components in the porphyrin plane. The orientation of the in-plane intemal electric field is exactly parallel to the field calculated from a point charge model where it is assumed that the principal contribution is from the deprotonated carboxy groups of the propionic acid side chains of the porphyrin ring. The magnitude is also accounted for if the local field factor (the multiplier that relates the externally applied electric field to the field felt by the porphyrin) is 2.1. The principle of hole-buming Stark spectroscopy is simple: a narrow-bandwidth laser photochemically alters the subensemble of probe molecules that absorb at the laser frequency creating a narrow hole in the inhomogeneously broadened absorption profile. These narrow holes serve as frequency markers in subsequent experiments that measure the effect of an externally applied electric field on excitation energy. The experimental and theoretical issues are thoroughly discussed in a recent review.6 The short lifetime of the excited state of the heme in myoglobin makes it impossible to burn holes that are sufficiently @
Abstract published in Advance ACS Absrracfs, November 1, 1995.
0022-365419512099-16527$09.0010
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16528 J. Phys. Chem., Vol. 99, No. 45, 1995
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Figure 1. Fluorescence excitation spectrum of protoporphyrin-IX myoglobin in 3: 1 glycero1:water glass at 77 K. Labels identify the 0-0 bands of transitions to the Qx state (16 122 cm-I), the Q, state (18 462 cm-') and the B states (B, at 23 282 cm-I, B, at 23 482 cm-I). We have arbitrarily assumed that the unresolved B, and B, components are split by 200 cm-' with the B, being lower in energy.9 Because the B state splitting is small compared to the excitation energies, the calculated profiles have no significant dependence on the value chosen for this splitting.
16138.9 16131 16131.1
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Figure 2. Fluorescence excitation scans at 1.2 K of photochemical holes burned into the Qx 0-0 band of protoporphyrin-IX myoglobin. Laser light polarized parallel to the applied field is shown at the left and laser light polarized perpendicular to the applied field is shown at the right; the lower scans are at zero extemal field and the upper scans are at 11.05 kV/cm. The smooth line through the noisy data is the profile calculated for local field factor 2.1 and internal field components E, = 29.9 MV/cm and E, = 38.6 MV/cm. Six additional profiles measured at different field strengths (three for light polarized parallel and three for light polarized perpendicular to the external electric field) are reproduced equally well with the same internal field components.
basis is restricted to all of the z-electron states in the fourorbital model,I2 namely, the Q and B (Soret) states. The x and y components of the electric field at the porphin center are given by the superposition of internal and extemal fields. As in our previous anal~sis,~ we assume that in the chromophore reference frame the internal electric field is fixed while, like the protein molecules themselves, the external field is randomly oriented. The zero-field energies are chosen so that diagonalization of eq 1 for zero external field (internal field only) gives the experimentally observed transition energies; the transition dipoles (0.102, 0.152, 1.104, and 1.151 D, respectively) come from theory.I3 Hole profiles for given internal and external electric fields are calculated in two steps. First, the excitation energy is calculated by diagonalizing eq 1 and weighted by the
fourth power of the projection of ,UQ, on the light polarization for all relative orientations of laboratory and molecular frames. This profile is then convolved with the zero extemal electric field hole profile. For a given local field factor, electric-field-induced change in a photochemical hole profile is determined by only two adjustable parameters, the x and y components of the internal field. These are sharply determined by fitting profiles for two polarizations (buming and observing light polarized parallel or perpendicular to the external field). We have found that as long as the externally applied fields are small compared to the internal field the orientation (relative sizes of the components) of the internal field that reproduces the measured profiles is independent of the local field factor that multiplies the externally applied field to give its value at the porphyrin but that the magnitude is inversely related. That is, it is the products of intemal field components times the local field factor that are sharply and unambiguously determined. Here we consider only data taken near the center of the inhomogeneously broadened band. Figure 2 compares typical measured and calculated profiles: the fit is exact. With the local field factor 2.1 eight profiles (four different external fields with light polarized parallel and four different external fields with light polarized perpendicular to the applied field) are simultaneously fit at the quantitative level shown in Figure 2 by the internal field components E, = 29.9 f 0.3 MV/cm and Ey = 38.6 f 0.7 MV/cm.I4 We now turn attention to the choice of local field factor. Since the porphyrin environment in myoglobin is hydrophobic (especially in the case of the free base), the fact that the internal field is more than an order of magnitude stronger than those found in n-alkane matrixes clearly indicates that there are point charges within a few angstroms of the porphin center. This is the case if the carboxyl groups on the propionic acid side chains are deprotonated, an idea that is supported by the neutron diffraction study on the closely related system sperm whale carbonmonoxymyoglobin.~5~'6 The contribution to the electric field at the center of the porphyrin ring (intersection of axes through diagonally opposite pyrrole nitrogens) from the deprotonated carboxy groups (represented as point charges) can be calculated using the X-ray structural coordinates of horse metmyoglobin:" the components along the two N-N diagonals are 29.4 and 36.1 MV/cm. In our initial analysis we used a local field factor of 0.78, which corrects for the effect of the glass capillary around the sample and the dielectric shielding by the glycerol/water glass'* but neglects the microscopic contributions inside a sphere that is large enough to justify the continuum assumption. There is a further multiplicative factor that comes from the polarization of protein units in the near neighborhood of the porphyrin. That these microscopic contributions are significant is evident in the fact that if they are neglected, the best fit x and y internal field components are unreasonably large (81.2 and 101.4 MV/cm, respectively). That the charged carboxy groups of the deprotonated propionic acid side chains make the principal contribution to the internal field is supported both by physical reasonableness and the orientation of the best-fit field (the orientation does not depend on local field factor). Choosing 2.7 for the microscopic contribution to the local field factor to get an overall local field factor of 2.1 gives best fit x and y intemal field components of 29.9 and 38.6 MV/cm, respectively, almost identical to the internal field components contributed by the deprotonated propionic acid side chains. Of course, other strongly polar groups, especially the water molecules solvating the protein, also contribute to the internal
Letters field. The crystal structure shows that the nearest water molecules are 7.9 8, from the porphyrin center. However, the maximum contribution from the 1.85 D dipole is 2.2 MV/cm. The cumulative effect of many water molecules is not proportional to their number since different, randomly oriented dipoles will interfere destructively. Thus, the total intemal electric field from solvent molecules cannot exceed a few MV/cm, so it is reasonable to expect the contribution from the propionic acid side chains to dominate. These conclusions can be definitively tested by experiments on myoglobin substituted with modified hemes where one of the propionic acid groups has been removed and by more detailed microscopic modeling analogous to that done for the octatetraeneln-alkane systems? this is in progress. Although this work is at a very early stage, we believe that it shows that using simple quantum mechanical models to quantitatively determine intemal electric fields at chromophores from the effects of extemal electric fields on photochemical hole profiles will have major impact on our understanding of charge separation in chromoproteins.
Acknowledgment. It is a pleasure to thank David Bocian for helpful and stimulating discussions. This research was supported by grants from the NIH (EY-06466) and the NSF (CHE-9523539). References and Notes (1) Gilson, M. K.; Straatsma, T. P.; McCammon, J. A.; Ripoll, D. R.; Faerman, C. H.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Science 1994, 263, 1276. (2) Tan, R. C.; Truong, T. N.; McCammon, J. A.; Sussman, J. L. Biochemistry 1993, 32, 401. (3) Grad], G.; Kohler, B. E.; Westerfield, C. J. Chem. Phys. 1992, 97, 6064. (4) Steffen, M. A,; Lao, K.; Boxer, S. G. Science 1994, 264, 810. (5) Kohler, B. E.; Woehl, J. C. J. Chem. Phys. 1995, 102, 7773. (6) Electric Field Effects In Molecular Systems Studied Via Persistent Hole Burning, Kohler, B. E., Personov, R. I., Woehl, J. C. In Laser Techniques in Chemistry (Techniques of Chemistry Series); Myers, A. B., Rizzo, T. R., Eds.; John Wiley: New York, 1995; Chapter 8. (7) Our preparation of apo-myoglobin followed the procedures of Teale, F. W. J. Biochim. Biophys. Acta 1959,35, 543. Wright, K. A,; Boxer, S. G. Biochemistry 1981, 20, 7546. The preparation of protoporphyrin-IX
J. Phys. Chem., Vol. 99, No. 45, 1995 16529 myoglobin (according to: Winterhalter, K. H.; Huehns, E. R. J. Biol. Chem. 1964, 239, 3699, on a Sephadex G25 column. Kaposi, A. D.; Fidy, J.: Stavrov, S. S.; Vanderkooi, J. M. J. Phys. Chem. 1993, 97, 6319) led to ca. 0.5 mM PPIX-Mb in 100 mM phosphate buffer, pH 7.0. (8) Volker, S.; van der Waals, J. H. Mol. Phys. 1976, 32, 1703. (9) Anex, B. G.; Umans, R. S. J. Am. Chem. SOC.1964, 86, 5026. (10) Gafert, J.; Friedrich, J.; Parak, F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2116. (1 1) There are some substantive differences. For example, we find no evidence for disorder in the intemal field and observe a systematic dependence of the Stark effect on bum frequency. These issues will be discussed in detail in a subsequent paper. (12) Gouterman, M.; Wagniere, G. H. J. Mol. Spectroscopy 1963, 11. 108.
(13) Baker, J. D.; Zemer, M. C. Chem. Phys. Lett. 1990, 175, 192. This paper reports f values: following instructions from Professor Zemer we calculated the RPA dipoles from the relation
p in Debye and B in cm-I.
(14) Even though there is a systematic variation of intemal field strength with bum frequency, the spread is less than 5%. This variation will be discussed in a subsequent paper. (15) Norvell, J. C.; Nunes, A. C.; Schoenbom, B. P. Science 1975,190, 568. (16) Cheng, X.; Schoenbom, B. P. J. Mol. Biol. 1991, 220, 381. (17) Evans, S. V.; Brayer, G. D. J. Mol. Biol. 1990, 213, 885-897. (18) The electric field Edle! in a dielectric (dielectric constant 61) contained in an infinitely long capillary (dielectric constant €2, inner and outer radii a and b, respectively) generated by a homogeneous extemal electric field Eo normal to the capillary axis is given by
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Thus, for our experiment where € 1 = 2.5, t? = 4.05, a = 0.44 mm, and b = 0.70 mm the local field factor that converts the externally applied field into the macroscopic cavity field in the glycerol/water glass is 0.78.
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