Proc. Nat. Acad. Sci. USA Vol. 69, No. 9, pp. 2622-2626, September 1972

Resonance Raman Spectra of Hemoglobin and Cytochrome c: Inverse Polarization and Vibronic Scattering (scattering tensor/porphyrin fluorescence)

THOMAS G. SPIRO AND THOMAS C. STREKAS Department of Chemistry, Princeton University, Princeton, New Jersey 08540

Communicated by Walter Kauzmann, July 11, 1972 Resonance Raman spectra of hemoglobin ABSTRACT and cytochrome c in dilute solution contain prominent bands that exhibit inverse polarization, i.e., the polarization vector of the incident radiation is rotated through 90' for 90' scattering, giving infinite depolarization ratios. This phenomenon is shown to require an antisymmetric molecular-scattering tensor. The antisymmetry, which is characteristic of resonance scattering, is associated with the form of a particular class of vibrations, A2,, of the tetragonal heme chromophores. The dependence of the resonance Raman spectra on the wavelength of the exciting radiation, as well as their polarization properties, demonstrates that the prominent bands correspond to vibronically active modes of the first electronic transition of the heme proteins, and provide confirmation of Albrecht's vibronic theory of Raman intensities.

The polarization data can be used to assign the symmetry species of the vibrational bands, and there is then an excellent correlation with assignments for free-base porphyrins that are available from polarized fluorescence spectra (3-5). These assignments, together with the dependence of Raman intensity on the wavelength of the exciting radiation, make it apparent that we are dealing with the molecular vibrations that are vibronically active in the electronic absorption spectrum of the heme proteins, and provide experimental support for the vibronic theory of Raman intensities, developed by Albrecht. (8). EXPERIMENTAL The Raman spectrometer was based on a Spex 1401 double monochromator. The light was detected by DC amplification of the photocurrent of a cooled ITT FW130 photomultiplier. Excitation was provided by several laser lines from a Coherent Radiation 52G Ar+ or Ar+/Kr+ laser. The scattered light was analyzed with a polaroid disk, followed by a polarization scrambler to equalize the spectrometer response to different polarization directions. Oxyhemoglobin was prepared from human whole blood by

Recently, we have obtained resonance Raman spectra of hemoglobin (1) and of cytochrome c (2a), using laser excitation at frequencies within the visible absorption envelopes of these heme proteins. Good quality spectra were obtained at protein concentrations ranging from 10-' to 10-' M. They exhibit a rich pattern of bands from 600 to 1700 cm-', arising from vibrational modes of the heme chromophores. Distinctive changes are observed, especially in relative intensities, as a result of changes in oxidation state or in the state of ligation. Resonance Raman spectroscopy offers promise as a structural probe for these and other heme proteins. In addition to strong resonance enhancement, the spectra we have obtained display a very unusual feature from the perspective of normal Raman spectroscopy. Several of the most prominent bands exhibit inverse polarization, i.e., for these bands the light scattered at 900 is found to be polarized perpendicular to the polarization of the incident light when the incident light polarization is perpendicular to the scattering direction. The depolarization ratio Pi = IjIl1 is infinity, or close to it. Of the remaining bands, almost all are depolarized (Pl = 3/4). Polarized bands (p, < 3/4) are weak or absent. This is in sharp contrast to the situation obtained for normal Raman spectra, and for previously reported resonance Raman spectra, in which the most prominent bands are polarized. The phenomenon of inverse-scattering polarization for an assembly of randomly oriented molecules was predicted on theoretical grounds nearly 40 years ago by Placzek (2). It requires that the scattering tensor be antisymmetric. The scattering tensor may become asymmetric as the exciting radiation approaches resonance with an electronic transition of the molecule. We believe that the spectra reported here contain the first experimental detection of this effect.

standard methods (24). Horse-heart cytochrome c (Sigma Type III) was used as purchased. Concentrations were determined spectrophotometrically from the known absorptivities (25, 26). Cytochrome c was kept in the reduced state with excess sodium dithionite, whose Raman scattering was negligible in relation to the spectrum of the protein. Spectra were obtained from diluted samples in 1-mm diameter quartz capillary tubes by use of transverse excitation. This geometry has the advantage of keeping the light path through the sample, and therefore the fraction of the light absorbed by the chromophore is kept to a minimum. Ammonium sulfate was added as an internal standard for intensity measurements. Peak areas were measured and compared with that of the vPSO4-1 band (983 cm-'), after correcting for variations in spectrometer response with wavelength by use of a response curve obtained with a standard quartziodine source. Sample concentrations and light-path lengths were sufficiently small that corrections due to absorption by the sample were negligible. RESULTS

Fig. 1 shows spectra obtained for oxyhemoglobin and reduced cytochrome c in parallel and perpendicular polarization. For both proteins, several prominent bands (labeled ip) are active in perpendicular, but absent (hemoglobin) or weak (cyto2622

Proc. Nat. Acad. Sci. USA 69

Resonance Raman Spectra of Hemoglobin and Cytochrome c

(1972)

2623

II

Ii

r

600

III

II, 1600

1500

1400

1300

1200

1100

1000 900

I I I

,

800

700

600

CMl1 FIG. 1. Resonance Raman spectra of oxyhemoglobin (bottom pair of curves) and ferrocytochrome c (top pair). The scattering geometry is shown schematically in the diagram at the top. Both the direction and the polarization vector of the incident laser radiation are perpendicular to the scattering direction. The scattered radiation is analyzed into components perpendicular (I1) and parallel (III) to the incident polarization vector. The exciting wavelength was 568.2 nm (5682 it) for HbO2 and 514.5 nm (5145 i) for cytochrome c. The slit width was about 10 cm-'. Concentrations about 0.5 mM for HbO2 and about 0.5 mM for cytochrome c.

chrome c) in parallel polarization. We estimate that the depolarization ratio exceeds 100 for the strongest bands. Almost all the remaining bands exhibit depolarization ratios close to the normal depolarized value, 3/4 (labeled dp). Only very weak bands are found that are active in parallel but not in perpendicular polarization (labeled p). Spectra have been obtained as well for several hemoglobin derivatives (1) and for oxidized cytochrome c (2a). Several spectral changes are observed, particularly in regard to relative intensities. Some of these differences may prove useful in probing for specific structural features (1). The overall polarization pattern remains the same for all the derivatives, however, and aids materially in correlating bands from one

derivative to another. The variation of relative intensity with exciting wavelength (excitation profile) for a number of the prominent bands of the ferrocytochrome c spectrum is presented in Fig. 2, and compared with the visible absorption spectrum of the molecule. DISCUSSION

Scattering Tensor Asymmetry and Anomtalous Polarization. The usual expression for the depolarization ratio for randomly

Chemistry: Spiro and StrekasPProc. Nat. Acad. Sci. USA 69

2624

I

(1972)

In his classic treatment of the Raman effect, Placzek (2) considered this problem and gave the following expression for the depolarization ratio:

[2]

P= (3g' + 5ga)/(10 o + 4w8) w

-J

where:

-J

9 9t

LI)

=

3rX2

=

1/3[(a=

-

Ca,,)2 + (at

-

aXzz)2 + (at,,

-

CaZZ)2]

'/2[(aCT + ax)2 + (axZ + aZX) 2 + (aZ,, + aY,)'] + (aTxza2X)' + (ayz,,- az) 2] 9= '/2,(a,, R- aTx) Z These three quantities are called, respectively, the isotropic, the quadrupole, and the magnetic dipole components of the +

0-

z z

TABLE 1. Vibrational assignments for porphyrin derivatives

w w -J

Fluorescencet

Resonance Raman*

LOG

ip(A2g)

Bi,

~~~~~0.5

M(nm)

720 W

(740) FIG. 2. Excitation profiles for the prominent Raman bands of ferrocytochrome c, and the electronic absorption spectrum ), both on a logarithmic scale. The points represent in( tensities of the indicated Raman band, measured relative to the vi sulfate peak from (NH4)2SO4 internal standard, with the available Ar + and Kr + laser lines. The profiles are displaced for clarity on an arbitrary log intensity scale. The available points were fit to the standard Gaussian curves displayed here by a DuPont 303 curve resolver.

955 M 974 VW

(985) 1014 VW

1057 W 1133 S (1132 M)

=

1176 M

1/2[(a

-

a1,)I +

392/(45r2 + 4y)

1/s(aC

+ a11

+ (a= (a,,,,

(1175 S)

[1]

+ azz)

-

az)2 a,,)']

+

3(az2,

+

[1 ] is valid only if the tensor is symmetric, i.e., aij

az,2 =

+

az'2)

aji.

Theory predicts that the scattering tensor may become asymmetric when the exciting radiation approaches resonance with an electronic transition of the molecule (2, 10-13). There

are, of course, as many tensors as processes. They are properly labeled *

(1150W) 1171 W

and 'y represents the anisotropy of the tensor: y2=

930 W 977 W (975 W) 996 W 1075 W 1085 W

(1135)

where a is the isotropic part of the molecular scattering tensor*: (X

756 S (753 M)

786 W (790)

1150W

oriented molecules is: (9): pi =

B2.)

604 W

520 530 540 550 560 570 580

510

dp(B1g and

155 VW (240) 310W

l

z 0

470 480 490 500

Ag

Resonance Rmn Raman*

there are scattering (aqi)mn, where i and j are coordinate axes in the molecular frame, and n and m are the initial and final states, respectively, of the molecule. Rayleigh scattering corresponds to m n, and Raman scattering corresponds to m n. In the present discussion, the subscripts m and n are omitted for clarity, and it is implicit that "the scattering tensor" refers to a specific transition (normally vibrational) of the molecule. =

1222 W

(1225) 1305 S (1313 VS) 1342 SV 1363 W 1395 VW (1400 S) 1431 W 1470 W

1319 M

(1345) 1361 W 1389 S 1409 VW

(1365 M)

1456 VW

1497 M 1533 W

(1550) 1589 VS (1585 VS)

1210 W 1225 M (1228 M)

1605 VS

1551 M (1547 M) 1571 M (1600 W)

(1585) 1615 VS

1638 M

(1622 M) * Data from this work for oxyhemoglobin

and, in parentheses,

ferrocytochrome c. ip = inverse polarized, dp = depolarized. t Data for porphine, from ref. 3, and, in parentheses, for protoporphyrin IX from ref. 7. t Symbols: W = weak, M = medium, S = strong, V = very.

Proc. Nat. Acad. Sci. USA 69

(1972)

Resonance Raman Spectra of Hemoglobin and Cytochrome c

scattering tensor. A direct connection with Eq. [1 ] can more conveniently be made if we redefine the quadrupole and magnetic dipole quantities slightly, as two anisotropy invariants of the tensor, a symmetric part: 'YS 2

=

a/29S

7as 2

=

3/29a

and an asymmetric part: Then 5yas2)/(45&2 + 4'S2)

[31 If the tensor is symmetric, then yS2 = 72, yas2 = 0, and Eq. [3] reduces to Eq. [1]. Eq. [3] can be derived by the usual averaging procedures (9), keeping track of all tensor elements individually. It is well known that Eq. [1], which applies far from resonance, predicts that pi can be no higher than 3/4, the depolarized values shared by all nontotally symmetric vibrational modes, for which a = 0. For totally symmetric modes, a has a finite value and Pi ranges between 3/4 and zero. It is easy to see from Eq. [3 ] how the polarization could become anomalous (pi > 3/4) near resonance, when the tensor is not symmetric. For nontotally symmetric modes: Pi = (3 yS2 +

PI = 3/4 +

5/4(yas2)/(7s2)

Since neither ya,2 nor 7y2 can be negative, the polarization must be anomalous if the scattering tensor is asymmetric. For totally symmetric modes y7as will be zero, since off-diagonal matrix elements are zero, if the molecular symmetry is Cn,, Dn} or higher (14). Otherwise, it is conceivable that even totally symmetric modes could exhibit anomalous polarization, the condition being that 7yas2 > 27/4 2. The phenomenon of inverse polarization, Pi = oo, requires that both a and yY be zero, while -as retains a finite value. This can only happen in the case of a nontotally symmetric vibration for which the scattering tensor is antisymmetric, i.e., atJ = - aji. Such modes are forbidden in nonresonance scattering. Porphyrin Optical Spectra and Assignment of Vibronic Miodes. The chromophore of both oxyhemoglobin and ferrocytochrome c is the dianion of protoporphyrin IX with a ferrous ion at its center (15). In hemoglobin, the iron-porphyrin complex (heme) is attached to the protein only by coordination of an imidazole side chain to an axial site on the iron atom (16). The other axial position is occupied by an oxygen molecule. In cytochrome c, the axial ligands are imidazole and methionine side chains, and, in addition, the heme is bound to the protein through thioether links at its periphery (17). Both molecules contain low-spin iron(II) and are diamagnetic. The optical spectra of all low-spin metalloporphyrins are similar, containing an intense absorption, called the Soret, or ,y band near 400 nm (4000 i) and a weaker pair of bands in the visible region, called Q bands, or a and ,3, in order of decreasing wavelength (15). In molecular orbital terms (18), these features are described as arising from two electronic transitions, both terminating in the lowest empty orbital, which is degenerate and of e. symmetry, if we assume a D4h model for the metalloporphyrin. The a transition originates in the highest filled orbital, which is of a2. symmetry, while the y transition originates in the next highest filled orbital, which

2625

is of a,. symmetry. The two transitions are both allowed by symmetry (E.), but they are strongly mixed by configuration interaction, with the y transition gaining most of the intensity. The (3 band is assigned to 0-1 vibrational components of the first electronic transition (6, 19, 20). According to Albrecht's development of Raman intensity theory (8), the vibrational modes that show greatest intensity increase as resonance is approached correspond to those that provide vibronic intensity in the electronic-absorption spectrum. Fig. 2 demonstrates that the excitation profiles of the most intense bands in the ferrocytochrome c spectrum fall within the envelope of the (3 band in the electronic-absorption spectrum. Moreover, the excitation profiles shift systematically to lower frequency with decreasing vibrational frequency, and the positions of their maxima are in satisfactory agreement with vo-, (marked by arrows), calculated by adding to the frequency of the a band (voo) the frequency shift of the Raman band in question. Although these frequency shifts correspond to ground-state vibrational frequencies, little difference is expected for the excited-state vibrational frequencies of these large molecules (3-5). It is evident that the observed Raman bands do in fact correspond to the individual vibronic components of the d band, which are not resolved in the electronic absorption spectrum. The vibrations that are capable of mixing the two electronic transitions and borrowing intensity from the y band are of B10, B2,, and A20 symmetry (6, 20). Accordingly, we expect to find these modes in the resonance Raman spectra of metalloporphyrins, and we should not be surprised that A,, modes, which give rise to polarized bands, are weak or absent, since Ala modes do not contribute appreciably to the vibronic intensity. The expected polarizations of the various symmetry classes can easily be determined by reference to tables of the forms of the scattering tensors. These have been constructed for various molecular-point groups by Ovander (21), and more recently (with corrections) by McClain (14). In D4h symmetry, Bi, modes have az = - ay,, while B20 modes have aer = ay.; all other tensor elements being zero. Therefore, p, = 3/4 for both symmetry classes. A20, modes (inactive in nonresonance scattering) have az, = -ay,, and therefore pi = a. We assign the inverse polarized bands in our resonance Raman spectra to A2. and the depolarized bands to B1i or B227.t Comparison with Fluorescence Data. These assignments of the vibrational modes of the heme proteins are strengthened by comparison with fluorescence data on porphine (3) and protoporphyrin IX (7), for which vibrational assignments have been made on the basis of fluorescence polarization. In these "free-base" porphyrins, a central metal ion is replaced by two protons, and the effective molecular symmetry is lowered from D4h to D2h. The correlation of vibrational modes is as follows: Both A2. and B2g (D4h) correlate with Bj,0 (D2h) and Bi, (D4h) correlates with A,(D2h). t The same statements apply to the A2, B,, and B2 modes in the C41, point group, which is actually more appropriate to hemoglobin and cytochrome c, since the two axial ligands are not the same. It is worth noting that Placzek (2), in predicting the appearance near resonance of new Raman lines with inverse

polarization, specifically cited the A2 mode of the C4v point group as an example.

2626

Chemistry: Spiro and StrekasPProc.. Nat. Acad. Sci. USA 69

Table 1 lists resonance Raman frequency shifts for oxyhemoglobin and (in parentheses) ferrocytochrome c, classified into inverse polarized (column 1) and depolarized (column 4) modes. For comparison, fluorescence-frequency intervals are listed for porphine and (in parentheses) for protoporphyrin IX, classified into B10 (column 2) and A, (column 3) modes. According to our assignments, the numbers in column 1 should correlate with those of column 2, while the numbers in column 4 should correlate with those in either column 2 or 3. The frequency match is highly satisfactory in view of the chemical differences among the derivatives being compared. Most of the heme-protein modes assigned to A4 do indeed find correspondence with Bl0 modes of the free-base porphyrins. Among the depolarized Raman bands, most find correspondence with An, modes of the porphyrins, suggesting that B10 modes predominate over B2, modes in the hemeprotein spectra. Lower Symmetry in Cytochrome c. Examination of the spectra in Fig. 1 shows that, while the bands labeled ip disappear almost completely in parallel polarization for hemoglobin, they retain appreciable intensity (notably at 1313 and 1400 cm-') in the parallel spectrum of cytochrome c, i.e., for hemoglobin the depolarization ratios of the anomalous bands have p co, while for cytochrome c these bands have 3/4 < p < co. As indicated above, the A2, modes in D4O symmetry have p = co, BiD and B2, modes have p = 3/4, and AI modes have p < 3/4. There are also E0-type Raman-active vibrational modes, and these could have anomalous polarization, since a,, $ a,. and ay1, $ ayi, all other tensor elements being zero (14). But these modes, which must have a component in the z direction, are forbidden by symmetry from mixing the x,y polarized r-;r * porphyrin electronic transitions. Consequently, their assignment to the intense, anomalous resonance Raman bands is excluded. There are two possibilities to account for depolarization ratios between 3/4 and co for cytochrome c. One is accidental degeneracy, with polarized or depolarized modes falling at frequencies within experimental error of inverse polarized modes. While accidental degeneracy is not uncommon in complicated molecules, it would be surprising to find it occurring for several of the cytochrome c modes but for none of the hemoglobin modes. The more likely possibility is that the effective heme symmetry of cytochrome c is lower than that of hemoglobin. For both chromophores the ideal 4-fold symmetry is broken by the arrangement of substituents on the periphery of the porphyrin ring, and by various van der Waals and ionic contacts with the polypeptide chain. In addition, the cytochrome c symmetry would be expected to be affected by its thioether links to the protein. Both electron-spin resonance (22) and polarized single-crystal absorption spectra (23) demonstrate substantial rhombic distortion for ferricytochrome c. Lowering of the effective symmetry to, say, D2h would produce depolarization ratios less than infinity. The A20 modes in D4O symmetry correlate with B,0 in D2h symmetry, for which a. $ - ay;, (14), and therefore 3/4 < p < co, ac-

(1972)

cording to Eq. [3 ]. Therefore, the retention of significant intensity for anomalous bands in the parallel spectrum of cytochrome c is consistent with the molecule having effective symmetry significantly lower than 4-fold. The observation suggests that resonance Raman depolarization ratios may provide a sensitive test for symmetry. This work was supported by U. S. Public Health Service Grant HE 12526 from the National Heart and Lung Institute. We thank Dr. D. Hatzenbuhler and the Aerochem Corp. for the use of their Ar+/Kr+ laser Raman spectrometer for recording some of our spectra.

1. Spiro, T. G. & Strekas, T. C. (1972) Biochim. Biophys. Acta 263, 830-833. 2. Placzek, G. (1934) in Rayleigh and Raman Scattering, UCRL Trans. No. 526 L from: "Handbuch der Radiologie," ed. Marx, E., Leipzig, Akademische Verlagsgesellechaft VI 2, 2a. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16.

17. 18. 19.

20.

21. 22. 23. 24. 25.

209-374; available from National Technical Information Service, U.S. Dept. of Commerce, Springfield, Va. Spiro, T. G. & Strekas, T. C. (1972) Biochim. Biophys. Acta 278, 188-192. Sevchenko, A. N., Solov'ev, K. N., Shkirman, S. F. & Sarzhevskaya, M. V. (1963) Proc. Acad. Sci. USSR Phys. Chem. Sect. (English trans.) 153, 1151-1155. Sevchenko, A. N., Mashenkov, V. A. & Solov'ev, K. N., (1968) Sov. Phys. Dokl. 13, 213-216. Sevchenko, A. N., Solov'ev, K. N., Mashenkov, V. A. & Shkirman, S. F. (1966) Sov. Phys. Dokl. 10, 778-780. Solov'ev, K. N., (1961) Opt. Spectroec. 10, 389-393. Litvin, F. F. & Personov, R. I. (1961) Sov. Phys. Dokl. 6, 134-136. Albrecht, A. C. (1961) J. Chem. Phys. 34, 1476-1484. Wilson, E. B., Decius, J. C. & Cross, P. C. (1955) Molecular Vibrations (McGraw-Hill, New York). Ovander, L. N. (1966) Opt. Spectrosc. Suppl. 2, 71-73. Ovander, L. N. (1964) Opt. Spectroec. 16, 401-402. Teller, E. (1939) in The Raman Effect and Its Chemical Applications, eds. Hibben, J. H., & Teller' E. (Reinhold, New York), A.C.S. Monograph Series, pp 120-121. Verlan, E. M. (1966) Opt. Spectroec. 20, 557-560. McClain, W. M. (1971) J. Chem. Phys. 55, 2789-2796. Smith, D. W. & Williams, R. J. P. (1970) in Structure and and Bonding (Springer-Verlag, New York), Vol. 7, pp 1-45. Perutz, M. F., Muirhead, H., Cox, J. M. & Goaman, L. C. G. (1968) Nature 219, 131-139. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L. & Margolaish, E. (1971) J. Biol. Chem. 246, 1511-1533. Gouterman, M. (1961) J. Mol. Spectrosc. 6, 138-163. Platt, J. R. (1956) in Radiation Biology (McGraw-Hill, New York), Vol. 3, chap. 2. Perrin, M. H., Gouterman, & Perrin, C. L. (1969) J. Chem. Phys. 50, 4137-4150. Ovander, L. N. (1960) Opt. Spectroec. 9, 302-304. Salmeen, I. & Palmer, G. (1968) J. Chem. Phys. 48, 20492052. Eaton, W. A. & Hochstrasser, R. M. (1971) J. Chem. Phys. 46, 2533-2539. Drabkin, D. L. (1966) J. Biol. Chem. 164, 703-723. Antonini, E. & Brunori, M. (1971) Hemoglobin and Myoglobin in Their Reaction with Ligands (North Holland

Publishing Co., Amsterdam).

26. Margolaish, E. & Frohwirt, N. (1959) Biochem. J. 71, 570572.