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Spectroscopic properties of crystals and monolayers of chlorophyll and related compounds
Spectroscopic Properties of Crystals and Monolayers of Chlorophyll and Related Compounds’
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HOLT,
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497
SPECTROSCOPY OF CHLOROPHYLL CRYSTALS
this may mean that the molecules are arranged perpendicularly to the layer plane instead of under a 55” angle to it (12.8jsin 55” E 16). The oblique stacking of t’he chlorophyllide molecules may be caused by the affinity of magnesium t,o water, or to polar groups in neighboring molecules. 3. PREPARATION OF MICROCRYSTSLS AXI MONOLAYERS, MEASUREMENT OF THEIR TRASSMISSIOK SPECTRil
AND
We prepared suspensions of microcrystals by diluting acetonic solutions with water, and measured their transmission spectra. The crystals became too opaque when their thickness approached 100 rnp. Above this limit (i.e., with crystals consisting of more than 100 molecular layersj, only reflection spectra can be observed. Chlorophyllide crystals with linear dimensions less than 100 rnp recrystallize in suspension into larger ones. By lowering the temperature, this transformation could be slowed down sufficiently to measure the absorption spectra in the different stages of crystal growth. For this purpose, 0.1 ml. of acetonic solution of ethyl chlorophyllide a (optical density at 660 rnk, -500) was cooled to 0°C. and diluted rapidly with 50 ml. of water, also at 0°C. The subsequent spectral changes were followed by means of a Beckman spectrophotometer (cf. Figs. 2 and 3).
WAVE LENGTH (M/d
FIG. 2. Transmission
spectra of et.hyl chlorophyllide microcrystals at different stages of growth. Curve 1: before addition of water to an acetonic solution of the pigment Curves 2-8: 1, 5, 10, 15, 20, 30, ant1 50 min. after the atltiition of water.
498
JACOBS,
HOLT,
KROMHOUT
AND
RABINOWITCH
WAVE LENGTH #.4/d
FIG. 3. Same as in Fig. 2. Curve 9: 80 min. after addition of water; curve 10: 120 min. after addition of water. Dashed line: contribution of scattering to curve 10 (long wavelength “tail” matched to that of the transmission spectrum).
In a separate experiment, a suspension of larger crystals was prepared by mixing 1 ml. of a more dilute pigment solution (optical density, log (1,/I) LX 50 at 600 mp), with 2 ml. of water; its absorption spectrum is shown in Fig. 4. Figure 5 is the reflection spectrum of a suspension of still larger crystals. Figure 6 shows the transmission spectrum of a suspension of microcrystals of methyl bacteriochlorophyllide. An improvement of Hanson’s (8) method for the preparation of monomolecular layers of chlorophyllide on water was described else-
WAVE LENGTH (M/d FIG. 4. Transmission spectra of “large” curve). The dashed curve is the contribution
chlorophyllide of scattering,
a microcrystals (solid “matched” as in Fig.
SPECTROSCOPY
OF
CHLOROPHYLL
WAVELENGTH
499
CRYSTALS
tt’nEJ)
FIG. 5. Reflection spectrum of largest chlorophyllide
crystals.
WAVE LENGTH (M,uu) FIG.
6. Transmission spectrum of methyl bacteriochlorophyllidemicrocrpstals Dashed line: bacteriochlorophyllide in molecular solution.
where (10). The transmission spectrum of a monomolecular layer of ethyl chlorophyllide a is shown in Fig. 7. The ordinate is the absolute optical density of a single monolayer, derived from determinations on stacks of 5-10 glass plates, each carrying one picked-up monolayer. Scattering
Correction
The transmission curves of suspensions must be corrected for scattering, which changes strongly in the neighborhood of absorption bands. Mie (II), Steubing (la), and Gans (13) have described this phenomenon in metallic
sols.
Similarly to the latter, pigment suspensions also show maxima of scattering on the low-frequency side of the absorption bands. We measured the fraction of a monochromatic beam scattered at right angle
500
JACOBS,
HOLT,
KROMHOUT
AND
RABINOWITCH
WAVE LENGTH (M&u) FIG.
7. Transmission
spectrum of a monomolecular layer of ethyl Dashed line: same pigment in solution.
chlorophyllide.
by a l-mm. layer of a dilute crystal suspension.2 The relative curves obtained in this way were “normalized” by matching their long wavelength “tails” with those of the transmission curves, on the plausible assumption that in this region opacity must be due entirely to scattering. The observed optical density of a suspension, D,,, , is related to the fractional absorption (A,) and fractional scatt’cring (AS’,) of a single particle, by the equation
L
1
Dsu’ = G In 1 - NaZ(A, + 8,)
(1)
where L = length of the absorption cell, 1 = average thickness of a particle, a = average cross section presented by a particle to the light beam, and N = number of particles per unit volume of suspension. Equation (1) was derived by Duysens (14) by means of probability considerations. In a simpler way, it can be obtained if one asks what light attenuation will be produced by L/l absorbing and scattering layers, each of which transmits the fraction [I - NaZ (A, + X,)] of the light falling on it. For Nal < 1 (i.e., a small ratio of aggregated volume of the particles to the total volume of the suspension) Eq. (1) is reduced to
D BUS = NaL (A, + f&l,
(14
2 More detailed measurements of selective scattering by pigment suspensions and live cells have been since carried out by Latimer in our laboratory (13~1).
I),,,, = .YflId III I (I -
(-I ,) + S,,) 1.
(1,))
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SPECTROSCOPY
OF
CHLOROPHYLL
TSBLE
I
Band Shifts in Pigment
Observed and Calculated
Red absorption peak
Compound
I
Band shift
transition
Ethyl chlorophyllide a Ethyl chlorophyllide b Methyl bacteriochlorophyllide Ethyl pheophorbide Ethyl pheophorbide
a b
n From x-ray data. b The transition dipole
Experimental in “large” crystals AE
cm.-1
cm.-’
x 10:
x102
1 1.1
0.38
1.9
1.9
I 1 1.1
0.28
1.4
1.i
i
1.1
0.79
4.4
1.9
0.89 0.89
0.31 0.27
1.2 1.1
1.3 1.1
710
-
’ 632
860
-
640
715 690
-
652 642
fiC is related
rheoretial, for tw dimensional crystals (Arrr)
648
mP
730
Crystals
I
n mono- isolated layers molecules (extrap.)
740
503
CRYSTALS
/
to f by the equation
47Gmcfi,Z j=--.--3e2hXo where XI, is the wavelength of “pure” The f values in Table I were calculated ether.
electronic transition in isolated by integration of the absorption
molecule. bands in
it may be a coincidence, that the band shift stops at the same size at which spontaneous recrystallization into larger crystals ceases. The absorption band of the smallest observed crystalline particles lay at 715 rnp (curves 2-7 in Fig. 2); but curves 2 and 3 suggest that still smaller crystals may occur, with absorption peaks between 675 and 715 mp. The shape of the red absorption band of the microcrystals is similar to that of the solution, but the band is about twice as wide (instead of much narrower, as in isocyanin micelles). Table I shows a comparison of the limiting band shifts in five pigments. The band positions of “isolated molecules” were extrapolated by Katz and Wassink’s procedure (15).
504
JrlCOBS,
HOLT,
KROMHOUT
5. ~UISCELLANEOUS
AND
RABINOWITCH
RESULTS
Amorphous Colloidal Form Figure 2 suggests that, immediately upon dilution, a colloidal suspension is formed, probably consisting of acetone droplets suspended in water. The pigment binds the acetone and prevents it from diffusing into water. In this state, the red absorption band lies at about 675 mp. Low-Temperature Spectra Absorption measurements at 77°K. were made on thin deposits of crystals, and on stacks of monomolecular layers. Only a slight narrowing, and small additional displacement of the peaks toward the red, could be noted. Fluorescence An attempt to detect the fluorescence of the crystals failed, although the measuring device was sensitive enough to discover yields down to 0.001% in the red or 0.10 % in the infrared, up to 1 p. (Fluorescence beyond 1 p could have remained undetected.) Weakening of the Blue Band in Crystals In a crystalline chlorophyllide monolayer, the molecules probably are anchored to the water surface by the polar (C=O) groups in ring V, so that their short axes are aligned parallel to the layer plane. A similar orientation can be postulated in three-dimensional crystals. On bhe basis of the analysis of Longuet-Higgins, Rector and Platt (9)) one can suggest that the transition dipole of the red band oscillates in the plane of the layer, and that of the blue band largely at a 55” angle to it. This explains why the blue band is weakened, relative to the red band, in large microcrystals and monolayers (cf. Figs. 3 and 7) : with the assumed dipole orientation, the absorption of the blue band must be weaker for light passing normally through a crystal plane than for light traversing a sheet of randomly oriented molecules. Very small crystals are traversed by light in all directions; but the larger, flat crystals transmit significantly only beams that have passed normally to their plane. (In experiments with monolayers, only light of normal incidence has been studied.) Shift of the Red Band in Crystals Iiatz and Wassink (15) have estimated the position of the red band of isolated chlorophyll a molecules as 648 mp, by extrapolation from band
SPECTROSCOPY
OF
CHLOROPHYLL
CRYSTALS
0'0' 3
positions in solvents with different indices of refraction. In solution, the red band of chlorophyll a is shifted to 660-670 rnH by inter&ion between the chlorophyll molecule ane t,he polar (or polarizable) solvent molecules. At concent,rntions above 1O-3 molar (i.e. intermolecular distances of 100 A. or less), a dipole-dipole coupling must develop betn-ten t,hc pigment, molecules. In the ground state, this produces the van der Waals mutually induced &poles, inversely proportional attraction energy of LLCO to the sixth power of t,he distance. In the excited state, the dipole is created by light (each molecule having an equal probability of being excited) ; the interaction bct\vecn any t,wo molecules of an excited crystal thus becomes that of two virtual dipoles, with energy proportional t,o the inverse third power of the distance. Because of this interaction, the energy difference bct,wecn the ground stat,e and the cwited stat,e becomes smaller in t#he condensed state; t,he absorption bands arc t,hrreforc shiftc~d t o\rard the longer UXVCS.IMimnt~es sho\v that t,his int,eract,ion is suflicicnt to csplnin t,he observed band shift, \vithout, rwoursc to cluantutu-nlt!~hanic:ll reso~nn~c ci’f~cls wus~d by ovtrlapping of t,hc cigcbtifunction of adjacent, molecules. E’rrilkel (16) aiid Peierls (17) first, in\,estigat)cd the st.:atw of :I system c~~n~posc~lof many identical molecules. More c~losely related to our (a:‘~ are t,he studies by Davydo\- (18), who developed a gcncral tJheory of energy states of molecular crystals and applied it to nnpht,hnlrnc. ITe assumed that the coupling was significant8 only betwcn nearest neighbors \vit,hin t,he plana,r netivork of t,hc crystal. The results were, however, not in good agreement viit,h the much more complicat,ed experimental findings.3 Heller and Marcus (19) and Dexter and Heller (20) computed the energy states of an ideal cubic molecular crystal, taking into account t,he dipole-dipole int,eraction with distant, as well as with the iiearest neighbors. For the energy of transition to the excited stat,e E, in an infinite cubic lattice, they fourld: AE= E, -
E. = 8, -
&
-
$$rno pn2 j0(2kR0)
+ .je(2kRo)
(2)
where S, - z,, is the energy of the transition in the isolated molecule; pn itz dipole moment.; R,, , the density of molecules; R. , the lattice constant, in t,he ground state; ?z,the wave number of the exciting radiat,ion; and ,i,, and j, , spherical Bessel functions. Since, for visible radiat,ion, 3 The theory was recent.ly improved by Craig (21).
506
JACOBS,
HOLT,
HROMHOUT
ASD
RABINOWITCH
2&R,, << 1, the quanta absorbed by the crystals should be reduced by about E = - (4~/3) no pn2. Heller and Marcus noted that this quantity is derived from an expression analogous to the classical formula for the interaction energy of a dipole with an infinite lattice of dipoles of average ++ magnitude pn and phase difference e--22uk R. A similar calculation, applied to an infinite isotropic crystalline sheet and normal incidence of light, gives an energy shift:
where n, is the number of molecules per unit area, and Ro , the lattice constant. Since n, g noRo , the absorption band shift for an infinite 31 of that for an infinite three-dimensional monomolecular layer is about ,4 crystal. Crystal Size E$ect The calculation of energy shifts for crystals of finite size is difficult because of the role played by dipoles located near the surface and therefore not uniformly surrounded by other dipoles. The interaction energy of the cendral atom in a spherical or circular array is, in the three-dimensional case: AEsD = --gin0 ~,~j~ (2kR) + j2 (2kR) - j. (2nkRo) - j, (2nkRd and in the two-dimensional
(4a)
case:
AEm = -n,pn2
(1 - (Ro/R))
(46)
where R is the radius of the array. These functions are shown in Fig. 9. In the three-dimensional model, the interaction energy would vanish, for any radius of the spherical array, if it were not for the phase factor, e-i2*‘d. Due to the latter, the interaction energy becomes finite as the radius approaches the wavelength of the incident radiation. Thus, in this case, the contribution of the nearest spherical shells is negligible, while that of more distant shells is relatively large. In a two-dimensional array, on the other hand, the phase factor is 1 everywhere, and the interaction energy is a rapidly converging function of the radius. The dipoles located near the surface (or circumference) of the array must broaden the absorption band toward higher frequencies and increase the radius needed for saturation. (The latter will occur only when
SPECTROSCOPY
OF
CHLOROPHYLL
CRYSTALS
50'7
R/Ro 0. Absorption band shifts in ethyl chlorophyllide crystals of different size. The two solid lines are theoretical curves for the central atom in a circular and in a spherical isotropic array of dipoles, respectively. The “TV-o-dimellsinllal” curve is related t,o the upper scale, the “three-dimensional” curve, to the lower scale. Open circles, experimental points referred to upper scale; hlnck circles, experimental points referred t,o levier scale. FIG.
the number of molecules with “snturat’ed” shifts xv-ill have 1)ecome much larger than that of the “non-saturated” ones; in a two-dimensional array, the interaction energy of the central molecule is nearly “saturated” if this molecule has neighbors up to ten lattice constants; but for such an array to contain ten times as many ‘kat,urated” as “non-saturated” molecules, its radius must be equal t’o 200 lattice constants.) A4s the radius increases, the one-sided broadening of the band, caused by surface-near molecules, will become less significant, and the band will approach a limiting shape as well as a limiting position. The limiting shifts me have observed in crystals can be interpret’ed by Eq. (3), describing the interaction in an infinite isotropic two-dimensional sheet, somewhat better than by Eq. (2)) describing the interact,ion in an infinite three-dimensional array (cf. Table I). The relative shifts of the chlorophyllides and the pheophorbides are as expected from their relative oscillator strengths; but considerable discrepancy remains in the case of bacteriochlorophyllide. The spectra of monomolecular layers confirm that a preponderant part of the interaction in crystals is due to forces within a single plane. For example, crystalline monolayers of ethyl chlorophyllide a show about 90 % of the band shift of three-dimensional crystals.
508
JACOBS, HOLT, KROMHOUT AND RABlNOWITCH
.!2further test can be made by comparing the functions AE = f(R), as derived for a circular and a spherical lattice, with the experimentally found dependence of the band shift on crystal size. The four points marked in Fig. 9 are for crystals with absorption maxima at 718, 722, 738, and 740 rnp, respectively. The lattice constant was assumed to be 10 A., average of the two constants of chlorophyllide within the basal layer (4 A. and 15 A.). The theoretical curve for a tivo-dimensional lattice represents fairly well the behavior of crystals with absorption maxima below 730 m,u. Growth in the third dimension could bc responsible for the final shift of the band, from 730 to 740 rnp, in the largest microcrystals (a shift not observed in monolayers). The experimental data suggest that the “nearest neighbors” account for about 50 % of the band shift; if the interaction were not predominantly confined to a planar network, the role of the nearest neighbors would be negligible. 6. EXERGY Y~IGRATIOS IS CHLOROPHYLLIUE
CRYSTALS
One of the problems of photosynthesis we had in mind, when studying pigment crystals, was that of excitation energy migration, postulated in certain kinetic theories of photosynthesis (“photosynthetic unit”). In linear polymers of isocyanine, rapid migration of electIronic excitation along the micelle leads to its uncoupling from molecular vibrations, thus causing extreme narrowing of the absorption band and appearance of resonance fluorescence. Kane of these characteristics is encountered in chlorophyllide or chlorophyll crystals (22). Their absorption bands have, in the limiting state, about t,wice the width of those in solution, indicating increased, rather than decreased coupling of elect’ronic excitation with vibrations (probably because of the superposition of lattice vibrations upon intramolecular vibrations) . iin increased coupling with vibrations must decrease the overlapping of the fluorescence and the absorption band, characteristic of chlorophyll in sohnion. The fluorescence band is thus pushed towards the infrared-how far, we do JJOt know, since 110 fluorescence could be obser\-ed in chlorophyllidc microcrystals up to h = 1 p. This could mean either a fluorescence yield 1 p. In the first case, the lifetime of excitation would be ~10-3 x 1 X lo-8 = 1 X lo-l1 sec. Since the width of the absorption band indicates coupling with vibrations with periods of the order of IO-l3 sec., the “visiting time” of excitation in each molecule must be >10-‘3 sec.; consequently, the number of energy exchanges during a
SPECTROSCOPY
OF CHLOROPHYLL
CRYSTALS
509
period <1 X lo-” sec. must be 1 p, overlapping would be absent and exchange of excitation energy practically impossible. The large interaction term, calculated above for the energy of the excit,ed state, does not imply, in this case, an efficient excitation energy exchange over t,he volume (or area) which contributes t’o t’his term [as suggested earlier by Rahinowitch et al. (23j]. After excitation has taken place, it soon becomes “trapped” in a lat’tice point by local lattice distortion. True, excitat,ion is equally probable in every lattice point; but after it has actually taken place, energy soon becomes trapped in a definit,c location by local lattice distortion. 7. APPLICATIOS
TO CHLOROPHYLL
IS VIVO
Chlorophyll in vice has an absorption peak at 675-680 mp. In artificially prepared chlorophyll-protein complexes, this peak lies at 670 rnp; the further shift in ZCOcould be due to interaction between pigment molecules-perhaps, in a monomolecular layer with a structure similar t,o that of the “colloidal” chlorophyll monolayers on water (IO), which, too, have a band at 675-680 mp. Elect’ron microscopy suggests that, in Go, chlorophyll molecules probably arc arranged in monolayers, on proteiwlipoproteid interfaces. The arca available for each chlorophyll molecule was estimat,ed as about, 1 sq. mp for higher plants [Rabinomitch (5)] and as 2.3 sq. rnp for Euglena [Wolken and Schn-ertz (al-)]. Thomas ct al. (25) found specific areas between 0.8 and 3.8 sq. nip for four species of plants and one species of bacteria (lvhile the chlorophyll content and the total area of the lamellae in a chloroplast’ varied by as much as a factor of 104). According to Eq. (3), the interaction energy in a monomolecular layer is proportional to l,‘R” . The calculated value of A,Q’ it1 Table I was for R,, ” 1 mp. For t’he band to lie at 675 mp in a similarlp regular array, ZZn\vould bar-c t’o be 3 mp (or larger, since a part of the shift may be due to association \vit,h proteins and lipides). This is a much looser packing t,han t,hc limited area of the chhloroplast Iamcllae permit,s. IIowevcr, imperfect alignment of the pigment dipoles, indicated by the optical properties of chloroplasts, could reduce the dipole interactions without much decrease in density. In “liquid” chlorophyll monolayers on water (lo), the surface density is about one molecule per 1 sq. mP; \-et the red band is locat,ed at 675 mp.; while in crystalline chlorophylli& monolayers, where R2 = 0.6 sq. ~1,the band lies at 730 mp! Obviously, the difference must be due to a less orderly arrangement of molecules,
510
JACOBS,
HOLT,
KHOMHOUT
AND
RABIXOWITCH
made possible by distention of the layer, rather than to the distention itself. The migration of excit’ation energy in chlorophyll monolayers in zliL!o could be made more, rather than less, likely, by looser arrangement, because this loosening reduces t,he coupling wit,h lattice vibrations. Such a reduction is indicated by a sizeable fluorescence yield, of the order of 2-Y % [Latimer et al. (as)]. The longer excitation lifetime could permit several hundred energy transfers, without uncoupling of the elect,ronic excit,ation from molecular vibrations. Thus, optical data are not incompatible with the assumption of such a (relat,irely) limit,ed migration of excit,ation energy in the chlorophyll monolayers in e+o. SUMMARY
Microcrystals of ethyl chlorophyllides and pheophorbides a and b, and of methyl bacteriochlorophyllide, have been prepared and their absorption spectra measured. The main long-wave bands are shifted toward longer waves by l-2 X 10Rcm.-’ compared to their (extrapolated) position in free molecules (i.e., by JO-80 rnp from their position in organic solvents). The shift is a function of the size of the microcrystals, reaching L’saturation” in crystals about 0.5 p in diamet’er. An only slightly smaller monolayers, indicating that the intcrshift is observed in “crystalline” actions responsible for the shift occur mainly in one crystallographic plane. In “liquid” monolayers, the shift, is much smaller-similar to that in amorphous colloidal solutions. A theory of the band shift is given, based on electrostatic interaction in an isotropic array of (virtual) dipoles, created by light absorption (without consideration of the overlapping of eigenfunctions and consequent resonance effects). The absolute value of the maximum shift in ‘
SPECTItOSCOPY
OF CHLOROPHYLL
CRYSTALS
511
1. OBREIMOV, 1. v., AND PRIKHOT'KO, ;2., Physik. %. Sowjetunion 1, 203 (1932); 9, 34, 48 (1936). 2. See, e, g., SHEPPARD, S. E., Reus. Ned. Phys. 14, 303 (1942). 3. JELLY, 13. IZ., Nature 138, 1009 (1936); 139, 631 (1937). 4. SCHEIBE,G., Anger. Chem.60,51,218 (1937). 5. RABINOWITCH, E. I., Ann. Reo. Plant Physiol. 3, 229 (1952). 6. HOLT, A.S., .~SD JACOBS,E.I':.,~~~.J. Rotany 41,710 (19543. 7. HoLT,~L.S.,ASD JACOBS,E.E:.,A~U. ,J.Botany 41, 718 (1954). 8. is.4NSOx, E. A., Rec. trau. bot. nirerl. 36,183 (1939). 9. LONGUET-HIGGISS, H. C.,RECTOR,C. W., AND PLANT, J.R.,J.Chm. Ph?ys. 18, 1174 (1950). 10. JACOBS,E.~.,HOLT, A. ~.,~IABINOWITCII, E. I.,./. Chem. Phys.22, 142 (1954). 11. MIE, G., Ann. Physik 26,377 (1908). 12. STEUBING, Ann. Physik 26,329 (1908). 13. GANS, R., Ann. Physik37, 881 (1912). 13~. LATIJIER, l’.,Thesis, Univ. of Illinois,1956; LATIMER, P., ANI) RABINOWITCH, E., J. Chem. Phys. 24, 180 (1956). 14. DUYSENS, L. N. M., Riochim. et Biophys. Acta 19, 1 (1956). 15. KATZ,~.,AND~~ASSINK,~~.C.,~~~Z~T)~~~~~~~~,~~(~~~~). 16. FRENKEL, J., Phys. Reu. 37, 17 (1931); 37, 1276 (1931) ; Physik. Z. Sowjetunion 9, 158 (1936). li. l'EIERLS,R.,A~nn. Physikl3,905 (1932). 18. DAVYDOV, A. S.,J.EzptZ. Theoret, Phys. (U.S.S.R.) 18,210 (1948). 19. HELLER,W.,ANDMARC~S,A., Phys.Rev.84,809(1949). 20. DEXTER,~. L., AND HELLER,W. R., Phys.Rev. 91,273 (1953). 21. CRAIG,D.P.,J. Chenl. Soc.539, 2309 (1955);C~~1G,n. P., AND WALSH,J., J. Chern. Phys. 24, 471 (1956). 22. JACOBS, E. E., VATTER, a., AND HOLT, A. S., Arch. Bioche?,l. Hiophys. 53, 228 (1954). 23. RABINOWITCR, E. I., JXOBS, 13:.E., HOLT, A. S., ASI) KROMIIOUT, R. K., 2. Physik 133,261(1952). 24. WOLKEN, J.J., AND SCIIWERTZ, F. A.,J.Gen. Physiol.20, 111 (1953). 25. THOMAS, J. B.,&~IXSAERT, K., AND~~LLERS, P. F., Actabot.neerl.6,315(1956). 26. LATIMER, P., BANNISTER, T.T., AXD RABINOWITCH, E.,Science124, 585 (1956).
-c!)(i
J.\(‘OIIS,
HOLT,
I
.1SI)
~~.\~~ISO\VIT~‘H
497
SPECTROSCOPY OF CHLOROPHYLL CRYSTALS
this may mean that the molecules are arranged perpendicularly to the layer plane instead of under a 55” angle to it (12.8jsin 55” E 16). The oblique stacking of t’he chlorophyllide molecules may be caused by the affinity of magnesium t,o water, or to polar groups in neighboring molecules. 3. PREPARATION OF MICROCRYSTSLS AXI MONOLAYERS, MEASUREMENT OF THEIR TRASSMISSIOK SPECTRil
AND
We prepared suspensions of microcrystals by diluting acetonic solutions with water, and measured their transmission spectra. The crystals became too opaque when their thickness approached 100 rnp. Above this limit (i.e., with crystals consisting of more than 100 molecular layersj, only reflection spectra can be observed. Chlorophyllide crystals with linear dimensions less than 100 rnp recrystallize in suspension into larger ones. By lowering the temperature, this transformation could be slowed down sufficiently to measure the absorption spectra in the different stages of crystal growth. For this purpose, 0.1 ml. of acetonic solution of ethyl chlorophyllide a (optical density at 660 rnk, -500) was cooled to 0°C. and diluted rapidly with 50 ml. of water, also at 0°C. The subsequent spectral changes were followed by means of a Beckman spectrophotometer (cf. Figs. 2 and 3).
WAVE LENGTH (M/d
FIG. 2. Transmission
spectra of et.hyl chlorophyllide microcrystals at different stages of growth. Curve 1: before addition of water to an acetonic solution of the pigment Curves 2-8: 1, 5, 10, 15, 20, 30, ant1 50 min. after the atltiition of water.
498
JACOBS,
HOLT,
KROMHOUT
AND
RABINOWITCH
WAVE LENGTH #.4/d
FIG. 3. Same as in Fig. 2. Curve 9: 80 min. after addition of water; curve 10: 120 min. after addition of water. Dashed line: contribution of scattering to curve 10 (long wavelength “tail” matched to that of the transmission spectrum).
In a separate experiment, a suspension of larger crystals was prepared by mixing 1 ml. of a more dilute pigment solution (optical density, log (1,/I) LX 50 at 600 mp), with 2 ml. of water; its absorption spectrum is shown in Fig. 4. Figure 5 is the reflection spectrum of a suspension of still larger crystals. Figure 6 shows the transmission spectrum of a suspension of microcrystals of methyl bacteriochlorophyllide. An improvement of Hanson’s (8) method for the preparation of monomolecular layers of chlorophyllide on water was described else-
WAVE LENGTH (M/d FIG. 4. Transmission spectra of “large” curve). The dashed curve is the contribution
chlorophyllide of scattering,
a microcrystals (solid “matched” as in Fig.
SPECTROSCOPY
OF
CHLOROPHYLL
WAVELENGTH
499
CRYSTALS
tt’nEJ)
FIG. 5. Reflection spectrum of largest chlorophyllide
crystals.
WAVE LENGTH (M,uu) FIG.
6. Transmission spectrum of methyl bacteriochlorophyllidemicrocrpstals Dashed line: bacteriochlorophyllide in molecular solution.
where (10). The transmission spectrum of a monomolecular layer of ethyl chlorophyllide a is shown in Fig. 7. The ordinate is the absolute optical density of a single monolayer, derived from determinations on stacks of 5-10 glass plates, each carrying one picked-up monolayer. Scattering
Correction
The transmission curves of suspensions must be corrected for scattering, which changes strongly in the neighborhood of absorption bands. Mie (II), Steubing (la), and Gans (13) have described this phenomenon in metallic
sols.
Similarly to the latter, pigment suspensions also show maxima of scattering on the low-frequency side of the absorption bands. We measured the fraction of a monochromatic beam scattered at right angle
500
JACOBS,
HOLT,
KROMHOUT
AND
RABINOWITCH
WAVE LENGTH (M&u) FIG.
7. Transmission
spectrum of a monomolecular layer of ethyl Dashed line: same pigment in solution.
chlorophyllide.
by a l-mm. layer of a dilute crystal suspension.2 The relative curves obtained in this way were “normalized” by matching their long wavelength “tails” with those of the transmission curves, on the plausible assumption that in this region opacity must be due entirely to scattering. The observed optical density of a suspension, D,,, , is related to the fractional absorption (A,) and fractional scatt’cring (AS’,) of a single particle, by the equation
L
1
Dsu’ = G In 1 - NaZ(A, + 8,)
(1)
where L = length of the absorption cell, 1 = average thickness of a particle, a = average cross section presented by a particle to the light beam, and N = number of particles per unit volume of suspension. Equation (1) was derived by Duysens (14) by means of probability considerations. In a simpler way, it can be obtained if one asks what light attenuation will be produced by L/l absorbing and scattering layers, each of which transmits the fraction [I - NaZ (A, + X,)] of the light falling on it. For Nal < 1 (i.e., a small ratio of aggregated volume of the particles to the total volume of the suspension) Eq. (1) is reduced to
D BUS = NaL (A, + f&l,
(14
2 More detailed measurements of selective scattering by pigment suspensions and live cells have been since carried out by Latimer in our laboratory (13~1).
I),,,, = .YflId III I (I -
(-I ,) + S,,) 1.
(1,))
x2
J.\(‘OI3’;
.\
b 1 1101,‘1’,
lil~OMMO1
‘I’
.\ \ I)
1:.\I~ISO\~I’I’(‘H
(‘
SPECTROSCOPY
OF
CHLOROPHYLL
TSBLE
I
Band Shifts in Pigment
Observed and Calculated
Red absorption peak
Compound
I
Band shift
transition
Ethyl chlorophyllide a Ethyl chlorophyllide b Methyl bacteriochlorophyllide Ethyl pheophorbide Ethyl pheophorbide
a b
n From x-ray data. b The transition dipole
Experimental in “large” crystals AE
cm.-1
cm.-’
x 10:
x102
1 1.1
0.38
1.9
1.9
I 1 1.1
0.28
1.4
1.i
i
1.1
0.79
4.4
1.9
0.89 0.89
0.31 0.27
1.2 1.1
1.3 1.1
710
-
’ 632
860
-
640
715 690
-
652 642
fiC is related
rheoretial, for tw dimensional crystals (Arrr)
648
mP
730
Crystals
I
n mono- isolated layers molecules (extrap.)
740
503
CRYSTALS
/
to f by the equation
47Gmcfi,Z j=--.--3e2hXo where XI, is the wavelength of “pure” The f values in Table I were calculated ether.
electronic transition in isolated by integration of the absorption
molecule. bands in
it may be a coincidence, that the band shift stops at the same size at which spontaneous recrystallization into larger crystals ceases. The absorption band of the smallest observed crystalline particles lay at 715 rnp (curves 2-7 in Fig. 2); but curves 2 and 3 suggest that still smaller crystals may occur, with absorption peaks between 675 and 715 mp. The shape of the red absorption band of the microcrystals is similar to that of the solution, but the band is about twice as wide (instead of much narrower, as in isocyanin micelles). Table I shows a comparison of the limiting band shifts in five pigments. The band positions of “isolated molecules” were extrapolated by Katz and Wassink’s procedure (15).
504
JrlCOBS,
HOLT,
KROMHOUT
5. ~UISCELLANEOUS
AND
RABINOWITCH
RESULTS
Amorphous Colloidal Form Figure 2 suggests that, immediately upon dilution, a colloidal suspension is formed, probably consisting of acetone droplets suspended in water. The pigment binds the acetone and prevents it from diffusing into water. In this state, the red absorption band lies at about 675 mp. Low-Temperature Spectra Absorption measurements at 77°K. were made on thin deposits of crystals, and on stacks of monomolecular layers. Only a slight narrowing, and small additional displacement of the peaks toward the red, could be noted. Fluorescence An attempt to detect the fluorescence of the crystals failed, although the measuring device was sensitive enough to discover yields down to 0.001% in the red or 0.10 % in the infrared, up to 1 p. (Fluorescence beyond 1 p could have remained undetected.) Weakening of the Blue Band in Crystals In a crystalline chlorophyllide monolayer, the molecules probably are anchored to the water surface by the polar (C=O) groups in ring V, so that their short axes are aligned parallel to the layer plane. A similar orientation can be postulated in three-dimensional crystals. On bhe basis of the analysis of Longuet-Higgins, Rector and Platt (9)) one can suggest that the transition dipole of the red band oscillates in the plane of the layer, and that of the blue band largely at a 55” angle to it. This explains why the blue band is weakened, relative to the red band, in large microcrystals and monolayers (cf. Figs. 3 and 7) : with the assumed dipole orientation, the absorption of the blue band must be weaker for light passing normally through a crystal plane than for light traversing a sheet of randomly oriented molecules. Very small crystals are traversed by light in all directions; but the larger, flat crystals transmit significantly only beams that have passed normally to their plane. (In experiments with monolayers, only light of normal incidence has been studied.) Shift of the Red Band in Crystals Iiatz and Wassink (15) have estimated the position of the red band of isolated chlorophyll a molecules as 648 mp, by extrapolation from band
SPECTROSCOPY
OF
CHLOROPHYLL
CRYSTALS
0'0' 3
positions in solvents with different indices of refraction. In solution, the red band of chlorophyll a is shifted to 660-670 rnH by inter&ion between the chlorophyll molecule ane t,he polar (or polarizable) solvent molecules. At concent,rntions above 1O-3 molar (i.e. intermolecular distances of 100 A. or less), a dipole-dipole coupling must develop betn-ten t,hc pigment, molecules. In the ground state, this produces the van der Waals mutually induced &poles, inversely proportional attraction energy of LLCO to the sixth power of t,he distance. In the excited state, the dipole is created by light (each molecule having an equal probability of being excited) ; the interaction bct\vecn any t,wo molecules of an excited crystal thus becomes that of two virtual dipoles, with energy proportional t,o the inverse third power of the distance. Because of this interaction, the energy difference bct,wecn the ground stat,e and the cwited stat,e becomes smaller in t#he condensed state; t,he absorption bands arc t,hrreforc shiftc~d t o\rard the longer UXVCS.IMimnt~es sho\v that t,his int,eract,ion is suflicicnt to csplnin t,he observed band shift, \vithout, rwoursc to cluantutu-nlt!~hanic:ll reso~nn~c ci’f~cls wus~d by ovtrlapping of t,hc cigcbtifunction of adjacent, molecules. E’rrilkel (16) aiid Peierls (17) first, in\,estigat)cd the st.:atw of :I system c~~n~posc~lof many identical molecules. More c~losely related to our (a:‘~ are t,he studies by Davydo\- (18), who developed a gcncral tJheory of energy states of molecular crystals and applied it to nnpht,hnlrnc. ITe assumed that the coupling was significant8 only betwcn nearest neighbors \vit,hin t,he plana,r netivork of t,hc crystal. The results were, however, not in good agreement viit,h the much more complicat,ed experimental findings.3 Heller and Marcus (19) and Dexter and Heller (20) computed the energy states of an ideal cubic molecular crystal, taking into account t,he dipole-dipole int,eraction with distant, as well as with the iiearest neighbors. For the energy of transition to the excited stat,e E, in an infinite cubic lattice, they fourld: AE= E, -
E. = 8, -
&
-
$$rno pn2 j0(2kR0)
+ .je(2kRo)
(2)
where S, - z,, is the energy of the transition in the isolated molecule; pn itz dipole moment.; R,, , the density of molecules; R. , the lattice constant, in t,he ground state; ?z,the wave number of the exciting radiat,ion; and ,i,, and j, , spherical Bessel functions. Since, for visible radiat,ion, 3 The theory was recent.ly improved by Craig (21).
506
JACOBS,
HOLT,
HROMHOUT
ASD
RABINOWITCH
2&R,, << 1, the quanta absorbed by the crystals should be reduced by about E = - (4~/3) no pn2. Heller and Marcus noted that this quantity is derived from an expression analogous to the classical formula for the interaction energy of a dipole with an infinite lattice of dipoles of average ++ magnitude pn and phase difference e--22uk R. A similar calculation, applied to an infinite isotropic crystalline sheet and normal incidence of light, gives an energy shift:
where n, is the number of molecules per unit area, and Ro , the lattice constant. Since n, g noRo , the absorption band shift for an infinite 31 of that for an infinite three-dimensional monomolecular layer is about ,4 crystal. Crystal Size E$ect The calculation of energy shifts for crystals of finite size is difficult because of the role played by dipoles located near the surface and therefore not uniformly surrounded by other dipoles. The interaction energy of the cendral atom in a spherical or circular array is, in the three-dimensional case: AEsD = --gin0 ~,~j~ (2kR) + j2 (2kR) - j. (2nkRo) - j, (2nkRd and in the two-dimensional
(4a)
case:
AEm = -n,pn2
(1 - (Ro/R))
(46)
where R is the radius of the array. These functions are shown in Fig. 9. In the three-dimensional model, the interaction energy would vanish, for any radius of the spherical array, if it were not for the phase factor, e-i2*‘d. Due to the latter, the interaction energy becomes finite as the radius approaches the wavelength of the incident radiation. Thus, in this case, the contribution of the nearest spherical shells is negligible, while that of more distant shells is relatively large. In a two-dimensional array, on the other hand, the phase factor is 1 everywhere, and the interaction energy is a rapidly converging function of the radius. The dipoles located near the surface (or circumference) of the array must broaden the absorption band toward higher frequencies and increase the radius needed for saturation. (The latter will occur only when
SPECTROSCOPY
OF
CHLOROPHYLL
CRYSTALS
50'7
R/Ro 0. Absorption band shifts in ethyl chlorophyllide crystals of different size. The two solid lines are theoretical curves for the central atom in a circular and in a spherical isotropic array of dipoles, respectively. The “TV-o-dimellsinllal” curve is related t,o the upper scale, the “three-dimensional” curve, to the lower scale. Open circles, experimental points referred to upper scale; hlnck circles, experimental points referred t,o levier scale. FIG.
the number of molecules with “snturat’ed” shifts xv-ill have 1)ecome much larger than that of the “non-saturated” ones; in a two-dimensional array, the interaction energy of the central molecule is nearly “saturated” if this molecule has neighbors up to ten lattice constants; but for such an array to contain ten times as many ‘kat,urated” as “non-saturated” molecules, its radius must be equal t’o 200 lattice constants.) A4s the radius increases, the one-sided broadening of the band, caused by surface-near molecules, will become less significant, and the band will approach a limiting shape as well as a limiting position. The limiting shifts me have observed in crystals can be interpret’ed by Eq. (3), describing the interaction in an infinite isotropic two-dimensional sheet, somewhat better than by Eq. (2)) describing the interact,ion in an infinite three-dimensional array (cf. Table I). The relative shifts of the chlorophyllides and the pheophorbides are as expected from their relative oscillator strengths; but considerable discrepancy remains in the case of bacteriochlorophyllide. The spectra of monomolecular layers confirm that a preponderant part of the interaction in crystals is due to forces within a single plane. For example, crystalline monolayers of ethyl chlorophyllide a show about 90 % of the band shift of three-dimensional crystals.
508
JACOBS, HOLT, KROMHOUT AND RABlNOWITCH
.!2further test can be made by comparing the functions AE = f(R), as derived for a circular and a spherical lattice, with the experimentally found dependence of the band shift on crystal size. The four points marked in Fig. 9 are for crystals with absorption maxima at 718, 722, 738, and 740 rnp, respectively. The lattice constant was assumed to be 10 A., average of the two constants of chlorophyllide within the basal layer (4 A. and 15 A.). The theoretical curve for a tivo-dimensional lattice represents fairly well the behavior of crystals with absorption maxima below 730 m,u. Growth in the third dimension could bc responsible for the final shift of the band, from 730 to 740 rnp, in the largest microcrystals (a shift not observed in monolayers). The experimental data suggest that the “nearest neighbors” account for about 50 % of the band shift; if the interaction were not predominantly confined to a planar network, the role of the nearest neighbors would be negligible. 6. EXERGY Y~IGRATIOS IS CHLOROPHYLLIUE
CRYSTALS
One of the problems of photosynthesis we had in mind, when studying pigment crystals, was that of excitation energy migration, postulated in certain kinetic theories of photosynthesis (“photosynthetic unit”). In linear polymers of isocyanine, rapid migration of electIronic excitation along the micelle leads to its uncoupling from molecular vibrations, thus causing extreme narrowing of the absorption band and appearance of resonance fluorescence. Kane of these characteristics is encountered in chlorophyllide or chlorophyll crystals (22). Their absorption bands have, in the limiting state, about t,wice the width of those in solution, indicating increased, rather than decreased coupling of elect’ronic excitation with vibrations (probably because of the superposition of lattice vibrations upon intramolecular vibrations) . iin increased coupling with vibrations must decrease the overlapping of the fluorescence and the absorption band, characteristic of chlorophyll in sohnion. The fluorescence band is thus pushed towards the infrared-how far, we do JJOt know, since 110 fluorescence could be obser\-ed in chlorophyllidc microcrystals up to h = 1 p. This could mean either a fluorescence yield
SPECTROSCOPY
OF CHLOROPHYLL
CRYSTALS
509
period <1 X lo-” sec. must be
TO CHLOROPHYLL
IS VIVO
Chlorophyll in vice has an absorption peak at 675-680 mp. In artificially prepared chlorophyll-protein complexes, this peak lies at 670 rnp; the further shift in ZCOcould be due to interaction between pigment molecules-perhaps, in a monomolecular layer with a structure similar t,o that of the “colloidal” chlorophyll monolayers on water (IO), which, too, have a band at 675-680 mp. Elect’ron microscopy suggests that, in Go, chlorophyll molecules probably arc arranged in monolayers, on proteiwlipoproteid interfaces. The arca available for each chlorophyll molecule was estimat,ed as about, 1 sq. mp for higher plants [Rabinomitch (5)] and as 2.3 sq. rnp for Euglena [Wolken and Schn-ertz (al-)]. Thomas ct al. (25) found specific areas between 0.8 and 3.8 sq. nip for four species of plants and one species of bacteria (lvhile the chlorophyll content and the total area of the lamellae in a chloroplast’ varied by as much as a factor of 104). According to Eq. (3), the interaction energy in a monomolecular layer is proportional to l,‘R” . The calculated value of A,Q’ it1 Table I was for R,, ” 1 mp. For t’he band to lie at 675 mp in a similarlp regular array, ZZn\vould bar-c t’o be 3 mp (or larger, since a part of the shift may be due to association \vit,h proteins and lipides). This is a much looser packing t,han t,hc limited area of the chhloroplast Iamcllae permit,s. IIowevcr, imperfect alignment of the pigment dipoles, indicated by the optical properties of chloroplasts, could reduce the dipole interactions without much decrease in density. In “liquid” chlorophyll monolayers on water (lo), the surface density is about one molecule per 1 sq. mP; \-et the red band is locat,ed at 675 mp.; while in crystalline chlorophylli& monolayers, where R2 = 0.6 sq. ~1,the band lies at 730 mp! Obviously, the difference must be due to a less orderly arrangement of molecules,
510
JACOBS,
HOLT,
KHOMHOUT
AND
RABIXOWITCH
made possible by distention of the layer, rather than to the distention itself. The migration of excit’ation energy in chlorophyll monolayers in zliL!o could be made more, rather than less, likely, by looser arrangement, because this loosening reduces t,he coupling wit,h lattice vibrations. Such a reduction is indicated by a sizeable fluorescence yield, of the order of 2-Y % [Latimer et al. (as)]. The longer excitation lifetime could permit several hundred energy transfers, without uncoupling of the elect,ronic excit,ation from molecular vibrations. Thus, optical data are not incompatible with the assumption of such a (relat,irely) limit,ed migration of excit,ation energy in the chlorophyll monolayers in e+o. SUMMARY
Microcrystals of ethyl chlorophyllides and pheophorbides a and b, and of methyl bacteriochlorophyllide, have been prepared and their absorption spectra measured. The main long-wave bands are shifted toward longer waves by l-2 X 10Rcm.-’ compared to their (extrapolated) position in free molecules (i.e., by JO-80 rnp from their position in organic solvents). The shift is a function of the size of the microcrystals, reaching L’saturation” in crystals about 0.5 p in diamet’er. An only slightly smaller monolayers, indicating that the intcrshift is observed in “crystalline” actions responsible for the shift occur mainly in one crystallographic plane. In “liquid” monolayers, the shift, is much smaller-similar to that in amorphous colloidal solutions. A theory of the band shift is given, based on electrostatic interaction in an isotropic array of (virtual) dipoles, created by light absorption (without consideration of the overlapping of eigenfunctions and consequent resonance effects). The absolute value of the maximum shift in ‘
SPECTItOSCOPY
OF CHLOROPHYLL
CRYSTALS
511
1. OBREIMOV, 1. v., AND PRIKHOT'KO, ;2., Physik. %. Sowjetunion 1, 203 (1932); 9, 34, 48 (1936). 2. See, e, g., SHEPPARD, S. E., Reus. Ned. Phys. 14, 303 (1942). 3. JELLY, 13. IZ., Nature 138, 1009 (1936); 139, 631 (1937). 4. SCHEIBE,G., Anger. Chem.60,51,218 (1937). 5. RABINOWITCH, E. I., Ann. Reo. Plant Physiol. 3, 229 (1952). 6. HOLT, A.S., .~SD JACOBS,E.I':.,~~~.J. Rotany 41,710 (19543. 7. HoLT,~L.S.,ASD JACOBS,E.E:.,A~U. ,J.Botany 41, 718 (1954). 8. is.4NSOx, E. A., Rec. trau. bot. nirerl. 36,183 (1939). 9. LONGUET-HIGGISS, H. C.,RECTOR,C. W., AND PLANT, J.R.,J.Chm. Ph?ys. 18, 1174 (1950). 10. JACOBS,E.~.,HOLT, A. ~.,~IABINOWITCII, E. I.,./. Chem. Phys.22, 142 (1954). 11. MIE, G., Ann. Physik 26,377 (1908). 12. STEUBING, Ann. Physik 26,329 (1908). 13. GANS, R., Ann. Physik37, 881 (1912). 13~. LATIJIER, l’.,Thesis, Univ. of Illinois,1956; LATIMER, P., ANI) RABINOWITCH, E., J. Chem. Phys. 24, 180 (1956). 14. DUYSENS, L. N. M., Riochim. et Biophys. Acta 19, 1 (1956). 15. KATZ,~.,AND~~ASSINK,~~.C.,~~~Z~T)~~~~~~~~,~~(~~~~). 16. FRENKEL, J., Phys. Reu. 37, 17 (1931); 37, 1276 (1931) ; Physik. Z. Sowjetunion 9, 158 (1936). li. l'EIERLS,R.,A~nn. Physikl3,905 (1932). 18. DAVYDOV, A. S.,J.EzptZ. Theoret, Phys. (U.S.S.R.) 18,210 (1948). 19. HELLER,W.,ANDMARC~S,A., Phys.Rev.84,809(1949). 20. DEXTER,~. L., AND HELLER,W. R., Phys.Rev. 91,273 (1953). 21. CRAIG,D.P.,J. Chenl. Soc.539, 2309 (1955);C~~1G,n. P., AND WALSH,J., J. Chern. Phys. 24, 471 (1956). 22. JACOBS, E. E., VATTER, a., AND HOLT, A. S., Arch. Bioche?,l. Hiophys. 53, 228 (1954). 23. RABINOWITCR, E. I., JXOBS, 13:.E., HOLT, A. S., ASI) KROMIIOUT, R. K., 2. Physik 133,261(1952). 24. WOLKEN, J.J., AND SCIIWERTZ, F. A.,J.Gen. Physiol.20, 111 (1953). 25. THOMAS, J. B.,&~IXSAERT, K., AND~~LLERS, P. F., Actabot.neerl.6,315(1956). 26. LATIMER, P., BANNISTER, T.T., AXD RABINOWITCH, E.,Science124, 585 (1956).