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The orientation of the triplet axes with respect to the optical transition moments in (bacterio) chlorophylls
19May 1995
CHEMICAL PHYSICS LETTERS ELSEVIER
Chemical Physics Letters 237 (1995) 493-501
The orientation of the triplet axes with respect to the optical transition moments in (bacterio)chlorophylls J. V r i e z e 1, A.J. H o f f * Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 6 February 1995; in final form 6 March 1995
Abstract
Chlorophyll a (Chl a), bacteriochlorophyll a (BChl a) and bacteriochlorophyll g (BChl g) were studied by absorbance-detected magnetic resonance (ADMR) and linear-dichroic (LD-) ADMR in zero magnetic field using two different glassy matrices. The microwave-induced triplet-minus-singlet absorbance-difference spectra were measured in the wavelength region 500-900 nm. With LD-ADMR the orientations of the near-infrared singlet-singlet and triplet-triplet transition moments relative to the in-plane triplet x and y axes were obtained. For all (B)Chls studied, the Qr- and near-infrared triplet-triplet transition moments are mutually parallel (within 5°), and oriented in the triplet xy plane. For BChl a and BChl g the Qr-transition moment was found to be oriented within 20 ° parallel to the triplet y axis. For Chl a the orientation of the Qy-transition moment with respect to the triplet y axis was found to be 35-45 °, the precise value depending on the solvent used.
1. Introduction
The optical properties of chlorophyll (Chl) and bacteriochlorophyll (BChl) can be reasonably well understood with the aid of molecular orbital theory. Calculations of the splitting of the near-infrared Qtransitions into the Qx- ($2 ~ So) and Qy- (S 1 ~ S 0) transitions, and the orientation of their transition moments in the molecular frame result in values that come close to those found experimentally [1-3]. The molecular x and y axes, x and y, are chosen
* Corresponding author. Present address: lnstitut ftir Experimentalphysik, Freie Universit~it Berlin, Arnimallee 14, 14195 Berlin, Germany.
parallel to the N]]-NIv and N [ - N m axes, respectively (Fig. 1), and the calculations predict an orientation of the Qr-transition moment, Qv, close to y. The Qx-transition moment, Qx, of BChl has been calculated to be oriented close (within 20 °) to x [3]. Experimentally, for Chl a and BChl a an angle of 20 ° between Qv and y has been found by lineardichroic absorption measurements in vesicles and liquid crystals [4,5], whereas a study of pyro-Chl a-substituted myoglobin indicated an orientation of Qv parallel to y [6]. For BChl a, Qx was reported to be oriented parallel to x [4]. Qualitatively, the relative intensities of Qx.y-bands of different (Mg)chlorins, Chl and BChl, and their splitting, can be explained by the 'four-orbital' model [7,8], in which only the two highest occupied molecular orbitals (HOMO) and the two lowest unoccupied
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J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
molecular orbitals (LUMO) of the w-electron system are considered. Only a few calculations have dealt with the triplet state of (B)Chls. Weiss [1] and Petke et al. [2] have calculated the energies of the four lowest triplet configurations, which are basically the same as those predicted with the 'four-orbital' model. The lowest excited triplet state of BChl and Chl was calculated to be a nearly pure 3(alu ~ egx) configuration. Experimentally, triplet states of BChls and Chls in vitro and in pigment-protein complexes have been studied with EPR and ODMR (reviewed in Refs. [9-11]). Although (B)Chls can be oriented in vesicles, to our knowledge no studies on the orientation of the triplet axes have been reported for oriented (B)Chls. Because Chl and BChl are nearly planar aromatic ,rrelectron systems, the triplet z axis, zx, is most likely oriented perpendicular to the molecular plane. This agrees with the observation that the triplet z-sublevel is the slowest decaying sublevel and that the angle between ZT and the (B)Chl plane exceeds the magic angle (54.7 °) [12]. Magnetophotoselection measurements on (B)Chls in a toluene/pyridine glass have shown that the triplet y axis is oriented along Qv for all (B)Chls to within 35 °, and the results were explained by a different sign of E for Chl and BChl [12]. The in-plane triplet axes, x T and Y'r, are commonly assumed to be parallel to the corresponding molecular axes. For Chl a-containing reaction centers of the plant photosystems, photosystem I and photosystem II, we recently measured an orientation of the S 1 ~ S O transition moment of the primary donor with respect to its triplet axes quite different from that found for BChl a- and b-containing reaction centers of purple bacteria [13-18]. It turned out to be impossible to explain the data with the assumption that Qr in Chl a in vivo is oriented parallel to YT [13,14]. The difficulty in reconciling our results on plant photosystems with the earlier data on Chl a in vitro [12], prompted us to obtain more accurate experimental data on the orientation of the triplet axes of (B)Chls in vitro. A better knowledge of the orientation of the triplet axes for monomeric BChl and Chl in the molecular frame will facilitate drawing conclusions about the structure of the (B)Chls in the protein complexes. In this work we present linear-dichroic ab-
sorbance-detected magnetic resonance (LD-ADMR) measurements in zero-magnetic field on (B)Chls in organic solvents. The (B)Chls of interest are Chl a, BChl g and BChl a, which form the primary electron donor and part of the antenna pigments in the plant photosystems, in Heliobacterium chlorum, and in a number of purple bacteria (e.g. Rhodobacter sphaeroides), respectively. Since the differences between these three (B)Chls in both their optical properties and the properties of the lowest excited triplet state are relatively large [11,19,20], these (B)Chls are suitable for a comparative study on the orientation of the triplet axes in the molecular frame, and for testing the theoretical predictions about the orientation of the triplet axes.
2. Experimental Chl a, BChl g and BChl a were isolated from spinach chloroplasts, Heliobacterium chlorum, and Rb. sphaeroides, respectively, using the procedure reported in Ref. [19]. The isolated (B)Chls were dissolved in ethanol/methanol (65%/35% vol/vol) or in toluene/pyridine (85%/15% vol/vol) to an optical density of = 0.3 per sample (which corresponds to a concentration of = 2 X 10 -5 M in a cell with a 2 mm path length) at the maximum of the Qr-absorption. Oxygen was removed from the ethanol/ methanol mixture by degassing the solvent by several freeze-thaw cycles. For the toluene/pyridine mixture this was not necessary since (B)Chls are effectively screened by the pyridine. The samples were cooled slowly to liquid nitrogen temperature to prevent cracking. All measurements were performed at = 1.5 K in a home-built four-window bath cryostat. The ADMR setup was basically the same as described in Refs. [21,22]. Time-resolved ADMR measurements were performed using short microwave pulses (25 ixs, repetition rate 33 Hz, 100 kHz frequency modulation), amplified with a broadband amplifier (IFI M5580). The signals were averaged with a Nicolet 527. For the LD-ADMR measurements a loop-gap cavity was used, with the direction of the microwave magnetic field perpendicular to the light beam [22]. A Glan-Thompson polarizer was placed between the sample and the monochromator to measure the change in light inten-
J. Vrieze,A.I. Hoff/ Chemical PhysicsLetters 237 (1995) 493-501
495
sity transmitted by the sample for the electric-field vector of the light wave parallel and perpendicular to the direction of the microwave magnetic field. a
BChl
a,b,g
3. Results Zhl
a
3.1. Absorbance spectra, zero-field splitting parameters and T -- S spectra
In Fig. 1 the low-temperature absorption spectra of Chl a, BChl a and BChl g are shown. The wavelength of the maximum at the red side, assigned as Qr, indicates that the (B)Chls are monomeric. The absorption bands of BChl g and Chl a in toluene/pyridine are narrower, better resolved, and located at slightly longer wavelengths than in ethanol/methanol, probably due to the strong coordination with pyridine, which results in biligated (B)Chls [23] and a better defined environment. The bands at 620 nm for Chl a, 698 nm for BChl g and 700 nm for BChl a, are ascribed to Qr-vibronic transitions that are about 1200-1300 cm-t higher in energy than the Qy-transition [20]. The zero-field transitions between the three components of the triplet state of BChl g in ethanol/methanol, optically detected at two different wavelengths within the Qr-absorption band, are shown in Fig. 2. As for all (B)Chls in vitro studied so far, only the I D I - IEI and IDI + IEI transitions were observed. The 2 I E I transition was not observed, probably due to almost equal steady-state population of the x- and y-triplet sublevels. The I D I
.
500
i
i
i
600
700
800
wavelength
.
.
.
900
(nr'n)
Fig. 1. Absorption spectra of Chl a and BChl g (at 1.5 K) in ethanol/methanol (solid line) and toluene/pyridine (dashed line). BChl a was measured in ethanol/methanol only. Inset: The structure of Chl a (left) and BChl a, b, and g (right). R l, R 2 is: COCH3, HCHE-CH 3 (BChl a); COCH3, = C H - C H 3 (BChl b); CH=CH2, = C H - C H 3 (BChl g). R is an esterifying alcohol
and I E[ parameters depend on the detection wavelength, indicating that the width of the absorption bands of BChl g in ethanol/methanol is largely due to site-heterogeneity. A similar heterogeneity has been observed earlier by ODMR for various (B)Chls [24-26] and can be explained by a variation in the environment of the (B)Chls resulting from differences in ligation of the solvent to the central Mg-atom and to polar groups attached to the rings (e.g. hydrogen-bonding to the carbonyl group(s)). The zero-field
Table 1 Zero-field splittings (MHz) and decay rates of the triplet sublevels of Chl a, BChl g, BChl a and BChl b.
Chl a BChl g BChl a
BChl b
[ D I - I EI
ID[ +IEI
725 a 730 b 480 a 470--490 b 516 a 516 c 495-505 b 492 c
955 a 955-970 b 925 a 930--960 b 858 a 865 c 890--900 b 835 c
kx
(s -1 )
ky
(s - 1 )
kz
(s -1 )
1100 + 300
1100 + 300
150 + 50
2700 + 500
3500 + 800
830 + 100
11950 + 700
15900 + 1300
1635 + 50
[25]
12400 + 900
14900 + 1300
1300 + 35
[25]
[26]
a In toluene/pyridine (85%/15% v / v ) . b In ethanol/methanol (65%/35% v / v ) . c In methyltetrahydrofuran. All zfs-parameters and decay rates were determined in zero-magnetic field. The decay rates were determined with the method described in Ref. [27].
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
496
frequencies of the (B)Chls determined by us are collected in Table 1, together with a number of data taken from the literature. The triplet-sublevel decay rates of BChl g have been measured by (partial) saturation of the ODMR line through application of a short microwave pulse [27]. At both the [D[ - [El and ID[ + [El transitions, a fast increase is followed by a slow decrease in the transmittance, shown in Fig. 2 (inset) for BChl g in ethanol/methanol. The fast increase in transmittance is attributed to transfer of the population pumped from the fast-decaying x- (or y-) sublevel into the slow-decaying z-sublevel, which is populated less. Thus, the cw-ADMR signals correspond to an increase in transmittance at the maximum of the Qr-absorption band, that is a decrease in ground-state absorbance, as generally observed for (B)Chls in organic solvents [25]. On varying the detection wavelength within the Qr,-band, or the microwave frequency within a zero-field transition, the same decay rates were obtained within the experimental error. The microwave-induced absorbance-difference spectra of Chl a, BChlg and BChl a in ethanol/
f
0
I
2oo
ti2e
,ms, 4
6
I
6oo 1ooo frequency (Ml-tz)
~4oo
Fig. 2. ADMR signals for BChl g in ethanol/methanol at 1.5 K. Spectrum of the change in transmittance A I as a function of the frequency of the amplitude-modulated microwaves at detection wavelengths of ( ) 760 and ( . . . . . ) 776 nm for BChl g in ethanol/methanol. Inset: The change in transmittance A I as a function of time due to a 25 p~s microwave pulse at a detection wavelength of 770 nm (see text). The central microwave frequency was set at 480 ([ D[ - [ E 1) and 930 MI-Iz (I D[ + I E I), with a width of 12 MHz. The dip in the signals occurring at --- 0.5 ms is due to an instrumental artifact.
ChJ
~
a
BCht g
< <::]
L
500
I
I
I
600
700
800
900
wavelength (nm) Fig. 3. Microwave-induced absorbance-difference spectra of Chl a, BChl g and BChl a in ethanol/methanol. Microwaves were set at 725 MIq_z (Chl a), 480 MHz (BChi g) and 900 MHz (BChl a). The microwaves were amplitude-modulated at a frequency of 277, 133 and 37 Hz, respectively. The dots indicate the wavelengths at which the linear dichroism was measured.
methanol are shown in Fig. 3. For facilitating comparison with literature data, the spectra are inverted compared to the spectra actually recorded. Comparison of the absorbance-difference spectra, which reflect the triplet-minus-singlet ( T - S) spectra, with the absorption spectra (Fig. 1), reveals a red-shift of the Qr-signal, particularly for BChl a and BChl g, with respect to the maximum in the absorption spectrum. Conceivably, (B)Chls that absorb in the longwavelength wing of the absorption band give rise to a higher triplet yield, a phenomenon possibly related with differences in structural conformations. The absorbance-difference spectrum of Chl a in ethanol/methanol shows a shoulder around 640 nm, similar to that observed in the absorption spectrum at low temperature. For the BChls, the band located at = 600 nm, assigned as Qx [20], is more pronounced and better resolved than for Chl a, and in the absorbance-difference spectra it is observed on top of a broad positive absorption. The bands with a negative
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
sign, corresponding with an increase in transmittance, reflect the bands observed in the ground-state absorption spectrum (Fig. 1). The broad features with positive sign in the absorbance-difference spectra, corresponding with a decrease in transmittance upon applying resonant microwaves, are ascribed to triplet-triplet absorptions from the lowest triplet state to higher excited triplet states. The large width of the triplet-triplet absorption may be due to closely spaced excited triplet states [2] combined with vibronic coupling between these states. The absorbance-difference spectra are in agreement with the flash-induced T - S spectra of Chl a and BChl a measured at room temperature [28]. The triplet-triplet absorptions are similar to those of the microwave-induced absorbance-difference spectra of Heliobacterium chlorum, Rhodobacter sphaeroides
and the plant photosystems [13,14,21,29]. 3.2. Linear-dichroic A D M R
The orientation of the optical transition moments in the triplet axes coordinate system were determined with LD-ADMR. We tentatively choose a positive E for BChl, whereas for Chl a E is taken negative, following Ref. [12]. Microwaves set at a frequency within the I D I + I E I (or ] D I - I E I ) zero-field transition select molecules that have x T (or YT) more or less oriented parallel to the microwave magnetic field if E is chosen positive, whereas for a negative sign of E the I Ol + I EI ( I O l - I E I ) transition corresponds with a y~ (x. r) selection. The orientation of the optical transition moment corresponding to the absorption band at which the absorbance-difference signal is detected, relative to the triplet axis system, can be determined from the relation between the ratio of the linear-dichroic to the isotropic absorbance-difference signal, R, and the angle between the optical transition moment and the triplet i axis, ozi, given by analogy to photoselection spectroscopy [30,31], Ri
A A I I - AA •
LD-(T - S)
AAII + AA ±
T- S
(3 c o s 2 a i - 1) (3+cos2ai) '
i
x, y, z
(1)
497
where AAII and AA± are the absorbance-difference signals parallel and perpendicular to the direction of the microwave magnetic field, respectively. Inspection of the slope d a i / d R i shows that the determination of a i is most accurate when a i has a value close to the magic angle (54.7°). The ratio of the linear-dichroic to the isotropic absorbance-difference signal becomes zero for high microwave powers, when all molecules have about equal probability of being excited, irrespective of their orientation. Therefore, for obtaining oti the absorbance-difference signals were measured as a function of the microwave power (10-0.01 mW at the source), for the polarization direction of the polarizer parallel and perpendicular to the direction of the microwave magnetic field, and extrapolated to zero-microwave power. For Chl a and BChl g the linear dichroism was determined at various wavelengths in the 500-900 nm region, in both e t h a n o l / m e t h a n o l and toluene/pyridine glasses. For BChl a the dichroism was determined in ethanol/methanol and at the Qytransition only, as for the other optical transitions in the absorbance-difference spectrum the signal-tonoise ratio was not sufficiently high to allow extrapolation to low microwave powers. The detection wavelengths at which the linear dichroism was determined are indicated in Fig. 3. We consider first the data for Chl a in an ethanol/methanol glass. The LD-(T - S) signal and T - S signal were measured as a function of the microwave power at the maximum of the Qr-band (675 nm), and plotted against each other in Fig. 4. The slope in Fig. 4 was fitted with a linear leastsquares method taking only the points at low microwave powers. The number of points participating in the fit was determined by increasing the number of points until the standard deviation of the fit increased significantly. From the fits we obtained for the I DI - I EI transition an angle of 48 ° ___3 ° between x T and Qy, and for the I Dt + I EI transition an angle of 4 5 ° + 2 ° between yv and Qv- These results, and those obtained for several other detection wavelengths, are summarized in Table 2. From this table, it appears that the triplet-triplet absorption above 690 nm has a polarization that is rotated by some 5 ° with respect to that of the Qr-absorption.
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
498
5
IDI-
H < q
IDI+IEI
q
<
/
<~ 4
I
I=
[
< <1 3
H < q
°°
H
/'j
R=o.o
R=O. 114•
q i
3
0
0
6
5
O
AA, +AAz
10
AAir +AA±
Fig. 4. •AII (solid circles) and A A ~ (open circles) measured as a function of the microwave power and plotted, together with the difference A A I I - A A L (squares), against AAII + A A ± , for Chl a in ethanol/methanol at a detection wavelength of 675 nm. The microwaves were set at 725 MHz ( [ D [ - [ E I) and 955 MHz ( I D I + [ E I). The R value for vanishing microwave power is given by the slope of the straight solid line, fitted through the squares with a linear least-squares method, yielding R values of 0.10 + 30% ( I D [ - [ E I) and 0.14 + 15% ( [ D [ + ] E I), corresponding with [ a x [ = 48 + 3 ° and [ O/y ] = 45 + 2 °.
In toluene/pyridine a different relative orientation is found, the difference being outside the experimental error: The Q~- and triplet-triplet transitions are rotated by 50-8 ° with respect to the corresponding transitions in ethanol/methanol. The mutual orientation of the triplet-triplet transition and the Qv-
transition moment is either almost parallel (5 °) or almost perpendicular (75 ° in toluene/pyridine and 85 ° in ethanol/methanol). Because a strong solvent dependence of the mutual orientation of the two transition moments seems unlikely, we prefer a parallel orientation.
Table 2 The orientation of the optical transition moments with respect to the triplet x and y axes for Chl a A,~t
Solvent
Assignment
Toluene/pyridine
530 560 620 640 665 a 670 678/675 700 a 730
a,b
Ethanol/methanol
I~xl
I%1
[~xl
I%1
(IDI-IEI)
(IDI+IEI)
(IDI-IE])
(IDI+IEI)
60 ° 47 ° 49 °
48 ° 47 ° 52 °
53 ° 58 ° 49 ° 49 ° 53° 52° 48° 55 ° 53 °
44 ° 37 ° 50 ° 43 ° 41° 48° 45° 41 ° 41 °
58 ° 52 °
36°
56 °
33 °
T, ~ T o c T, ~ T o vibronic Q r
Qx/vibronic Qy Qr Qr Qr T, ~ T O T, ~ T o
a The polarizations at this wavelength were determined via an extrapolation to low power as in Fig. 4, resulting in an error of at most _+3 °. All other angles are obtained at high microwave power ( ~ 50 mW) and normalized on the R value measured at the wavelength of the Qv-maximum at the same microwave power, resulting in an error of approximately + 10 °. b The detection wavelength was set at 678 n ~ for toluene/pyridine and at 675 nm for ethanol/methanol. c 1", ,-- To: transitions between the lowest triplet state and higher excited triplet states.
J. Vrieze, A.L Hoff / Chemical PhysicsLetters 237 (1995) 493-501
499
Table 3 The orientation of the optical m o m e n t s with respect to the triplet axes for BChl g Adet
Assignment
Solvent Ethanol/methanol
Toluene/pyridine
605/595 b 630 7 8 0 / 7 7 0 a,b 840
la/I (IDt+IEI)
lavl (IDI-IEI)
0 ! 15 ° 70±15 ° 80 ± 10 ° 70 ± 15 °
90 _+ 15 ° 20_+10 ° 15 _+ 10 ° 0 ± 15 °
I~xl (IDI+qEI)
I%1 (IDI-IEI) Qx T.~T
77 _+ 10 °
Oc
Qy
19 ± 10 °
T. ~ T 0
a The polarizations at 7 7 0 / 7 8 0 n m were determined via an extrapolation to lower p o w e r as in Fig. 4. The r e m a i n i n g angles are obtained at high m i c o w a v e r p o w e r ( = 5 0 m V ) and normalized on the R value measured at the Qv-absorption b a n d at the s a m e m i c r o w a v e power. b The detection w a v e l e n g t h w a s set at 605 (Qx) and 7 8 0 nm ( Q r ) for t o l u e n e / p y r i d i n e , and at 770 nm (QY) for e t h a n o l / m e t h a n o l . c ,in , - To: triplet-triplet transitions.
The results for BChl g are summarized in Table 3. In contrast with the data obtained for Chl a, the linear dichroism of this molecule at the I D I + I EI transition has a sign opposite to that at the I D I - I EI transition, resulting in an orientation of Qy almost perpendicular to x r. In toluene/pyridine we obtained, within the experimental error, values similar to those found in ethanol/methanol. The larger errors in a for BChl g compared to those for Chl a mainly arise because the values of a for the former compound differ more from the magic angle (see above). A variation of the microwave frequency within the zero-field transitions (over a range of about 7 MHz for the I D I - I E I transition and 20 MHz for the I D ] + t EI transition) a n d / o r within the Q r-absorption band (770-780 nm) did not change the results, within the experimental error. As for Chl a, the triplet-triplet absorption of BChl g has approximately the same polarization as the Qr-transition. The results for the Qr-transitions of Chl a, BChl a and BChl g are collected in Table 4.
4. Discussion For both Chl a and BChl g, Q r , to within the accuracy of the experiment, is found to lie approximately in the plane spanned by x T and YT (Table 2), which presumably coincides with the molecular xy plane (see Fig. 1). This tallies with the observation that the triplet z-sublevel is the slowest decaying and less populated sublevel. We have not been able to obtain an accurate determination of YT of BChl a, but the values in Table 4 suggest that also for this BChl x x and YT are approximately oriented in the molecular xy plane. For BChl g, we could perform a LD-ADMR experiment on the Qx-band, and we observed an orientation of Q x with respect to x x and Yx close to 0 ° and 90 °, respectively (Table 3). Therefore, the mutual angle of the Q-bands in this molecule is 70°-90 °, as found for BChl a with linear-dichroic absorption measurements [4]. From a simulation of the L D - ( T - S) spectra of
Table 4 The orientation o f the Q r - t r a n s i t i o n m o m e n t with respect to the triplet x and y axes for Chl a, BChl g and BChl a
Chl a BChl g BChl a
Ade, (nm)
Solvent
I O~x I
I O/y I
675 678 770 780 780
ethanol/methanol toluene/pyridine ethanol/methanol toluene/pyridine ethanol/methanol
48 52 77 80 73
45 36 19 15 20
___3 ° _+ 2 ° ± 10 ° _+ 5 ° _+ 10 °
+ + + ± ±
2° 3° 10 ° 10 ° 15 °
500
J. Vrieze,A.J. Hoff/ ChemicalPhysicsLetters237 (1995)493-501
Rhodopseudomonas (Rps.) viridis, monitored by
Y, YT and x T are directed close to y and x, respec-
LD-ADMR, it was concluded that the orientation of the near-infrared triplet-triplet transition is perpendicular to Qr [16]. In this work, however, we show that for BChl g and Chin in vitro the triplet-triplet transition moment is almost parallel to Qv, just as for the Chl a-containing reaction centers of photosystem I and II [13,14]. Although for BChl a we have not been able to determine the orientation of the triplet-triplet transition moment with respect to the triplet axes, we have no reason to assume that for this BChl Qv and the triplet-triplet transition moment would not be mutually parallel. A parallel configuration is supported by the polarization of the broad triplet-triplet absorption in the T - S spectrum of Rb. sphaeroides [32]. When regarding the orientations found here for the near-infrared triplettriplet transitions, and in view of the width generally found for triplet-triplet absorptions, it seems likely that the narrow absorption at 870 nm in the T - S spectrum of Rps. viridis [16], and at 820 nm in the T - S spectrum of Rb. sphaeroides [33], which were ascribed to a triplet-triplet transition, are effectively singlet-singlet Qr-transitions. This view is corroborated by recent LD-ADMR results on genetically modified reaction centers, and for a further discussion we refer to Refs. [18,34]. In order to determine the orientation of the triplet axes in the molecular coordinate system, knowledge is required of the orientation of Qv in this frame, as from LD-ADMR only relative angles are obtained. From linear-dichroic absorbance spectroscopy on Chl a in vesicles and BChl a in liquid crystals, Bauman and Wrobel [4] and Fragata et al. [5] concluded to similar polarizations of the Qr-transitions of Chl a and BChl a, with an angle of = 20 ° between Qr and y. The orientations found here for Q r in the triplet axes frame agree with the sign of the magneto-photoselection signals observed by Thurnauer and Norris for (B)Chls in a toluene/pyridine glass [12]. These authors have concluded that for all (B)Chls Qr makes an angle of less than 35 ° with YT, and that the sign of E for the Chls is opposite to that for the BChls. A remarkable result of the present study is that the in-plane triplet axes of Chl a seem to be 'rotated' with respect to those of BChl. Accepting that for all (B)Chls Qv is approximately parallel to
tively, for BChl a and BChl g, and close to the bisectrices of these axes for Chl a. Although the orientation of Qv with respect to the in-plane triplet axes depends somewhat on the solvent (see Tables 2 and 4), the overall difference in orientation between Chl a and the two BChls is much too large for a solvent effect. For BChl a and b in reaction centers of Rb. sphaeroides and Rps. viridis, respectively, whose structures have been solved by X-ray crystallography, the EPR and the LD-ADMR results could only be explained when it was assumed that YT of a localized triplet state and Q r are both oriented close to y (to within 10 °) [16,17]. These analyses agree well with our results for BChl a and g. The electron density in the triplet state of both Chl and BChl, as calculated by Petke et al. [2,3], comes close to that of a pure 3(alu --~ eg x) configuration. On the basis of the symmetry of the conjugated macrocycle of (B)Chl, which is approximately D2h for BChl and C2v for Chl, x T and YT should run through the NII-NIv and NI-NII I axes, respectively, in agreement with our results for BChl a and g, but not for Chl a. It has been suggested previously that the temperature and solvent dependence of the linewidth of the cation of Chl a measured by EPR could be explained by some admixture of the a2u first excited doublet configuration to the alu doublet ground-state configuration [35]. If mixing of the alu and a2u HOMOs occurs in Chl a, then a similar mixing might occur in the triplet state and cause a rotation of the in-plane triplet axes with respect to the molecular frame. Nevertheless, the Qv ($1 ~ So) and the triplet-triplet excitations apparently remain y polarized, just as observed for BChl for which the orbital mixing is assumed to be less than for Chl [8]. Different types of ring substituents are not expected to have a large effect on the orientation of the triplet axes. In fact, for BChl a and BChl g the difference in ring substituents is larger than for BChl a and Chl a, yet the difference in triplet axes orientation between BChl a and BChl g is smaller than that between the BChls and Chl a. Therefore, it seems plausible that the difference between BChl and Chl a is due to the presence of extra "rr-electron density on ring II in Chl a compared to BChl, which leads to a reduction of
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
the energy gap between the a2u and alu orbitals [1,8] and, consequently, to a greater sensitivity to perturbations induced by ring substituents. The results of magnetophotoselection experiments on (B)Chls in vitro were earlier interpreted as 3Chl a and 3BChl a having opposite signs of E [12]. Accordmgly, we assumed for Chl a a negatwe sign of E. The results presented here on the angle between Q r and the in-plane triplet axes of Chl a (close to 45°), however, preclude a definite conclusion about the sign of E. •
3
•
•
Acknowledgement We thank Erik-Jan van de Meent and Christof Francke for isolating the (B)Chls and for preparing some of the samples, and Professor J.H. van der Waals for helpful discussion. This work was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organisation for Scientific Research (NWO).
References [1] C. Weiss, J. Molec. Spectry. 44 (1972) 37. [2] J.D. Petke, G.M. Maggiora, L. Shipman and R.E. Christoffersen, Photochem. Photobiol. 30 (1979) 203. [3] J.D. Petke, G.M. Maggiora, L.L. Shipman and R.E. Christoffersen, Photochem. Photobiol. 32 (1980) 399. [4] D. Bauman and D. Wrobel, Biophys. Chem. 12 (1980) 83. [5] M. Fragata, B. Nord6n and T. Kurucsev, Photochem. Photobiol. 47 (1988) 133. [6] R.S. Moog, A. Kuki, M.D. Fayer and S.G. Boxer, Biochem. 23 (1984) 1564. [7] M. Gouterman, in: The porphyrins, Vol. 3, ed. D. Dolphin, (Academic Press, New York, 1978) ch. 1. [8] L.K. Hanson, in: The chlorophylls, ed. H. Scheer (CRC Press, Boca Raton, 1991) p. 993. [9] D.E. Budil and M.C. Thurnauer, Biochim. Biophys. Acta 1057 (1991) 1. [10] T.J. Schaafsma, in: Triplet state ODMR spectroscopy, ed. R.H. Clarke (Wiley-Interscience, New York, 1982) p. 291. [11] A. Angerhofer, in: The chlorophylls, ed. H. Scheer (CRC Press, Boca Raton, 1991) p. 945. [12] M.C. Thurnauer and J.R. Norris, Chem. Phys. Letters 47 (1977) 100.
501
[13] R. van der Vos, P.J. van Leeuwen, P. Braun and A.J. Hoff, Biochim. Biophys. Acta 1140 (1992) 184. [14] J. Vrieze, P. Gast and A.J. Hoff, in: Research in photosynthesis, Vol. 1, ed. N. Murata (Kluwer, Academic Publishers, Dordrecht, 1992) p. 553. [15] H.A. Frank, J. Bolt, R. Friesner and K. Sauer, Biochim. Biophys. Acta 547 (1979) 502. [16] E.J. Lous and A.J. Hoff, Proc. Natl. Acad. Sci. US 84 (1987) 6147. [17] J.R. Norris, D.E. Budil, P. Gast, C.-H. Chang, O. El-Kabbani and M. Schiffer, Proc. Natl. Acad. Sci. US 86 (1989) 4335. [18] J. Vrieze and A.J. Hoff, submitted for publication. [19] M. Kobayashi, E.J. van de Meent, C. Erkelens, J. Amesz, I. Ikegami and T. Watanabe, Biochim. Biophys. Acta 1057 (1991) 89. [20] A.J. Hoff and J. Amesz, in: The chlorophylls, ed. H. Scheer (CRC Press, Boca Raton, 1991) p. 723. [21] H.J. den Blanken and A.J. Hoff, Biochim. Biophys. Acta 681 (1982) 365. [22] H.J. den Blanken, R.F. Meiburg and A.J. Hoff, Chem. Phys. Letters 105 (1984) 336. [23] I. Renge and R. Avarmaa, Photochem. Photobiol. 42 (1985) 253. [24] S. Hotchandani, R.M. Leblanc, R.H. Clarke and M. Fragata, Photochem. Photobiol. 36 (1982) 235. [25] H.J. den Blanken and A.J. Hoff, Chem. Phys. Letters 96 (1983) 343. [26] A. Angerhofer, J.U. von Schlitz and H.C. Wolf, Chem. Phys. Lett. 151 (1988) 195. [27] W.G. van Dorp, W.H. Schoemaker, M. Soma and J.H. van der Waals, Mol. Phys. 30 (1975) 1701. [28] N. Periasiamy and H. Linschitz, J. Am. Chem. Soc. 101 (1979) 1056. [29] J. Vrieze, E.J. van de Meent and A.J. Hoff, in: The photosynthetic bacterial reaction center. Structure, spectroscopy and dynamics, eds. J. Breton and A. Verm6glio (Plenum Press, New York, London 1992) p. 67. [30] A.J. Hoff, in: Antennas and reaction centers of photosynthetic bacteria, ed. M.E. Michel-Beyerle (Springer Verlag Berlin, 1985)p. 150. [31] A. Verm6glio, J. Breton, G. Paillotin and R. Cogdell, Biochim. Biophys. Acta 501 (1978) 514. [32] A. Angerhofer, D. Beese, A.J. Hoff, E.J. Lous and H. Scheer, in: Proceedings of the Indo-US workshop on Applications of Molecular Biology in Bioenergetics of Photosynthesis, New Delhi (1988). [33] J.A. Dijkman, H.J. den Blanken and A.J. Hoff, Israel J. Chem. 28 (1988) 141. [34] J. Vrieze, J.C. Williams, J.P. Allen and A.J. Hoff, Biochim. Biophys. Acta, submitted. [35] M.S. Davis, A. Forman and J. Fajer, Proc. Natl. Acad. Sci. US 76 (1979) 4170.
CHEMICAL PHYSICS LETTERS ELSEVIER
Chemical Physics Letters 237 (1995) 493-501
The orientation of the triplet axes with respect to the optical transition moments in (bacterio)chlorophylls J. V r i e z e 1, A.J. H o f f * Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 6 February 1995; in final form 6 March 1995
Abstract
Chlorophyll a (Chl a), bacteriochlorophyll a (BChl a) and bacteriochlorophyll g (BChl g) were studied by absorbance-detected magnetic resonance (ADMR) and linear-dichroic (LD-) ADMR in zero magnetic field using two different glassy matrices. The microwave-induced triplet-minus-singlet absorbance-difference spectra were measured in the wavelength region 500-900 nm. With LD-ADMR the orientations of the near-infrared singlet-singlet and triplet-triplet transition moments relative to the in-plane triplet x and y axes were obtained. For all (B)Chls studied, the Qr- and near-infrared triplet-triplet transition moments are mutually parallel (within 5°), and oriented in the triplet xy plane. For BChl a and BChl g the Qr-transition moment was found to be oriented within 20 ° parallel to the triplet y axis. For Chl a the orientation of the Qy-transition moment with respect to the triplet y axis was found to be 35-45 °, the precise value depending on the solvent used.
1. Introduction
The optical properties of chlorophyll (Chl) and bacteriochlorophyll (BChl) can be reasonably well understood with the aid of molecular orbital theory. Calculations of the splitting of the near-infrared Qtransitions into the Qx- ($2 ~ So) and Qy- (S 1 ~ S 0) transitions, and the orientation of their transition moments in the molecular frame result in values that come close to those found experimentally [1-3]. The molecular x and y axes, x and y, are chosen
* Corresponding author. Present address: lnstitut ftir Experimentalphysik, Freie Universit~it Berlin, Arnimallee 14, 14195 Berlin, Germany.
parallel to the N]]-NIv and N [ - N m axes, respectively (Fig. 1), and the calculations predict an orientation of the Qr-transition moment, Qv, close to y. The Qx-transition moment, Qx, of BChl has been calculated to be oriented close (within 20 °) to x [3]. Experimentally, for Chl a and BChl a an angle of 20 ° between Qv and y has been found by lineardichroic absorption measurements in vesicles and liquid crystals [4,5], whereas a study of pyro-Chl a-substituted myoglobin indicated an orientation of Qv parallel to y [6]. For BChl a, Qx was reported to be oriented parallel to x [4]. Qualitatively, the relative intensities of Qx.y-bands of different (Mg)chlorins, Chl and BChl, and their splitting, can be explained by the 'four-orbital' model [7,8], in which only the two highest occupied molecular orbitals (HOMO) and the two lowest unoccupied
0009-2614/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved
SSDI 0 0 0 9 - 2 6 1 4 ( 9 5 ) 0 0 3 5 4 - 1
494
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
molecular orbitals (LUMO) of the w-electron system are considered. Only a few calculations have dealt with the triplet state of (B)Chls. Weiss [1] and Petke et al. [2] have calculated the energies of the four lowest triplet configurations, which are basically the same as those predicted with the 'four-orbital' model. The lowest excited triplet state of BChl and Chl was calculated to be a nearly pure 3(alu ~ egx) configuration. Experimentally, triplet states of BChls and Chls in vitro and in pigment-protein complexes have been studied with EPR and ODMR (reviewed in Refs. [9-11]). Although (B)Chls can be oriented in vesicles, to our knowledge no studies on the orientation of the triplet axes have been reported for oriented (B)Chls. Because Chl and BChl are nearly planar aromatic ,rrelectron systems, the triplet z axis, zx, is most likely oriented perpendicular to the molecular plane. This agrees with the observation that the triplet z-sublevel is the slowest decaying sublevel and that the angle between ZT and the (B)Chl plane exceeds the magic angle (54.7 °) [12]. Magnetophotoselection measurements on (B)Chls in a toluene/pyridine glass have shown that the triplet y axis is oriented along Qv for all (B)Chls to within 35 °, and the results were explained by a different sign of E for Chl and BChl [12]. The in-plane triplet axes, x T and Y'r, are commonly assumed to be parallel to the corresponding molecular axes. For Chl a-containing reaction centers of the plant photosystems, photosystem I and photosystem II, we recently measured an orientation of the S 1 ~ S O transition moment of the primary donor with respect to its triplet axes quite different from that found for BChl a- and b-containing reaction centers of purple bacteria [13-18]. It turned out to be impossible to explain the data with the assumption that Qr in Chl a in vivo is oriented parallel to YT [13,14]. The difficulty in reconciling our results on plant photosystems with the earlier data on Chl a in vitro [12], prompted us to obtain more accurate experimental data on the orientation of the triplet axes of (B)Chls in vitro. A better knowledge of the orientation of the triplet axes for monomeric BChl and Chl in the molecular frame will facilitate drawing conclusions about the structure of the (B)Chls in the protein complexes. In this work we present linear-dichroic ab-
sorbance-detected magnetic resonance (LD-ADMR) measurements in zero-magnetic field on (B)Chls in organic solvents. The (B)Chls of interest are Chl a, BChl g and BChl a, which form the primary electron donor and part of the antenna pigments in the plant photosystems, in Heliobacterium chlorum, and in a number of purple bacteria (e.g. Rhodobacter sphaeroides), respectively. Since the differences between these three (B)Chls in both their optical properties and the properties of the lowest excited triplet state are relatively large [11,19,20], these (B)Chls are suitable for a comparative study on the orientation of the triplet axes in the molecular frame, and for testing the theoretical predictions about the orientation of the triplet axes.
2. Experimental Chl a, BChl g and BChl a were isolated from spinach chloroplasts, Heliobacterium chlorum, and Rb. sphaeroides, respectively, using the procedure reported in Ref. [19]. The isolated (B)Chls were dissolved in ethanol/methanol (65%/35% vol/vol) or in toluene/pyridine (85%/15% vol/vol) to an optical density of = 0.3 per sample (which corresponds to a concentration of = 2 X 10 -5 M in a cell with a 2 mm path length) at the maximum of the Qr-absorption. Oxygen was removed from the ethanol/ methanol mixture by degassing the solvent by several freeze-thaw cycles. For the toluene/pyridine mixture this was not necessary since (B)Chls are effectively screened by the pyridine. The samples were cooled slowly to liquid nitrogen temperature to prevent cracking. All measurements were performed at = 1.5 K in a home-built four-window bath cryostat. The ADMR setup was basically the same as described in Refs. [21,22]. Time-resolved ADMR measurements were performed using short microwave pulses (25 ixs, repetition rate 33 Hz, 100 kHz frequency modulation), amplified with a broadband amplifier (IFI M5580). The signals were averaged with a Nicolet 527. For the LD-ADMR measurements a loop-gap cavity was used, with the direction of the microwave magnetic field perpendicular to the light beam [22]. A Glan-Thompson polarizer was placed between the sample and the monochromator to measure the change in light inten-
J. Vrieze,A.I. Hoff/ Chemical PhysicsLetters 237 (1995) 493-501
495
sity transmitted by the sample for the electric-field vector of the light wave parallel and perpendicular to the direction of the microwave magnetic field. a
BChl
a,b,g
3. Results Zhl
a
3.1. Absorbance spectra, zero-field splitting parameters and T -- S spectra
In Fig. 1 the low-temperature absorption spectra of Chl a, BChl a and BChl g are shown. The wavelength of the maximum at the red side, assigned as Qr, indicates that the (B)Chls are monomeric. The absorption bands of BChl g and Chl a in toluene/pyridine are narrower, better resolved, and located at slightly longer wavelengths than in ethanol/methanol, probably due to the strong coordination with pyridine, which results in biligated (B)Chls [23] and a better defined environment. The bands at 620 nm for Chl a, 698 nm for BChl g and 700 nm for BChl a, are ascribed to Qr-vibronic transitions that are about 1200-1300 cm-t higher in energy than the Qy-transition [20]. The zero-field transitions between the three components of the triplet state of BChl g in ethanol/methanol, optically detected at two different wavelengths within the Qr-absorption band, are shown in Fig. 2. As for all (B)Chls in vitro studied so far, only the I D I - IEI and IDI + IEI transitions were observed. The 2 I E I transition was not observed, probably due to almost equal steady-state population of the x- and y-triplet sublevels. The I D I
.
500
i
i
i
600
700
800
wavelength
.
.
.
900
(nr'n)
Fig. 1. Absorption spectra of Chl a and BChl g (at 1.5 K) in ethanol/methanol (solid line) and toluene/pyridine (dashed line). BChl a was measured in ethanol/methanol only. Inset: The structure of Chl a (left) and BChl a, b, and g (right). R l, R 2 is: COCH3, HCHE-CH 3 (BChl a); COCH3, = C H - C H 3 (BChl b); CH=CH2, = C H - C H 3 (BChl g). R is an esterifying alcohol
and I E[ parameters depend on the detection wavelength, indicating that the width of the absorption bands of BChl g in ethanol/methanol is largely due to site-heterogeneity. A similar heterogeneity has been observed earlier by ODMR for various (B)Chls [24-26] and can be explained by a variation in the environment of the (B)Chls resulting from differences in ligation of the solvent to the central Mg-atom and to polar groups attached to the rings (e.g. hydrogen-bonding to the carbonyl group(s)). The zero-field
Table 1 Zero-field splittings (MHz) and decay rates of the triplet sublevels of Chl a, BChl g, BChl a and BChl b.
Chl a BChl g BChl a
BChl b
[ D I - I EI
ID[ +IEI
725 a 730 b 480 a 470--490 b 516 a 516 c 495-505 b 492 c
955 a 955-970 b 925 a 930--960 b 858 a 865 c 890--900 b 835 c
kx
(s -1 )
ky
(s - 1 )
kz
(s -1 )
1100 + 300
1100 + 300
150 + 50
2700 + 500
3500 + 800
830 + 100
11950 + 700
15900 + 1300
1635 + 50
[25]
12400 + 900
14900 + 1300
1300 + 35
[25]
[26]
a In toluene/pyridine (85%/15% v / v ) . b In ethanol/methanol (65%/35% v / v ) . c In methyltetrahydrofuran. All zfs-parameters and decay rates were determined in zero-magnetic field. The decay rates were determined with the method described in Ref. [27].
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
496
frequencies of the (B)Chls determined by us are collected in Table 1, together with a number of data taken from the literature. The triplet-sublevel decay rates of BChl g have been measured by (partial) saturation of the ODMR line through application of a short microwave pulse [27]. At both the [D[ - [El and ID[ + [El transitions, a fast increase is followed by a slow decrease in the transmittance, shown in Fig. 2 (inset) for BChl g in ethanol/methanol. The fast increase in transmittance is attributed to transfer of the population pumped from the fast-decaying x- (or y-) sublevel into the slow-decaying z-sublevel, which is populated less. Thus, the cw-ADMR signals correspond to an increase in transmittance at the maximum of the Qr-absorption band, that is a decrease in ground-state absorbance, as generally observed for (B)Chls in organic solvents [25]. On varying the detection wavelength within the Qr,-band, or the microwave frequency within a zero-field transition, the same decay rates were obtained within the experimental error. The microwave-induced absorbance-difference spectra of Chl a, BChlg and BChl a in ethanol/
f
0
I
2oo
ti2e
,ms, 4
6
I
6oo 1ooo frequency (Ml-tz)
~4oo
Fig. 2. ADMR signals for BChl g in ethanol/methanol at 1.5 K. Spectrum of the change in transmittance A I as a function of the frequency of the amplitude-modulated microwaves at detection wavelengths of ( ) 760 and ( . . . . . ) 776 nm for BChl g in ethanol/methanol. Inset: The change in transmittance A I as a function of time due to a 25 p~s microwave pulse at a detection wavelength of 770 nm (see text). The central microwave frequency was set at 480 ([ D[ - [ E 1) and 930 MI-Iz (I D[ + I E I), with a width of 12 MHz. The dip in the signals occurring at --- 0.5 ms is due to an instrumental artifact.
ChJ
~
a
BCht g
< <::]
L
500
I
I
I
600
700
800
900
wavelength (nm) Fig. 3. Microwave-induced absorbance-difference spectra of Chl a, BChl g and BChl a in ethanol/methanol. Microwaves were set at 725 MIq_z (Chl a), 480 MHz (BChi g) and 900 MHz (BChl a). The microwaves were amplitude-modulated at a frequency of 277, 133 and 37 Hz, respectively. The dots indicate the wavelengths at which the linear dichroism was measured.
methanol are shown in Fig. 3. For facilitating comparison with literature data, the spectra are inverted compared to the spectra actually recorded. Comparison of the absorbance-difference spectra, which reflect the triplet-minus-singlet ( T - S) spectra, with the absorption spectra (Fig. 1), reveals a red-shift of the Qr-signal, particularly for BChl a and BChl g, with respect to the maximum in the absorption spectrum. Conceivably, (B)Chls that absorb in the longwavelength wing of the absorption band give rise to a higher triplet yield, a phenomenon possibly related with differences in structural conformations. The absorbance-difference spectrum of Chl a in ethanol/methanol shows a shoulder around 640 nm, similar to that observed in the absorption spectrum at low temperature. For the BChls, the band located at = 600 nm, assigned as Qx [20], is more pronounced and better resolved than for Chl a, and in the absorbance-difference spectra it is observed on top of a broad positive absorption. The bands with a negative
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
sign, corresponding with an increase in transmittance, reflect the bands observed in the ground-state absorption spectrum (Fig. 1). The broad features with positive sign in the absorbance-difference spectra, corresponding with a decrease in transmittance upon applying resonant microwaves, are ascribed to triplet-triplet absorptions from the lowest triplet state to higher excited triplet states. The large width of the triplet-triplet absorption may be due to closely spaced excited triplet states [2] combined with vibronic coupling between these states. The absorbance-difference spectra are in agreement with the flash-induced T - S spectra of Chl a and BChl a measured at room temperature [28]. The triplet-triplet absorptions are similar to those of the microwave-induced absorbance-difference spectra of Heliobacterium chlorum, Rhodobacter sphaeroides
and the plant photosystems [13,14,21,29]. 3.2. Linear-dichroic A D M R
The orientation of the optical transition moments in the triplet axes coordinate system were determined with LD-ADMR. We tentatively choose a positive E for BChl, whereas for Chl a E is taken negative, following Ref. [12]. Microwaves set at a frequency within the I D I + I E I (or ] D I - I E I ) zero-field transition select molecules that have x T (or YT) more or less oriented parallel to the microwave magnetic field if E is chosen positive, whereas for a negative sign of E the I Ol + I EI ( I O l - I E I ) transition corresponds with a y~ (x. r) selection. The orientation of the optical transition moment corresponding to the absorption band at which the absorbance-difference signal is detected, relative to the triplet axis system, can be determined from the relation between the ratio of the linear-dichroic to the isotropic absorbance-difference signal, R, and the angle between the optical transition moment and the triplet i axis, ozi, given by analogy to photoselection spectroscopy [30,31], Ri
A A I I - AA •
LD-(T - S)
AAII + AA ±
T- S
(3 c o s 2 a i - 1) (3+cos2ai) '
i
x, y, z
(1)
497
where AAII and AA± are the absorbance-difference signals parallel and perpendicular to the direction of the microwave magnetic field, respectively. Inspection of the slope d a i / d R i shows that the determination of a i is most accurate when a i has a value close to the magic angle (54.7°). The ratio of the linear-dichroic to the isotropic absorbance-difference signal becomes zero for high microwave powers, when all molecules have about equal probability of being excited, irrespective of their orientation. Therefore, for obtaining oti the absorbance-difference signals were measured as a function of the microwave power (10-0.01 mW at the source), for the polarization direction of the polarizer parallel and perpendicular to the direction of the microwave magnetic field, and extrapolated to zero-microwave power. For Chl a and BChl g the linear dichroism was determined at various wavelengths in the 500-900 nm region, in both e t h a n o l / m e t h a n o l and toluene/pyridine glasses. For BChl a the dichroism was determined in ethanol/methanol and at the Qytransition only, as for the other optical transitions in the absorbance-difference spectrum the signal-tonoise ratio was not sufficiently high to allow extrapolation to low microwave powers. The detection wavelengths at which the linear dichroism was determined are indicated in Fig. 3. We consider first the data for Chl a in an ethanol/methanol glass. The LD-(T - S) signal and T - S signal were measured as a function of the microwave power at the maximum of the Qr-band (675 nm), and plotted against each other in Fig. 4. The slope in Fig. 4 was fitted with a linear leastsquares method taking only the points at low microwave powers. The number of points participating in the fit was determined by increasing the number of points until the standard deviation of the fit increased significantly. From the fits we obtained for the I DI - I EI transition an angle of 48 ° ___3 ° between x T and Qy, and for the I Dt + I EI transition an angle of 4 5 ° + 2 ° between yv and Qv- These results, and those obtained for several other detection wavelengths, are summarized in Table 2. From this table, it appears that the triplet-triplet absorption above 690 nm has a polarization that is rotated by some 5 ° with respect to that of the Qr-absorption.
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
498
5
IDI-
H < q
IDI+IEI
q
<
/
<~ 4
I
I=
[
< <1 3
H < q
°°
H
/'j
R=o.o
R=O. 114•
q i
3
0
0
6
5
O
AA, +AAz
10
AAir +AA±
Fig. 4. •AII (solid circles) and A A ~ (open circles) measured as a function of the microwave power and plotted, together with the difference A A I I - A A L (squares), against AAII + A A ± , for Chl a in ethanol/methanol at a detection wavelength of 675 nm. The microwaves were set at 725 MHz ( [ D [ - [ E I) and 955 MHz ( I D I + [ E I). The R value for vanishing microwave power is given by the slope of the straight solid line, fitted through the squares with a linear least-squares method, yielding R values of 0.10 + 30% ( I D [ - [ E I) and 0.14 + 15% ( [ D [ + ] E I), corresponding with [ a x [ = 48 + 3 ° and [ O/y ] = 45 + 2 °.
In toluene/pyridine a different relative orientation is found, the difference being outside the experimental error: The Q~- and triplet-triplet transitions are rotated by 50-8 ° with respect to the corresponding transitions in ethanol/methanol. The mutual orientation of the triplet-triplet transition and the Qv-
transition moment is either almost parallel (5 °) or almost perpendicular (75 ° in toluene/pyridine and 85 ° in ethanol/methanol). Because a strong solvent dependence of the mutual orientation of the two transition moments seems unlikely, we prefer a parallel orientation.
Table 2 The orientation of the optical transition moments with respect to the triplet x and y axes for Chl a A,~t
Solvent
Assignment
Toluene/pyridine
530 560 620 640 665 a 670 678/675 700 a 730
a,b
Ethanol/methanol
I~xl
I%1
[~xl
I%1
(IDI-IEI)
(IDI+IEI)
(IDI-IE])
(IDI+IEI)
60 ° 47 ° 49 °
48 ° 47 ° 52 °
53 ° 58 ° 49 ° 49 ° 53° 52° 48° 55 ° 53 °
44 ° 37 ° 50 ° 43 ° 41° 48° 45° 41 ° 41 °
58 ° 52 °
36°
56 °
33 °
T, ~ T o c T, ~ T o vibronic Q r
Qx/vibronic Qy Qr Qr Qr T, ~ T O T, ~ T o
a The polarizations at this wavelength were determined via an extrapolation to low power as in Fig. 4, resulting in an error of at most _+3 °. All other angles are obtained at high microwave power ( ~ 50 mW) and normalized on the R value measured at the wavelength of the Qv-maximum at the same microwave power, resulting in an error of approximately + 10 °. b The detection wavelength was set at 678 n ~ for toluene/pyridine and at 675 nm for ethanol/methanol. c 1", ,-- To: transitions between the lowest triplet state and higher excited triplet states.
J. Vrieze, A.L Hoff / Chemical PhysicsLetters 237 (1995) 493-501
499
Table 3 The orientation of the optical m o m e n t s with respect to the triplet axes for BChl g Adet
Assignment
Solvent Ethanol/methanol
Toluene/pyridine
605/595 b 630 7 8 0 / 7 7 0 a,b 840
la/I (IDt+IEI)
lavl (IDI-IEI)
0 ! 15 ° 70±15 ° 80 ± 10 ° 70 ± 15 °
90 _+ 15 ° 20_+10 ° 15 _+ 10 ° 0 ± 15 °
I~xl (IDI+qEI)
I%1 (IDI-IEI) Qx T.~T
77 _+ 10 °
Oc
Qy
19 ± 10 °
T. ~ T 0
a The polarizations at 7 7 0 / 7 8 0 n m were determined via an extrapolation to lower p o w e r as in Fig. 4. The r e m a i n i n g angles are obtained at high m i c o w a v e r p o w e r ( = 5 0 m V ) and normalized on the R value measured at the Qv-absorption b a n d at the s a m e m i c r o w a v e power. b The detection w a v e l e n g t h w a s set at 605 (Qx) and 7 8 0 nm ( Q r ) for t o l u e n e / p y r i d i n e , and at 770 nm (QY) for e t h a n o l / m e t h a n o l . c ,in , - To: triplet-triplet transitions.
The results for BChl g are summarized in Table 3. In contrast with the data obtained for Chl a, the linear dichroism of this molecule at the I D I + I EI transition has a sign opposite to that at the I D I - I EI transition, resulting in an orientation of Qy almost perpendicular to x r. In toluene/pyridine we obtained, within the experimental error, values similar to those found in ethanol/methanol. The larger errors in a for BChl g compared to those for Chl a mainly arise because the values of a for the former compound differ more from the magic angle (see above). A variation of the microwave frequency within the zero-field transitions (over a range of about 7 MHz for the I D I - I E I transition and 20 MHz for the I D ] + t EI transition) a n d / o r within the Q r-absorption band (770-780 nm) did not change the results, within the experimental error. As for Chl a, the triplet-triplet absorption of BChl g has approximately the same polarization as the Qr-transition. The results for the Qr-transitions of Chl a, BChl a and BChl g are collected in Table 4.
4. Discussion For both Chl a and BChl g, Q r , to within the accuracy of the experiment, is found to lie approximately in the plane spanned by x T and YT (Table 2), which presumably coincides with the molecular xy plane (see Fig. 1). This tallies with the observation that the triplet z-sublevel is the slowest decaying and less populated sublevel. We have not been able to obtain an accurate determination of YT of BChl a, but the values in Table 4 suggest that also for this BChl x x and YT are approximately oriented in the molecular xy plane. For BChl g, we could perform a LD-ADMR experiment on the Qx-band, and we observed an orientation of Q x with respect to x x and Yx close to 0 ° and 90 °, respectively (Table 3). Therefore, the mutual angle of the Q-bands in this molecule is 70°-90 °, as found for BChl a with linear-dichroic absorption measurements [4]. From a simulation of the L D - ( T - S) spectra of
Table 4 The orientation o f the Q r - t r a n s i t i o n m o m e n t with respect to the triplet x and y axes for Chl a, BChl g and BChl a
Chl a BChl g BChl a
Ade, (nm)
Solvent
I O~x I
I O/y I
675 678 770 780 780
ethanol/methanol toluene/pyridine ethanol/methanol toluene/pyridine ethanol/methanol
48 52 77 80 73
45 36 19 15 20
___3 ° _+ 2 ° ± 10 ° _+ 5 ° _+ 10 °
+ + + ± ±
2° 3° 10 ° 10 ° 15 °
500
J. Vrieze,A.J. Hoff/ ChemicalPhysicsLetters237 (1995)493-501
Rhodopseudomonas (Rps.) viridis, monitored by
Y, YT and x T are directed close to y and x, respec-
LD-ADMR, it was concluded that the orientation of the near-infrared triplet-triplet transition is perpendicular to Qr [16]. In this work, however, we show that for BChl g and Chin in vitro the triplet-triplet transition moment is almost parallel to Qv, just as for the Chl a-containing reaction centers of photosystem I and II [13,14]. Although for BChl a we have not been able to determine the orientation of the triplet-triplet transition moment with respect to the triplet axes, we have no reason to assume that for this BChl Qv and the triplet-triplet transition moment would not be mutually parallel. A parallel configuration is supported by the polarization of the broad triplet-triplet absorption in the T - S spectrum of Rb. sphaeroides [32]. When regarding the orientations found here for the near-infrared triplettriplet transitions, and in view of the width generally found for triplet-triplet absorptions, it seems likely that the narrow absorption at 870 nm in the T - S spectrum of Rps. viridis [16], and at 820 nm in the T - S spectrum of Rb. sphaeroides [33], which were ascribed to a triplet-triplet transition, are effectively singlet-singlet Qr-transitions. This view is corroborated by recent LD-ADMR results on genetically modified reaction centers, and for a further discussion we refer to Refs. [18,34]. In order to determine the orientation of the triplet axes in the molecular coordinate system, knowledge is required of the orientation of Qv in this frame, as from LD-ADMR only relative angles are obtained. From linear-dichroic absorbance spectroscopy on Chl a in vesicles and BChl a in liquid crystals, Bauman and Wrobel [4] and Fragata et al. [5] concluded to similar polarizations of the Qr-transitions of Chl a and BChl a, with an angle of = 20 ° between Qr and y. The orientations found here for Q r in the triplet axes frame agree with the sign of the magneto-photoselection signals observed by Thurnauer and Norris for (B)Chls in a toluene/pyridine glass [12]. These authors have concluded that for all (B)Chls Qr makes an angle of less than 35 ° with YT, and that the sign of E for the Chls is opposite to that for the BChls. A remarkable result of the present study is that the in-plane triplet axes of Chl a seem to be 'rotated' with respect to those of BChl. Accepting that for all (B)Chls Qv is approximately parallel to
tively, for BChl a and BChl g, and close to the bisectrices of these axes for Chl a. Although the orientation of Qv with respect to the in-plane triplet axes depends somewhat on the solvent (see Tables 2 and 4), the overall difference in orientation between Chl a and the two BChls is much too large for a solvent effect. For BChl a and b in reaction centers of Rb. sphaeroides and Rps. viridis, respectively, whose structures have been solved by X-ray crystallography, the EPR and the LD-ADMR results could only be explained when it was assumed that YT of a localized triplet state and Q r are both oriented close to y (to within 10 °) [16,17]. These analyses agree well with our results for BChl a and g. The electron density in the triplet state of both Chl and BChl, as calculated by Petke et al. [2,3], comes close to that of a pure 3(alu --~ eg x) configuration. On the basis of the symmetry of the conjugated macrocycle of (B)Chl, which is approximately D2h for BChl and C2v for Chl, x T and YT should run through the NII-NIv and NI-NII I axes, respectively, in agreement with our results for BChl a and g, but not for Chl a. It has been suggested previously that the temperature and solvent dependence of the linewidth of the cation of Chl a measured by EPR could be explained by some admixture of the a2u first excited doublet configuration to the alu doublet ground-state configuration [35]. If mixing of the alu and a2u HOMOs occurs in Chl a, then a similar mixing might occur in the triplet state and cause a rotation of the in-plane triplet axes with respect to the molecular frame. Nevertheless, the Qv ($1 ~ So) and the triplet-triplet excitations apparently remain y polarized, just as observed for BChl for which the orbital mixing is assumed to be less than for Chl [8]. Different types of ring substituents are not expected to have a large effect on the orientation of the triplet axes. In fact, for BChl a and BChl g the difference in ring substituents is larger than for BChl a and Chl a, yet the difference in triplet axes orientation between BChl a and BChl g is smaller than that between the BChls and Chl a. Therefore, it seems plausible that the difference between BChl and Chl a is due to the presence of extra "rr-electron density on ring II in Chl a compared to BChl, which leads to a reduction of
J. Vrieze, A.J. Hoff / Chemical Physics Letters 237 (1995) 493-501
the energy gap between the a2u and alu orbitals [1,8] and, consequently, to a greater sensitivity to perturbations induced by ring substituents. The results of magnetophotoselection experiments on (B)Chls in vitro were earlier interpreted as 3Chl a and 3BChl a having opposite signs of E [12]. Accordmgly, we assumed for Chl a a negatwe sign of E. The results presented here on the angle between Q r and the in-plane triplet axes of Chl a (close to 45°), however, preclude a definite conclusion about the sign of E. •
3
•
•
Acknowledgement We thank Erik-Jan van de Meent and Christof Francke for isolating the (B)Chls and for preparing some of the samples, and Professor J.H. van der Waals for helpful discussion. This work was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organisation for Scientific Research (NWO).
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