Triplet excitation of precursors of spirilloxanthin bound to the chromatophores of Rhodospirillum rubrum as detected by transient Raman spectroscopy

Journal of Molecular Structure, 242 (1991) 15-26 Elsevier Science Publishers B.V.. Amsterdam

15

TRIPLET EXCITATION OF PRECURSORS OF SPIRILLOXANTHIN BOUND TO THE CHROMATOPHORES OF Rhodospirilluna rubrun AS DETECTED BY TRANSIENT RAMAN SPECTROSCOPY*

MITSURU NARUSE, HIDEKI YASUSHI KOYAMA**

HASHIMOTO,

MICHITAKA

KUKI and

Faculty of Science, Kwansei Gakuin University, Uegahara, Nishinomiya 662 (Japan) (Received

10 May 1990)

ABSTRACT Analysis by HPLC, and by subsequent mass spectrometry, of the carotenoid extract of the chromatophores prepared from the cells of a 3-day culture of Rhodospirillum rubrum Sl shows the presence of precursors of spirilloxanthin (rhodovibrin, anhydrorhodovibrin, and 3,4-dihydrospirilloxanthin) as minor components in addition to the major component, spirilloxanthin. Only spirilloxanthin is found in the extract of the chromatophores from the cells of a g-day culture. Transient Raman spectra of the carotenoids bound to the above two different kinds of chromatophore have been recorded by using 532 nm, N 100 ps mode-locked (76 MHz) and Q-switched (800 Hz) pulses; a T, Raman spectrum has been obtained by a one-color, pump-and-probe technique. The T, Raman spectra of rhodovibrin and spirilloxanthin free in tetrahydrofuran solution have also been recorded by using 337 nm pump and 532 nm probe N 10 ns pulses (10 Hz); a T, Raman spectrum has been obtained by a two-color, pump and probe technique (delay 1.8 p) using anthracene as a sensitizer. Spectral comparison indicates that the precursors as well as spirilloxanthin bound to the chromatophores can be excited to the TI state. The result strongly suggests that the precursors are tightly bound to the light-harvesting complex and are involved in energy transfer.

INTRODUCTION

Carotenoids in photosynthetic bacteria have dual functions of light-harvesting and photo-protection [ 11. Natural selection of carotenoid configurations, i.e. all-tran.s by the light-harvesting complex (LH) and 15-ck by the reaction center (RC ), has been found [ 2-91, and possible implications of the configurational selections have been discussed in relation to the excited-state properties of isomeric carotenoids [ 91. Since the symmetry-forbidden 2lA; (S,)*‘A; (S,) transition and the spin-forbidden 3B,+ (Tl)*lAg (So) *Dedicated to Professor Masamichi Tsuboi on the occasion of his 65th birthday. **Author to whom all correspondence should be addressed.

0022-2860/91/$03.50

0 1991-

Elsevier Science Publishers

B.V.

16

transition are utilized in the functions of light-harvesting and photo-protection, respectively, overlap of a pair of molecular orbitals of adjacent pigment molecules plays a crucial role in the above energy transfer functions through the electron-exchange mechanism. Therefore, the location as well as the molecular structures of the carotenoids in the excited states are of primary importance. Resonance Raman spectroscopy has proved to be powerful in elucidating the excited-state structures and vibrations of carotenoids bound to the photosynthetic systems. Lutz et al. [lo] first applied transient Raman spectroscopy to the T, state of a cis-carotenoid bound to the RC of Rhodobacter spheroides 2.4.1 and Ga; the Raman spectra of the T, states which were trapped at low temperatures were completely different from those of isomeric T1 carotenoids free in solution at room temperature [ 11,121. Hayashi et al. [ 341 applied picosecond time-resolved Raman spectroscopy to carotenoids bound to the chromatophores of Chromatium uinosum; TI Raman lines of carotenoids bound to the LH were detected at room temperature. Kuki et al. [ 131 and Hashimoto and Koyama [ 351 applied picosecond transient Raman spectroscopy to carotenoids bound to the chromatophores of Rhodobacter spheroides 2.4.1 and spinach chloroplasts, respectively; the presence of the 2lA; state as the S, state was demonstrated, and the vibronic coupling of the particular state with the ‘A; (S,) state th rough an (A,) C=C stretching vibration was shown to be weakened by intermolecular interaction with the apo-complex. In addition, the TI Raman spectra of carotenoids bound to the LH, which were essentially the same as those of free all- trans carotenoids, were presented. The LH of Rhodospirillum rubrum has a unique property in binding all- trans carotenoids; it binds not only the final product of carotenogenesis but also its precursors. Lozano et al. [ 141 and Schwerzmann and Bachofen [ 151 have proposed that the composition of carotenoids including spirilloxanthin and precursors in young cultures is approximately the same for LH, RC and the chromatophores (e.g. the carotenoids are equilibrated). The precursors have been assigned tentatively, based on spectral comparison, to rhodovibrin, anhydrorhodovibrin and other xanthophylls [ 151, although Koyama et al. [ 91 extracted 3,4-dihydrospirilloxanthin from the LH. Lozano et al. [ 141 used the CD and fluorescence excitation spectra to show that the precursors are tightly bound and involved in singlet-singlet energy transfer from a carotenoid to a bacteriochlorophyll (BChl). They also showed that the activity of the photoprotective function (triplet-triplet energy transfer from a BChl to a carotenoid) of a mixture of the precursors and spirilloxanthin was not much lower than that of pure spirilloxanthin. Schwerzmann and Bachofen [ 151 used the 880 nm absorption of BChl to show that its interaction with carotenoids should be independent of the carotenoid composition. Thus, some evidence for the carotenoid precursor-BChl interaction has been obtained. Pigment and peptide analyses of the LH of Rhodospirillum rubrum have

17

established that four BChls and two carotenoids are bound to two peptide subunits, a and p [ 16-181. The relative arrangement of the pigments is still to be elucidated. Here, a precursor, having spectral properties different from those of spirilloxanthin, functions potentially as a probe for the above energy transfer. In the present investigation, we have attempted to detect by picosecond transient Raman spectroscopy the T1 state of the precursors bound to the chromatophores of Rhodospirillum rubrum in order to prove that the precursor(s) are tightly bound to the LH and are involved in energy transfer. EXPERIMENTAL

Chromatophores (25 000-100 000 xg fraction) were prepared by sonication of the cells of Rhodospirillum rubrum Sl which were grown semi-anaerobically in the medium of Ormerod et al. [ 191 for 3 days or 6 days, and they were suspended in 0.1 M Tris-HCl (pH 7.5). Analysis of carotenoids which were extracted from the chromatophores with tetrahydrofuran (THF) was performed by reversed-phase high-pressure liquid chromatography (HPLC) [20]; THF (1.5 ml) was added to the chromatophore suspension (0.1 ml) and shaken, and then the THF layer was collected. The HPLC used a column 4 mm in diameter~30 cm in length packed with Chemcosorb 5 ODS-H, and the eluent consistedof solution A (methanol/water/ ion pair solution, 18: 1: 1, v/v) and solution B (ethyl acetate). The ion pair solution consisted of tetrabutylammonium hydroxide (10 ml of 0.5 M solution) and ammonium acetate (7.7 g) made up to 100 ml with distilled water. The solution was neutralized with acetic acid to pH 7.1. A linear gradient was applied, from pure solution A to pure solution B in 20 min, which was followed by isocratic hold with solution B for 5 min. The flow rate was 1 ml min-’ and the detection wavelength was 500 nm. The major component, spirilloxanthin, was identified by co-chromatography with the authentic sample, which was obtained by the method described elsewhere [ 91. The second and the third major components were collected from the THF extract (saponificated with 6% potassium hydroxide) of the chromatophores prepared from the cells of the 3-day culture. They were then subjected to electron-impact mass spectrometry using an Hitachi M-80 mass spectrometer, and were identified as rhodovibrin and anhydrorhodovibrin. Analysis of 3,4-dihydrospirilloxanthin was performed as described previously [9]; carotenoids were extracted with acetone, transferred to n-hexane, and subjected to HPLC using a Ca( OH), column and 10% acetone in benzene as eluent. Spirilloxanthin and 3,4-dihydrospirilloxanthin were identified by this HPLC. Transient Raman spectra of the carotenoids bound to the chromatophores (the concentration of the suspension, 0Dss2= 2) were recorded by using the 532 nm, mode-locked (76 MHz) and Q-switched (800 Hz) pulse trains (duration N 100 ps) from a Nd: YAG laser (Quantronix 416); a difference spec-

18

trum of high-power minus low-power was taken. The S, Raman spectra were recorded using low power. Experimental details have been described elsewhere [21-231. The T, Raman spectra of spirilloxanthin (5~ lop5 M) and rhodovibrin ( - 2 x low5 M) in THF solutions were recorded by pumping the sensitizer, anthracene (5 x 10e3 M), with 337 nm pulses from a Nz laser (NDC JLlOOOL), and by probing with 532 nm pulses from a Nd: YAG laser (Quantel YG480); the repetition rate was 10 Hz and the delay time was 1.8 ps. Experimental details have been described elsewhere [ 121. Accumulation of the above spectral data on an image-intensified diode-array detector (Princeton Instruments IRY-700) was for 6-14 min; no spectral smoothing was performed. RESULTS AND DISCUSSION

Figure 1 shows reversed-phase HPLC elution profiles for the THF extracts of the chromatophores from the cells of, (a) the 3-day and (b) the 6-day cultures. The elution profile for the 3-day culture shows three carotenoid components (the peak denoted B is due to BChl): component 2 was assigned to spirilloxanthin by co-chromatography with the authentic sample of spirilloxanthin. The electron-impact ionization mass spectrum of component 1 (data not shown) gave the molecular-ion peak at m/z 584 as well as the fragment peak of M - 106 (characteristic of the carotenoid backbone) at m/z 478. It also gave fragment peaks characteristic of one half of the spirilloxanthin skeleton, (a)

(b)

3 A.lb

12

16 Retention

20

0

?!2= ,,

Time

12

I

16

20

min

Fig. 1. Reversed-phase HPLC elution profiles of the THF extracts of the chromatophoresprepared from the cells of, (a) a 3-day culture and (b) a 6-day culture. Assignment of the peaks: B, bacteriochlorophyll a; 1, rhodovibrin; 2, spirilloxanthin; 3, anhydrorhodovibrin. Carotenoids were extracted with tetrahydrofuran. HPLC conditions are described in the text.

19

i.e. one at m/z 73 (cleavage of the l-2 bond) and the other at m/z 91. Thus, component 1 was assigned to rhodovibrin. The mass spectrum of component 3 gave the molecular-ion peak at m/z 566 and the fragment peak of M - 106 at m/z 460; it also gave fragment peaks at m/z 73 and 91. Thus, component 3 was assigned to anhydrorhodovibrin. (The mass spectra of rhodovibrin and anhydrorhodovibrin obtained in the present investigation were in general agreement with those reported earlier [ 241, although peaks due to unknown contaminants were also detected.) The elution profile for the 6-day culture shows virtually only component 2, i.e. spirilloxanthin. 3,4-Dihydrospirilloxanthin was identified for the chromatophores from the 3-day culture by another type of HPLC analysis using a calcium hydroxide column, as reported previously [9] (data not shown). In the above reversedphase HPLC, the peak of 3,4dihydrospirilloxanthin was not resolved from that of spirilloxanthin. The amount of 3,4-dihydrospirilloxanthin was negligible for the 6-day culture. Figure 2 compares the chemical structures of spirilloxanthin and the precursors, i.e. 3,4dihydrospirilloxanthin, rhodovibrin and anhydrorhodovibrin. As indicated by square brackets, all the precursors have exactly the same chromophoric skeleton consisting of a conjugated polyene-chain with twelve C=C bonds and six methyl groups attached to it. The only structural difference among the precursors is the end group on the right-hand-side of the figure. On the other hand, spirilloxanthin has a conjugated polyene-chain with thirteen C=C bonds, to which six methyl groups are attached to form the center of symmetry. Table 1 lists the wavelengths of the vibrational structures of the ‘AZ c and of the precursors in ‘A; and ‘B,+ +lA- absorptions of spirilloxanthin THF solutions. Reflicting the same chromophoric skeleton, all the precursors give the same absorption peaks within the limits of experimental error. Based on the above results, we assumed the same E values of 144 000 for all

(b)

Fig. 2. Chemical structures of, (a) spirilloxanthin, (b) 3,4-dihydrospirilloxanthin, (c) rhodovibrin, and (d) anhydrorhodovibrin. The chromophoric skeletons of spirilloxanthin and the precursors, which are responsible for the resonance F&man process, are indicated by square brackets.

20 TABLE 1 Electronic absorption peaks of spirilloxanthin

and its precursors (in THF solution) %++‘A, ”

‘A; +‘A,

Spirilloxanthin 3,4-Dihydrospirilloxanthin Rhodovibrin Anhydrorhodovibrin

l+O

oto

2+0

1-O

o+o

373 362 363 362

393 378 376 378

475 464 464 464

503 491 491 490

538 524 524 523

532 nm

.,

I

I

400

500 Wavelength

600

I

nm

Fig. 3. Absorption spectra of the chromatophores prepared from the cells of, (a) the 3-day culture (--- ) and (b) the g-day culture (-). Spectra are normalized at the Soret absorption of bacteriochlorophyll. The probing wavelength of 532 nm is shown with a vertical line.

the precursors to calculate the carotenoid composition for the extract of the chromatophores from the 3-day culture. The average of the E value (149 000 [=I) o f s P iri11oxanthin (having thirteen conjugated C=C bonds) and that (139000 [26]) ofp- carotene (having eleven conjugated C=C bonds) was taken. Corrections were made for relative absorption at the detection wavelength (500 nm) in the particular eluent, when the area under each peak in the chromatogram was used in the calculation. The molar ratio of spirilloxanthin: 3,4dihydrospirilloxanthin : rhodovibrin : anhydrorhodovibrin was calculated to be 6:1.5:2:1. Figure 3 shows the absorption spectra of the chromatophores from the cells

21

of, (a) the 3-day culture (broken line) and (b) the 6-day culture (solid line); the spectra are normalized at the Soret absorption of BChl. As the culture became older and the density of the cells increased, (1) the sharpening, (2) the red-shift and (3) the intensity increase of the vibrational structures of the ‘B + c ‘A - carotenoid absorption took place. The spectral change is explained in t”,rms o! transformation during growth from the precursors (having the same spectral properties) to spirilloxanthin, since the former has II,,, in the shorter wavelength region and is supposed to have a smaller E value. Figure 3 provides information concerning the relative resonance conditions between the precursors and spirilloxanthin bound to the chromatophores from the 3-day culture. Deconvolution of the spectrum, by taking account of the molar ratio of the precursors (3 ) versus spirilloxanthin (4 ) , and of the E values of the precursors (144 000) and spirilloxanthin (149 000), suggests that spirilloxanthin is under better resonance condition than the precursors, when the probing wavelength of 532 nm (shown by a vertical weak line in the figure ) is used. Nevertheless, the precursors and spirilloxanthin bound to the chromatophores as well as those free in THF solution (see Table 1) constitute similar rigorous resonance conditions. Since the relative intensity of the carotenoid Raman lines is not strongly dependent on the probing wavelength [4], the above similar resonance conditions facilitate comparison of Raman spectra of those different carotenoids in different environments. Figure 4 shows the So Raman spectra of the chromatophores from the cells of, (a) the 3-day culture and (b) the 6-day culture, and those of, (c) rhodovibrin and (d) spirilloxanthin in THF solution. Since the resonance Raman spectrum of a precursor reflects only the vibrations which take place in the chromophoric skeleton mentioned above (here, the ‘B,+ t ‘A; transition is under the resonance condition), rhodovibrin is considered to stand for all the precursors having the same chromophoric skeleton. The Raman spectra of the two different chromatophores are similar to one another. The only difference noted concerns the splitting of the 1186 (1187) and 1145 cm -’ lines; it is clearer for the 6-day culture. The Raman spectra of the free carotenoids are also similar to one another except for the frequency of the C=C stretching line. It is higher for rhodovibrin (1515 cm-‘) than for spirilloxanthin (1507 cm-l), reflecting the difference in the number of conjugated double bonds. The fact that the frequency of the C=C stretching line in the spectrum of the chromatophores (1507 cm-‘) from the 3-day culture coincides with that of spirilloxanthin supports the above idea that spirilloxanthin is under better resonance condition and that its spectral contribution is predominant. The most important information obtained from the spectra of the chromatophores is that the bound carotenoids are in the all-trans configuration. The Raman spectra of the carotenoids in THF solutions show that they are also in the all-tram configuration. (See Koyama et al. [ 271) for the dependence of the Raman spectra on the ck-trans configurations.) It is to be

22

noted that a Raman line at 1347 cm-l, which can be assigned either to the CH in-plane bending coupled with the C-C stretching or to the methyl symmetric deformation, is clearly identified not for the free carotenoids but for the bound carotenoids. This observation suggests a distortion in the configuration and/or in the electronic structure of the polyene skeleton. The T,+T, absorption of spirilloxanthin bound to the chromatophores has been shown by Nuijs et al. [ 281 to appear around 570 nm (see below). Assuming the same difference in the wavelength of the A,,, between rhodovibrin and spirilloxanthin in the S, state in THF solution (12 nm), the T,, e Tl absorption of the precursors is estimated to be around 558 nm. Thus, when probed at 532 nm, the Tl precursors bound to the chromatophores are expected to be under better resonance condition than T, spirilloxanthin. Figure 5 shows the T1 Raman spectra of the chromatophores from the cells

(b)

(b)

Cc)

Cd) 1500 Roman

1000 shift

/ cm-l

1500 Ramon

1000 shift

/

cm-l

Fig. 4. S, Raman spectra of the chromatophores prepared from the cells of, (a) the 3-day culture and (b) the 6-day culture. The So Raman spectra of, (c) rhodovibrin and (d) spirilloxanthin in THF solution are also shown for comparison. The spectra were recorded using the 532 nm, - 100 ps mode-locked and Q-switched pulse trains with low-power. Fig. 5. The transient Raman spectra of the chromatophores prepared from the cells of, (a) the 3day culture and (b) the 6-day culture. The Tl Raman spectra of, (c) rhodovibrin and (d) spirilloxanthin in THF solution are shown for comparison. See text for experimental details.

23

of, (a) the 3-day culture and (b) the g-day culture, and those of, (c) rhodovibrin and (d) spirilloxanthin in THF solution. The T1 precursors bound to the chromatophores for the 3-day culture are expected to be detected more easily, when the better resonance conditions as well as the molar ratio of precursors : spirilloxanthin = 3 : 4 are considered. Actually, the transient Raman spectrum of the chromatophores from the 3-day culture is definitely different from that of the chromatophores from the 6-day culture, and the former is explained in terms of an overlap of the T, Raman spectra of both the precursors and spirilloxanthin. Firstly, the spectrum of the chromatophores for the 3-day culture is characterized by a broader profile around 1265 cm-’ (peaks at 1274 and 1255 cm-l ) and also by the presence of a weak line at 972 cm-l. These characteristics are ascribable to the presence of the precursors, since the spectrum of rhodovibrin is characterized by a broader profile around 1265 cm-l and also by the presence of a line at 968 cm-‘. (By contrast, spirilloxanthin gives a sharper line at 1266 cm-l, and does not give any line around 970 cm-‘.) Secondly, the spectrum of the chromatophores for the 3-day culture is characterized by the higher frequencies of the Raman lines at 1175,112O and 1009 cm-‘; the corresponding lines appear at 1167, 1118 and 1003 cm-’ for the chromatophores from the 6-day culture. The characteristic is in accord with that of rhodovibrin; the corresponding Raman lines of rhodovibrin appear at 1177,1125 and 1006 cm-l, respectively, while those of spirilloxanthin appear at 1170,112l and 1003 cm-‘. The above comparisons lead us to the conclusion that the precursors as well as spirilloxanthin bound to the chromatophores can be excited to the T1 state. The T1 carotenoids are considered to be bound in the “all-tram” configuration. Binding of spirilloxanthin to the chromatophores affects the frequencies and the intensities of the T, Raman lines (see Figs. 5b and 5d). Low-frequency shifts of 1481-+1477, 1266+1260, 1170+1167 and 1121-+1118 cm-‘, and a high-frequency shift of 1344-+1347 cm-’ are seen; no frequency shift is seen for the 1003 cm-l line. Relative intensities of the 1481 and 1344 cm-’ Raman lines are strongly enhanced by the binding (1477 and 1347 cm-l ). The changes in frequencies and in intensities upon binding should be ascribed to changes in the geometry and in the electronic structure of the polyene skeleton. Parallel changes in intensities seem to take place for the precursors also (Figs. 5a and 5c). In the present investigation, the key Raman line of the 2lA; (S, ) state, such as the 1766 cm-’ line of spheroidene bound to the chromatophores of Rhodobatter spheroides 2.4.1 [ 131 or the 1753 cm-’ line of p-carotene bound to spinach chloroplasts [35] has not been detected for spirilloxanthin, even though exactly the same pump and probe pulses were applied. The result should be ascribed to the difference in the resonance condition as well as the lifetime of the S, state. Nuijs et al. [28] recorded the transient absorption of spirillox-

24

anthin bound to the chromatophores of Rhodospirillum rubrum by using a 35 ps, 532 nm pulse for excitation. They ascribed a broad transient absorption with a maximum near 610 nm to the S, state of the carotenoid, the lifetime of which was estimated to be less than 1 ps. (The lifetime for spirilloxanthin in n-hexane/isopropanol(97 : 3, v/v) was determined to be 0.6 + 0.1 ps. ) We used N 100 ps, 532 nm pulses for both pumping and probing. Therefore, the duration of the pump-and-probe pulse is about two orders of magnitude longer than the lifetime of bound spirilloxanthin. Further, the probing wavelength is much shorter than the S n t S, absorption. Thus, the S, state of the carotenoids should not be detected under the present conditions. Nuijs et al. [28] showed that the T1 state of spirilloxanthin bound to the chromatophores of Rhodospirillum rubrum is generated through a singlet homofission (and possibly heterofission) process, when a 532 nm, 35 ps pulse is used for direct excitation. The process was first demonstrated by Kingma et al. [ 29,301 based on the magnetic field effects on the emission of S, BChl and on the transient absorption of spirilloxanthin bound to the LH (chromatophores) of Rhodospirillum rubrum. Nuijs et al. [28] did not observe the generation of T1 spirilloxanthin through energy transfer from T1 BChl, the rise time of which had been determined by Monger et al. [31] to be N 20 ns by using a 532 nm, 15 ns pulse. They detected, instead, the decay of S1 BChl within 3 ns and also a non-linear loss of S, BChl at high laser power for excitation. They ascribed the observation to singlet-singlet annihilation of S, BChl. They found that the formation of T, carotenoid is proportional to the formation of S, carotenoid; here, no non-linear loss was observed. Thus, the process which includes intersystem crossing of BChl and subsequent triplet energy transfer from BChl to carotenoid was excluded. Further, appreciable dichroism of the T, c Tl absorption of the carotenoid excluded the possibility of depolarization steps such as energy transfer among BChls. The high peak-power of the picosecond pulses facilitates singlet homofission (heterofission) using S, carotenoid, while it causes quenching of S, BChl through singlet-singlet annihilation. In the present Raman measurements, we used N 100 ps pulses which were focused onto the sample solution. The photon density is estimated to be of the order of 1017photons cmM2,which compares to 1014-1016photons cmm2used by Nuijs et al. [ 281. Therefore, the T, carotenoids (spirilloxanthin and the precursors) detected by the present transient Raman spectroscopy should be generated through the singlet homofission (heterofission) process. Singlet homofission indicates that two carotenoid molecules taken from spirilloxanthin and the precursors are tightly bound together in the LH complex, while singlet heterofission indicates that a carotenoid and a BChl molecule are tightly bound together. In the present investigation, three different types of precursors were identified in the chromatophores prepared from the cells of the 3-day culture. Rhodovibrin and anhydrorhodovibrin, the last and the second last precursors of

25

spirilloxanthin in the pathway of carotenogenesis proposed by Liaaen-Jensen et al. [32], have been shown to occur by mass spectrometry. 3,4-Dihydrospirilloxanthin, the last precursor of spirilloxanthin in the pathway of carotenogenesis proposed by Malhotra et al. [ 331, were identified by mass spectrometry in a previous investigation [ 91. The precursors identified strongly support the above two different pathways of carotenogenesis. The biological implications of those precursors still remain to be elucidated. However, the present investigation has established that those precursors (unspecified) can be excited to the T, state, and are involved in energy transfer as the final product, spirilloxanthin. ACKNOWLEDGMENTS

The authors are indebted to Dr. Kayoko Saiki of Kobe Women’s College of Pharmacy for recording the mass spectra of the precursors. Thanks are also due to Dr. Hidenori Hayashi of the Faculty of Science, the University of Tokyo, for provision of a copy of the manuscript [ 341 prior to publication.

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