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Fluorescence-ODMR of chlorophyll in photosynthetic bacteria. Reaction centers of Rhodopseudomonas sphaeroides R 26
FLUORESCENCE-ODMR REACTION
14 January 1983
CHEhlJCAL PHYSICS LETTERS
Volume 94;number 2
CENTERS
OF CHLOROPHYLL
IN PHOTOSYNTHETIC
OF RHODOPSEUDOMONAS
J. BECK, J-U_ VON SCmZ
SPHAEROIDES
BACTERIA. R 26
and H-C. WOLF
PIiwikaliscltesInstitut. Teilinstitut 3. UniversitiitStuttgart, PfaffenwaIdring
5 7. D-7000
Sncttgarr-80,
West Germany
Received 19 October 1982
Fluorescence-detected ODMR measurements in zero field on isolated reaction centers and whole cells of the photosynthetic bacteria Rhodopseudomonas sphaeroides R 26 are reported. On reaction center preparations the triplet state could be found on the emission of Pat md on the fluorescence of other contaminating pigment compleses, all identified by escitation spectroscopy. Usinghigh-resolution EEDOR type double resonance, two triplet stateswith slightly different E values have been observed in all reaction centers.
1. Introduction The triplet state of chlorophyll in photosynthetic systems (plants and bacteria) has been used in the past with great success as an internal probe which allows the application of spin resonance methods to a study of the electronic and geometrical structure of the different chlorophyll-protein complexes which are active in the process of photosynthesis [ 1- 12]_ In bacteria, the process occurs in the photosynthetic apparatus which contains the light harvesting complexes forming the antenna system and the reaction centers. It is in these reaction centers where after the arrival of light excitation the charge separation is induced. When the quinone, which is also part of the reaction center is reduced or removed, the excitation decays via several radical pair states to the triplet state of the primary donor P8h5 and then to the ground state [ 13--2O]_ In this way an investigation of the triplet state is made possible_ In recent years Clarke and Hoff 12-81 reported ODMR experiments performed on a variety of photosynthetic bacteria in which the ODMR transitions were monitored via the emission of the antenna complexes in whole cells and chromatophores or via the absorption of the primary donor P865 in reaction center preparations [9,10] _In their work, the ODMR transi0 009-2614/83/0000-0000/S
03.00
0 1983 North-Holland
tions were attributed to one triplet state solely belonging to Pgb5_ The differences in the signs of the signals detected on the fluorescence of the antenna pigments and the absorption ofP86s respectively were explained with the model of the Vredenberg-Duysens relation [5-8,211 for the singlet energy transfer between the reaction centers and the antenna complexes. In this paper we report on F-ODMR experiments [22] detecting the ODMR transitions at several wavelengths of the fluorescence, the origin of which was analysed by means of excitation spectroscopy [23]_ On reaction center preparations we have been able to observe the ODMR signals of the triplet via the prompt fluorescence of P,,, directly and additionally via the emission of other contaminating pigment complexes. First reports of this work have been given elsewhere [24]_ Related experiments ofHoff et al_ [25] confirm our results. Using high-resolution EEDOR (electron-electron double resonance) experiments we could further prove the existence of two slightly different triplet states on whole cells as well as on two different reaction center preparations. 2. Materials and methods In our esperiments
we used a reaction center prepa141
CHEMICAL
ration from Rps. sphaeroides R 26 with the quinone reduced by dithionite which was a kind gift of Dr. J. Norris and another preparation with the quinone removed given IO us by Professor H. Scheer. The concentration of the samples was lo-35 fi1. Whole ceils of Rps. sphaeroides R 26 were reduced with didlionite [ 12.13]. The optical density was 1.0 31 600 mn (I CJJJ cuvette). Quartz tubes with an inner dkuneter of 2 mm were filled to 25 mm. Ail F-ODM R and EEDOR [ 261 esperimen ts were performed at T = f 2 IL The apparatus is described in rcf_ I?]_ Additionally we used for the optical excitation at 596 nni a cw dye laser system (Spectra Physics 11\0&1 164-07 sr1d rllodcl 375).
3. Results
Fig. I _shows the fluorescence spectra of ~c~l~lrolc 311d ot‘tl~e resction center preparations used for ,~ur ~spcrinwnts. The emission band of whole cells with rhc iwxiuiurn a1 910 rim represents the emission 01‘ JIJC Lt I-I antenna complexes [3S-301. The bands LIJ x~v&~~g~l~s shorter than S50 nm belong to precurWJS ()I‘ rhr bactcriocf~loropl~yll (BChl) biosynthesis 127.- Z I 1. 3’1~ near-infrared emission of the reaction cc111cr preparations have a masimum at %920 nm. The
CIY/S
PHYSICS
14 January
LETTERS
1983
blue side of this is more pronounced in the preparation wiviththe quinone removed_ The bands between 750 and 950 nm are due to “free” BChl and bacteriopheophytin (BPh) molecules and those around 690 nm due to 2-divinyL2-acetylchlorophyll as proved with excitation and emission-spectroscopy 1231. For the F-ODMR experiments on reaction center preparations we used several different wavelengths of the fluorescence for detection. The signals of the quinone-free preparation are represented in fig. 2. Exactly the same results are obtained on preparations with the quinone removed [31]. When monitoring the F-ODMR in the spectral region from 935 to 915 nm (fig. 2) we obtain positive ODMR signals, solely representing the D + E and the D - E transitions between the triplet sublevels. The signals are broad (30MHz) andhave a lineshape which points towards the assumption that there are two signals superimposed_ This aspect will be examined later in connection with the EEDOR e.uperiments. When the blue side (905-S95 nm) of the broad emission band with a maximum at 920 nm is used for detection the sign of the F-ODMR response changes and we observe negative F-ODMR signals which have the same frequencies as above. Unexpectedly the same transitions could also be detected on the emission at
780 2fL-3fs LOO
500
700
600
v/MHz
-
I.‘&. 2. FODhfR signals from isolated reaction centers of Rhodopseudomonas sphaeroides R 26 with quinone reduced by dithionite as a function of the wavelength of detection. T= 1-S K, bandwidth of detection 1 ntn. The signals are normalized to the same iteight.
Volume 94. number 2
14 January 1983
CHEMICAL PHYSICS LETTERS
830 nm with a negative sign and at 780 mn With a positive sign_ The F-ODMR signals detected on whole cells using the emission of the III-1 antenna system with a maximum at 910 nm, appeared at the same frequencies as those of the reaction center preparations detected at 895 nm but are negative in sign_The frequencies, linewidths and lineshapes of these signals are independent of the wavelengths within the fluorescence band. This is also demonstrated by F-MDR experiments in which the amplitude modulated rf is fsed at the resonance frequency of one transition while the optical wavelength is scanned over the fluorescence spectrum with lock-in detection_ The observed F-MDR spectrum is identical with the fluorescence spectrum of all antenna pigments showing that the modulation of the reaction center triplet concentration influences the fluorescence of the antenna complexes. We turn now to the more detailed investigation of the inhomogeneously broadened ODMR transitions. In reaction center preparations as well as in whole cells the signals have a half-width of lo-30 MHz and holeburning experiments resulted in holes with a half-width of ~2 MHz. In order to find a correlation between distinct frequency regions within the inhomogeneous D + E and D - E transitions we used EEDOR experiments_ For that purpose we saturated of the signals with one microwave source fiied in frequency and monitored the corresponding 3E transitions_ These 3E signals, which do not show without EEDOR rf are represented in fig. 3. They were detected with different saturation frequencies applied to the D + E and
D - E transitions
of whole cells and reaction center preparations detected at 935 nm. In both samples the variation of the saturation frequency within each of the inhomogeneous transitions yielded two different 2Esignalsatabout 190and215MHzinisolated reaction centers and at about 183 and 209 MHz in whole cells indicating that in both cases there exist two slightly different triplet states. Within experimental accuracy the two 2E signals measured in whole cells and reaction center preparation are identical_ These experiments allow us to correlate welldefined sections of the inhomogeneous D + E transition to respective sections of the D - E transition via one of the two 2E signals. In fig. 4 the correlated sec-
tions are represented schematically. It can be seen that the 2E signals at 190 MHz (RC) and 183 MHz (whole
RC 670
RC 657 RC
451
RC 472 WC 6.m
WC 6’70 /J-
WC L75
-7l
WC 453 100
150
200
250
300
v/MHz
-
Fig. 3. Comparison of the 2E s&n& of isolated reaction centers (1 C) (quinone reduced) detected on the fluorescenac at 935 nr&and of whole cells (WC) (quinone reduced). The saturation fl .quencies used for the EEDOR experiments are shown on ‘he left. All signals are normalized to the same height.
cells) - attrib:ted to triplet I- belong to the highfrequency region of the D -E and to the low-frequency region of the D + E signal. The 2E transition at 2 15 to triplet II corresponds to the low-frequency side of the
MHz (RC) and 209 MHz (whole cells) attributed
2lE:
IDI- IEI
IDI . IEI
v/MHz
LOO
500
6&l V/Mnz
7ilO -
Fig. 4. Schematic representation of the correlation between the two 2E s&n& r;nd the sections of the D + E and D - E transitions shown at the signals of isolated reaction centers
(RC) and whole cells (WC).
Surnm:rr~~ t*ti ;:!I rcsonancc ticqucncies n’JtaIncd by conventional i’-ODBIR and of al1 frequency sections which could be correlated IV rhc 1.1 I)OR c\pcrimcn;s for triplcr 1 and triplet II. Error limits: AD, AE = 0.5 X lob4 cm-‘. The EEDOR csperiments were pcrl;*lrllctf in Ihc folkwing manner: In all casts the 2E frequency was swept and the saturation frequency was applied within the I‘rcclur~:~~ ~angcs of t!w II + I:‘ or D - E sigals. Those regions in which the saturation frequencies have results in the E value of ~riplcr 1 mdicate the ccc&us of the U + E and D - E signals which can be correlated with triplet I. The same procedure eshibited lhc ‘.aIrr&t4 sections of triplet II. The valuers pircu in parcnthcses are calculated from the esperhncntal data ..~~ ._~._____ --..._ 2iEI (31112)
rriplcr
It
correlated sections
209
I:‘ and to the high-frequency side of the D + E SipId. ‘I’fwarms oi’ the correlating regions as given in fig. 4 I;lr the I--ODMI~ signals of wltolc cells demonstrate
I)
dr;~sGcally 0131 the signals of triplet 1 dominate those t)t- I riplet II. This is also seen in fig. 3 where the 21;’ IIansirions of triplet I are detectable via all saturation I’rcquencirs within the D + If and the D - E signals in i‘c~ntrast io fhc 2/:‘signals of triplet II which are only ~~I~rvec! in vev narrow regions as a superposition \r;itli ~lrosc 01 triplet I. Table 1 summarizes all transilion! l~rcqucncies
and zfs parameters.
f. IIiscussion ‘1I1e origin of t11r reduction of the zfs parameters dcleclsti in whole cells and reaction center preparations rr‘l:!tivr’ to Ihe lKh1 monomer is discussed extcnbivcl>. ~II the literature [ 2-81. The ODhlR frequenzics mci~surcd in our expcrimcnts are comparable with lhobe frclni Clarke und I Id1 [Z-S] so we focus our a:tcnlion OII the clijfiwnr r&s observed on whole cells :IIIJ on reaction center preparations at several wavelengths ofthe sl[qi:s’lrf[~* cIij~2rc~Jlr
rspe’imCn
ts.
fluorescence rri~Jkr
SIUICS
(fig. 2) and to the IW~ discovered
in our
EEDOR
D - E (MHz)
D-f-E (MHz)
IDI (low4
cm-‘)
IEI (10m4 cm-‘)
-
450-484
655-688
460-472 (467)
(650-662) 657
183.1-187.1
31.2
451-460 (463)
(666-675) 678
184.3-187.2
35.6
452-478
640-666
453175
(639-653)
180.2-190.2
30.8
662-670 (660-666)
18-7.6-I
34.6
(453-461) 453-469
86.6
For an explanation of the sign of the F-ODhlR signals it is unavoidable to consider the energy-transfer processes which are assumed in the photosynthetic apparatus. For that purpose we have to identify at first the molecule or the complex from which the monitored fluorescence originates. In the case of the rcaction center preparations this was done by fluorescence excitation spectroscopy 1231. So we could prove that the region from 935 to 9 15 nm of the near infrared emission can be attributed to the fluorescence of PgG5 [23]_ This means that the F-ODMR signals observed there are detected directly on the fluorescence of the primary donor where the triplet excitation is localized. The positive sign of the signals at direct detection of the fluorescence and the negative sign (equal in frequency) at detection via the antenna emission in whole cells C0JlfirJJZ fltC JJlOdd of a singlet-singlet
energy
transfer
in the photosynthetic
apparatus [ 5,7,8,2 11. It can be verified experimentally that the F-ODMR transitions of the LH-I antenna complexes are not identical with those of the reaction centers. It is shown in the following paper that the F-ODhlR transitions of the antenrta complexes detected on the antenna fluorescence are different from those reported here. Thus in the photosynthetic apparatus of whole cells
Volume 94. numbrr 2
CHEMICAL
PHYSICS LETTERS
the application of resonant microwaves between the triplet sublevels decreases the population of the triplet state T, and so increases the population of the singlet ground state St, - in agreement with the A-DMR experiments of Hoff [9,10] - and as a result the fluorescence of P,,, increases under steady-state conditions. The antenna fluorescence, on the other hand, decreases due to the couphng between the singlet states of the antenna and reaction center complexes_ This coupling is known as Vredenberg-Duysens relation [21]. Further insight into the energy-transfer processes in whole cells is given by the F-MDR spectrum which is identical with the fluorescence of the antenna system_ From this we conclude that the modulation of rite excited triplet comennation in PgGs affects all fluorescent antenna pigments. This works only if the absorbed excitation energy is delocalized in the whole antenna system before being trapped by the reaction center or emitted by fluorescence, analogous to the ideas of Campillo [33]_ The fact that the F-ODMR spectra are observed even at T = 1.2 K demands that the singlet energy levels of the antenna pigments are almost degenerate, having a broad homogeneous linewidth of *ZOO cm-l. Shifting the wavelength of detection to the blue side of the near-infrared emission (895-905 nm) the excitation spectra show an increase of the 596 nm band of lo- 15% relative to the excitation band of the BP11(530-540 nm) of the reaction center. This indicates that there is a fraction of additional BChl fluorescence in this spectral region. The only BChl complexes except reaction centers, which are known to emit at these wavelengths, are the LH-I antenna complexes_ From this we conclude that in the region from S95 to 90s run the superposition of the fluorescence from “pure” reaction centers and from a contamination of antenna complexes aggregated with reaction centers is observed_ This is confirmed by the negative sign of the F-ODMR signals indicating the same singlet-singlet energy transfer as in whole cells. The emission bands at 780 and 830 nm in fig. 1 arc attributed to monomeric BChl and BPh molecules which are not involved in the reaction center protein complex. Concerning the excitation spectra, we assume that these pigments originate from denaturated reaction centers [23]. The F-ODMR transition frequencies detected on
these emission bands are the same as those of the 895 nm emission and therefore different from monomeric BChl and BPh molecules even when they are bound to protein [31]. So we attribute these signals to reaction center triplets which are observed via si&et energy transfer from the BChl and BPh molecules to *he reaction center complexes. Due to the high concentration of the sample we are not able to decide whether the singlet energy transfer is only an effect of re-absorption or a Fiirster-type transfer_ The different signs of the F-ODhIR signals observed at 780 and 830 nm are consistent with the model of a singlet-singlet energy transfer only when the experimentally determined alterations of the absorption changes influenced by microwave resonance transitions in the region of the spectral overlap between donor and acceptor is regarded_ This absorption difference spectrum as published by Hoff [ lo] shows a decrease in absorption at 780 nm and an increase at S30 nm. With this a positive F-ODMR signal at 780 nrn and a negative one at 830 nm are predicted which is in agreement with our experiments (fig. 2). Up to now the ODhlR transitions of the reaction center were attributed to one triplet state belonging to P,,, only. Our high-resolution EEDOR experiments (fig. 3) demonstrate that in isolated reaction centers as well as in reaction centers of whole cells always a superposition of two slightly different triplet states is observed. This leads to the conclusion that the ODOR sigiials of rencrion cmlers are iI1general 5 wperposiriorl ofrltcse two triplets. The variation in half-widths and lineshapes of ODNR lines observed by several authors [3-9,X] can be explained by different ratios of triplet I relative to triplet II. So all inconsistencies in the zfs parameters detected with F-ODMR in whole cells and F-ODXlR and A-DMR in isolated reaction centers as published by Hoff et al. [25] disappear. A comparison of the zfs parameters of triplet I and II shows that their D values are nearly identical (table 1) and that there is only a difference in E (3 1 and 35 X 10e4 cm- I)_ From this we assume that the IWOtriplets belong to two slightly different geonietrical configurations of Pa5 possibly generated by a change in the mutual orientation of the two molecules constituting the dimer. Preparative artefacts and an influence of the detergent as an origin of the two conBgurations can be es145
CHEMICAL
94, number 2
V0h1me
eluded. because the two triplets are also observed in whole cells. The fact that a preparation with reduced rluinone and one with the quinone removed show the SCUIW results eliminates an influence of quinone and dithionite. A possible esplanation would be the existence of
further triplet states as required by Schenk et al. [ I 5, lb] for the decay rates of the radical pair. These states ;1re espected to have very short lifetimes and therefore it is unreasonable that they are detected in our experiments. We think that at least at low temperature Ilvo geutrtcrrical cmjijp-ariota of PgGs are allowed. The fact that the intensity of triplet II is lower in systems consisting of reaction center proteins combined with antenna complexes (whole cells and the signals detected on the reaction center fluorescence at 895-905 nm) is a hint for the influence of the surrounding of the reaction
center
proteins.
“pure
isolated”
reaction
cen-
ter proteins exhibit both triplet states whereas the association with antenna proteins leads to a lower intensity of triplet
181A.H.
Hoff, Physics Rept. 54 (1979)
1983
75.
191 H.J. den Blanken. G.P. van der Zwet and A.H. Hoff, Chem. Phys. Letters 85 (1982)
335.
[lOI H.J. den Blanken and A.H. Hoff, Biochim. Biophys. IllI 1121
1131 1141 [ISI [I61 1171
It81 [I91 I201
Il.
Acknowledgement We are very Srateful IO Dr. J. Norris (Argonne Sarional Laboratory) and Professor H. Scheer (UniversitZt Xliinchen) for their generous gifts of reaction center preparations. A. Angerhofen took the excitation spectra which were of great value for the interpretations. This work was supported by the Deutsche l~orscltungsgenieinscliaft.
14 January
PHYSICS LETTERS
1251
Acta (1982), to be published. G.H. van Brakel, Dissertation, Landbouwhogeschool Wageningen (1982). T-J. Schaafsma, in: Triplet state ODMR spectroscopy, ed. R.H. Clarke (Wiley, New York, 1982) ch. 8, p_ 346. V.A. Shuvalov and W-IV_ Parson, Proc. Natl. Acad. Sci. US 78 (1981) 957. V.A. Shuvalov and W-W. Parson, Biochii. Biophys. Acta 639 (1981) 50. CC. Schenk. RX. Blankenship and W-W. Parson, Biochim. Biophys. Acta 680 (1982) 44. CC. Schenk, W-W. Parson, D. Holten and hi-W_ Windsor, Biochim. Biophys. Acta 635 (I 981) 383. R. Haberkom and WE. Michel-Beyerle, FEBS Letters 75 (1977) 5. A. Oprodnik, H-W_ Kruger, H. Orthuber, R. Haberkorn, ME. Michel-Bcyerle and H. Scheer. Biophys. J. (1982). to be published. AH. Hoff, P. Cast and J.C. Romihn. FEBS Letters 73 (1977) 18.5. M.K. Bowman. D.E. Budil, G.L. Gloss. A.C. Kosta. CA. Wraight and J.R. Norris, Proc. NatI. Acad. Sci. US 78 (1981) 3305. WM. Vredenberg and L.N.M_ Duysens, Nature 197 (1963) 355. W.G. van Dorp, T.J. Schaafsma. W. Soma and J.H. van der Waals. Chem. Phgs. Letters 21 (1973) 221. A. Angerhofer. Diplomarbeit, Universitit Stuttgart (1982). J. Beck, J.U. van Schiitr and H.C. Wolf, Photosynthese. Rundgespr%zh der Deurschcn Forschungsgemcinschaft Whirburg, November 1981; DPG-Friihjshrstagung 1982, Verhandl. DPG 4 (I 982) 394. H.J. den Blanken, G.P. van der Zwct and A.H. Hoff, Bio-
chim. Biophys. Acta (1982). to be published. 1351 TS. Kuan, D.S. Tinti and MA. El-Sayed. Chem. Phys. Letters 4 (1970)
507.
I271 J. Beck, G.H. Kaiser, J.U. von Schiitz and H.C. Wolf,
References
Biochim. Biophys. Acta 634 (1981)
165.
1281 G.H. Kaiser, J. Beck, J.U. von Schiitz and ILC. Wolf, P.L. Dutton. J.S. Leigh and M. Seibert, Biochem. phys. Rrs. Commun. 46 (197 1) 406. K.11. Clarke. K.E. Canners, J.R. Norris and MC.
Bio-
‘ffmrnaucr. J. Am. Chem. Sec. 97 (1975) 7178. R.11. Clarke and RI. Connors. Chem. Phys. Letters 42 (1976) 69. R.11. CLrRc. R.E. Connors. H.A. Frank and J.C. Hoch, Chcm. Phys. Letters 45 (1977) 52X A.H. ffoffand H. Carter de Vrics. Biochim. Biophys. A~CX503 (1978) 94. A.ti. Hoff. Biochim. Biophys. Acta 440 (1976) 765. A.H. Hoff. in: Triplet state ODMR spectroscopy. ed. KM. Clarke (Wiley, New York, 1982) ch. 9, p. 415.
Biochim. Biophys. Acta 634 (1981)
153.
1’91 G.H. Kaiser, J. Beck, J-U. van Schlitz and H.C. Wolf, in:
Photosynthesis. Proceedings of the 5th International Photosynthesis Congress, Halkidiki, Greece, Vol. 1. ed. G. Akoyunoglou (Balaban. International Science Services, Philadelphia. 1981) p_ 69. [ 301 G.H. Kaiser. Dissertation. UniversitZt Stuttgart (1980). 1311 J. Beck. Dissertation. Univcrsitit Stuttgart (1982). [ 321 A-J. Campillo. R-C!. Hyer. T.G. hlongcr. W.W. Parson and S.L. Shapiro, Proc. Natl. Acad. Sci. US 74 (1977) 1997.
14 January 1983
CHEhlJCAL PHYSICS LETTERS
Volume 94;number 2
CENTERS
OF CHLOROPHYLL
IN PHOTOSYNTHETIC
OF RHODOPSEUDOMONAS
J. BECK, J-U_ VON SCmZ
SPHAEROIDES
BACTERIA. R 26
and H-C. WOLF
PIiwikaliscltesInstitut. Teilinstitut 3. UniversitiitStuttgart, PfaffenwaIdring
5 7. D-7000
Sncttgarr-80,
West Germany
Received 19 October 1982
Fluorescence-detected ODMR measurements in zero field on isolated reaction centers and whole cells of the photosynthetic bacteria Rhodopseudomonas sphaeroides R 26 are reported. On reaction center preparations the triplet state could be found on the emission of Pat md on the fluorescence of other contaminating pigment compleses, all identified by escitation spectroscopy. Usinghigh-resolution EEDOR type double resonance, two triplet stateswith slightly different E values have been observed in all reaction centers.
1. Introduction The triplet state of chlorophyll in photosynthetic systems (plants and bacteria) has been used in the past with great success as an internal probe which allows the application of spin resonance methods to a study of the electronic and geometrical structure of the different chlorophyll-protein complexes which are active in the process of photosynthesis [ 1- 12]_ In bacteria, the process occurs in the photosynthetic apparatus which contains the light harvesting complexes forming the antenna system and the reaction centers. It is in these reaction centers where after the arrival of light excitation the charge separation is induced. When the quinone, which is also part of the reaction center is reduced or removed, the excitation decays via several radical pair states to the triplet state of the primary donor P8h5 and then to the ground state [ 13--2O]_ In this way an investigation of the triplet state is made possible_ In recent years Clarke and Hoff 12-81 reported ODMR experiments performed on a variety of photosynthetic bacteria in which the ODMR transitions were monitored via the emission of the antenna complexes in whole cells and chromatophores or via the absorption of the primary donor P865 in reaction center preparations [9,10] _In their work, the ODMR transi0 009-2614/83/0000-0000/S
03.00
0 1983 North-Holland
tions were attributed to one triplet state solely belonging to Pgb5_ The differences in the signs of the signals detected on the fluorescence of the antenna pigments and the absorption ofP86s respectively were explained with the model of the Vredenberg-Duysens relation [5-8,211 for the singlet energy transfer between the reaction centers and the antenna complexes. In this paper we report on F-ODMR experiments [22] detecting the ODMR transitions at several wavelengths of the fluorescence, the origin of which was analysed by means of excitation spectroscopy [23]_ On reaction center preparations we have been able to observe the ODMR signals of the triplet via the prompt fluorescence of P,,, directly and additionally via the emission of other contaminating pigment complexes. First reports of this work have been given elsewhere [24]_ Related experiments ofHoff et al_ [25] confirm our results. Using high-resolution EEDOR (electron-electron double resonance) experiments we could further prove the existence of two slightly different triplet states on whole cells as well as on two different reaction center preparations. 2. Materials and methods In our esperiments
we used a reaction center prepa141
CHEMICAL
ration from Rps. sphaeroides R 26 with the quinone reduced by dithionite which was a kind gift of Dr. J. Norris and another preparation with the quinone removed given IO us by Professor H. Scheer. The concentration of the samples was lo-35 fi1. Whole ceils of Rps. sphaeroides R 26 were reduced with didlionite [ 12.13]. The optical density was 1.0 31 600 mn (I CJJJ cuvette). Quartz tubes with an inner dkuneter of 2 mm were filled to 25 mm. Ail F-ODM R and EEDOR [ 261 esperimen ts were performed at T = f 2 IL The apparatus is described in rcf_ I?]_ Additionally we used for the optical excitation at 596 nni a cw dye laser system (Spectra Physics 11\0&1 164-07 sr1d rllodcl 375).
3. Results
Fig. I _shows the fluorescence spectra of ~c~l~lrolc 311d ot‘tl~e resction center preparations used for ,~ur ~spcrinwnts. The emission band of whole cells with rhc iwxiuiurn a1 910 rim represents the emission 01‘ JIJC Lt I-I antenna complexes [3S-301. The bands LIJ x~v&~~g~l~s shorter than S50 nm belong to precurWJS ()I‘ rhr bactcriocf~loropl~yll (BChl) biosynthesis 127.- Z I 1. 3’1~ near-infrared emission of the reaction cc111cr preparations have a masimum at %920 nm. The
CIY/S
PHYSICS
14 January
LETTERS
1983
blue side of this is more pronounced in the preparation wiviththe quinone removed_ The bands between 750 and 950 nm are due to “free” BChl and bacteriopheophytin (BPh) molecules and those around 690 nm due to 2-divinyL2-acetylchlorophyll as proved with excitation and emission-spectroscopy 1231. For the F-ODMR experiments on reaction center preparations we used several different wavelengths of the fluorescence for detection. The signals of the quinone-free preparation are represented in fig. 2. Exactly the same results are obtained on preparations with the quinone removed [31]. When monitoring the F-ODMR in the spectral region from 935 to 915 nm (fig. 2) we obtain positive ODMR signals, solely representing the D + E and the D - E transitions between the triplet sublevels. The signals are broad (30MHz) andhave a lineshape which points towards the assumption that there are two signals superimposed_ This aspect will be examined later in connection with the EEDOR e.uperiments. When the blue side (905-S95 nm) of the broad emission band with a maximum at 920 nm is used for detection the sign of the F-ODMR response changes and we observe negative F-ODMR signals which have the same frequencies as above. Unexpectedly the same transitions could also be detected on the emission at
780 2fL-3fs LOO
500
700
600
v/MHz
-
I.‘&. 2. FODhfR signals from isolated reaction centers of Rhodopseudomonas sphaeroides R 26 with quinone reduced by dithionite as a function of the wavelength of detection. T= 1-S K, bandwidth of detection 1 ntn. The signals are normalized to the same iteight.
Volume 94. number 2
14 January 1983
CHEMICAL PHYSICS LETTERS
830 nm with a negative sign and at 780 mn With a positive sign_ The F-ODMR signals detected on whole cells using the emission of the III-1 antenna system with a maximum at 910 nm, appeared at the same frequencies as those of the reaction center preparations detected at 895 nm but are negative in sign_The frequencies, linewidths and lineshapes of these signals are independent of the wavelengths within the fluorescence band. This is also demonstrated by F-MDR experiments in which the amplitude modulated rf is fsed at the resonance frequency of one transition while the optical wavelength is scanned over the fluorescence spectrum with lock-in detection_ The observed F-MDR spectrum is identical with the fluorescence spectrum of all antenna pigments showing that the modulation of the reaction center triplet concentration influences the fluorescence of the antenna complexes. We turn now to the more detailed investigation of the inhomogeneously broadened ODMR transitions. In reaction center preparations as well as in whole cells the signals have a half-width of lo-30 MHz and holeburning experiments resulted in holes with a half-width of ~2 MHz. In order to find a correlation between distinct frequency regions within the inhomogeneous D + E and D - E transitions we used EEDOR experiments_ For that purpose we saturated of the signals with one microwave source fiied in frequency and monitored the corresponding 3E transitions_ These 3E signals, which do not show without EEDOR rf are represented in fig. 3. They were detected with different saturation frequencies applied to the D + E and
D - E transitions
of whole cells and reaction center preparations detected at 935 nm. In both samples the variation of the saturation frequency within each of the inhomogeneous transitions yielded two different 2Esignalsatabout 190and215MHzinisolated reaction centers and at about 183 and 209 MHz in whole cells indicating that in both cases there exist two slightly different triplet states. Within experimental accuracy the two 2E signals measured in whole cells and reaction center preparation are identical_ These experiments allow us to correlate welldefined sections of the inhomogeneous D + E transition to respective sections of the D - E transition via one of the two 2E signals. In fig. 4 the correlated sec-
tions are represented schematically. It can be seen that the 2E signals at 190 MHz (RC) and 183 MHz (whole
RC 670
RC 657 RC
451
RC 472 WC 6.m
WC 6’70 /J-
WC L75
-7l
WC 453 100
150
200
250
300
v/MHz
-
Fig. 3. Comparison of the 2E s&n& of isolated reaction centers (1 C) (quinone reduced) detected on the fluorescenac at 935 nr&and of whole cells (WC) (quinone reduced). The saturation fl .quencies used for the EEDOR experiments are shown on ‘he left. All signals are normalized to the same height.
cells) - attrib:ted to triplet I- belong to the highfrequency region of the D -E and to the low-frequency region of the D + E signal. The 2E transition at 2 15 to triplet II corresponds to the low-frequency side of the
MHz (RC) and 209 MHz (whole cells) attributed
2lE:
IDI- IEI
IDI . IEI
v/MHz
LOO
500
6&l V/Mnz
7ilO -
Fig. 4. Schematic representation of the correlation between the two 2E s&n& r;nd the sections of the D + E and D - E transitions shown at the signals of isolated reaction centers
(RC) and whole cells (WC).
Surnm:rr~~ t*ti ;:!I rcsonancc ticqucncies n’JtaIncd by conventional i’-ODBIR and of al1 frequency sections which could be correlated IV rhc 1.1 I)OR c\pcrimcn;s for triplcr 1 and triplet II. Error limits: AD, AE = 0.5 X lob4 cm-‘. The EEDOR csperiments were pcrl;*lrllctf in Ihc folkwing manner: In all casts the 2E frequency was swept and the saturation frequency was applied within the I‘rcclur~:~~ ~angcs of t!w II + I:‘ or D - E sigals. Those regions in which the saturation frequencies have results in the E value of ~riplcr 1 mdicate the ccc&us of the U + E and D - E signals which can be correlated with triplet I. The same procedure eshibited lhc ‘.aIrr&t4 sections of triplet II. The valuers pircu in parcnthcses are calculated from the esperhncntal data ..~~ ._~._____ --..._ 2iEI (31112)
rriplcr
It
correlated sections
209
I:‘ and to the high-frequency side of the D + E SipId. ‘I’fwarms oi’ the correlating regions as given in fig. 4 I;lr the I--ODMI~ signals of wltolc cells demonstrate
I)
dr;~sGcally 0131 the signals of triplet 1 dominate those t)t- I riplet II. This is also seen in fig. 3 where the 21;’ IIansirions of triplet I are detectable via all saturation I’rcquencirs within the D + If and the D - E signals in i‘c~ntrast io fhc 2/:‘signals of triplet II which are only ~~I~rvec! in vev narrow regions as a superposition \r;itli ~lrosc 01 triplet I. Table 1 summarizes all transilion! l~rcqucncies
and zfs parameters.
f. IIiscussion ‘1I1e origin of t11r reduction of the zfs parameters dcleclsti in whole cells and reaction center preparations rr‘l:!tivr’ to Ihe lKh1 monomer is discussed extcnbivcl>. ~II the literature [ 2-81. The ODhlR frequenzics mci~surcd in our expcrimcnts are comparable with lhobe frclni Clarke und I Id1 [Z-S] so we focus our a:tcnlion OII the clijfiwnr r&s observed on whole cells :IIIJ on reaction center preparations at several wavelengths ofthe sl[qi:s’lrf[~* cIij~2rc~Jlr
rspe’imCn
ts.
fluorescence rri~Jkr
SIUICS
(fig. 2) and to the IW~ discovered
in our
EEDOR
D - E (MHz)
D-f-E (MHz)
IDI (low4
cm-‘)
IEI (10m4 cm-‘)
-
450-484
655-688
460-472 (467)
(650-662) 657
183.1-187.1
31.2
451-460 (463)
(666-675) 678
184.3-187.2
35.6
452-478
640-666
453175
(639-653)
180.2-190.2
30.8
662-670 (660-666)
18-7.6-I
34.6
(453-461) 453-469
86.6
For an explanation of the sign of the F-ODhlR signals it is unavoidable to consider the energy-transfer processes which are assumed in the photosynthetic apparatus. For that purpose we have to identify at first the molecule or the complex from which the monitored fluorescence originates. In the case of the rcaction center preparations this was done by fluorescence excitation spectroscopy 1231. So we could prove that the region from 935 to 9 15 nm of the near infrared emission can be attributed to the fluorescence of PgG5 [23]_ This means that the F-ODMR signals observed there are detected directly on the fluorescence of the primary donor where the triplet excitation is localized. The positive sign of the signals at direct detection of the fluorescence and the negative sign (equal in frequency) at detection via the antenna emission in whole cells C0JlfirJJZ fltC JJlOdd of a singlet-singlet
energy
transfer
in the photosynthetic
apparatus [ 5,7,8,2 11. It can be verified experimentally that the F-ODMR transitions of the LH-I antenna complexes are not identical with those of the reaction centers. It is shown in the following paper that the F-ODhlR transitions of the antenrta complexes detected on the antenna fluorescence are different from those reported here. Thus in the photosynthetic apparatus of whole cells
Volume 94. numbrr 2
CHEMICAL
PHYSICS LETTERS
the application of resonant microwaves between the triplet sublevels decreases the population of the triplet state T, and so increases the population of the singlet ground state St, - in agreement with the A-DMR experiments of Hoff [9,10] - and as a result the fluorescence of P,,, increases under steady-state conditions. The antenna fluorescence, on the other hand, decreases due to the couphng between the singlet states of the antenna and reaction center complexes_ This coupling is known as Vredenberg-Duysens relation [21]. Further insight into the energy-transfer processes in whole cells is given by the F-MDR spectrum which is identical with the fluorescence of the antenna system_ From this we conclude that the modulation of rite excited triplet comennation in PgGs affects all fluorescent antenna pigments. This works only if the absorbed excitation energy is delocalized in the whole antenna system before being trapped by the reaction center or emitted by fluorescence, analogous to the ideas of Campillo [33]_ The fact that the F-ODMR spectra are observed even at T = 1.2 K demands that the singlet energy levels of the antenna pigments are almost degenerate, having a broad homogeneous linewidth of *ZOO cm-l. Shifting the wavelength of detection to the blue side of the near-infrared emission (895-905 nm) the excitation spectra show an increase of the 596 nm band of lo- 15% relative to the excitation band of the BP11(530-540 nm) of the reaction center. This indicates that there is a fraction of additional BChl fluorescence in this spectral region. The only BChl complexes except reaction centers, which are known to emit at these wavelengths, are the LH-I antenna complexes_ From this we conclude that in the region from S95 to 90s run the superposition of the fluorescence from “pure” reaction centers and from a contamination of antenna complexes aggregated with reaction centers is observed_ This is confirmed by the negative sign of the F-ODMR signals indicating the same singlet-singlet energy transfer as in whole cells. The emission bands at 780 and 830 nm in fig. 1 arc attributed to monomeric BChl and BPh molecules which are not involved in the reaction center protein complex. Concerning the excitation spectra, we assume that these pigments originate from denaturated reaction centers [23]. The F-ODMR transition frequencies detected on
these emission bands are the same as those of the 895 nm emission and therefore different from monomeric BChl and BPh molecules even when they are bound to protein [31]. So we attribute these signals to reaction center triplets which are observed via si&et energy transfer from the BChl and BPh molecules to *he reaction center complexes. Due to the high concentration of the sample we are not able to decide whether the singlet energy transfer is only an effect of re-absorption or a Fiirster-type transfer_ The different signs of the F-ODhIR signals observed at 780 and 830 nm are consistent with the model of a singlet-singlet energy transfer only when the experimentally determined alterations of the absorption changes influenced by microwave resonance transitions in the region of the spectral overlap between donor and acceptor is regarded_ This absorption difference spectrum as published by Hoff [ lo] shows a decrease in absorption at 780 nm and an increase at S30 nm. With this a positive F-ODMR signal at 780 nrn and a negative one at 830 nm are predicted which is in agreement with our experiments (fig. 2). Up to now the ODhlR transitions of the reaction center were attributed to one triplet state belonging to P,,, only. Our high-resolution EEDOR experiments (fig. 3) demonstrate that in isolated reaction centers as well as in reaction centers of whole cells always a superposition of two slightly different triplet states is observed. This leads to the conclusion that the ODOR sigiials of rencrion cmlers are iI1general 5 wperposiriorl ofrltcse two triplets. The variation in half-widths and lineshapes of ODNR lines observed by several authors [3-9,X] can be explained by different ratios of triplet I relative to triplet II. So all inconsistencies in the zfs parameters detected with F-ODMR in whole cells and F-ODXlR and A-DMR in isolated reaction centers as published by Hoff et al. [25] disappear. A comparison of the zfs parameters of triplet I and II shows that their D values are nearly identical (table 1) and that there is only a difference in E (3 1 and 35 X 10e4 cm- I)_ From this we assume that the IWOtriplets belong to two slightly different geonietrical configurations of Pa5 possibly generated by a change in the mutual orientation of the two molecules constituting the dimer. Preparative artefacts and an influence of the detergent as an origin of the two conBgurations can be es145
CHEMICAL
94, number 2
V0h1me
eluded. because the two triplets are also observed in whole cells. The fact that a preparation with reduced rluinone and one with the quinone removed show the SCUIW results eliminates an influence of quinone and dithionite. A possible esplanation would be the existence of
further triplet states as required by Schenk et al. [ I 5, lb] for the decay rates of the radical pair. These states ;1re espected to have very short lifetimes and therefore it is unreasonable that they are detected in our experiments. We think that at least at low temperature Ilvo geutrtcrrical cmjijp-ariota of PgGs are allowed. The fact that the intensity of triplet II is lower in systems consisting of reaction center proteins combined with antenna complexes (whole cells and the signals detected on the reaction center fluorescence at 895-905 nm) is a hint for the influence of the surrounding of the reaction
center
proteins.
“pure
isolated”
reaction
cen-
ter proteins exhibit both triplet states whereas the association with antenna proteins leads to a lower intensity of triplet
181A.H.
Hoff, Physics Rept. 54 (1979)
1983
75.
191 H.J. den Blanken. G.P. van der Zwet and A.H. Hoff, Chem. Phys. Letters 85 (1982)
335.
[lOI H.J. den Blanken and A.H. Hoff, Biochim. Biophys. IllI 1121
1131 1141 [ISI [I61 1171
It81 [I91 I201
Il.
Acknowledgement We are very Srateful IO Dr. J. Norris (Argonne Sarional Laboratory) and Professor H. Scheer (UniversitZt Xliinchen) for their generous gifts of reaction center preparations. A. Angerhofen took the excitation spectra which were of great value for the interpretations. This work was supported by the Deutsche l~orscltungsgenieinscliaft.
14 January
PHYSICS LETTERS
1251
Acta (1982), to be published. G.H. van Brakel, Dissertation, Landbouwhogeschool Wageningen (1982). T-J. Schaafsma, in: Triplet state ODMR spectroscopy, ed. R.H. Clarke (Wiley, New York, 1982) ch. 8, p_ 346. V.A. Shuvalov and W-IV_ Parson, Proc. Natl. Acad. Sci. US 78 (1981) 957. V.A. Shuvalov and W-W. Parson, Biochii. Biophys. Acta 639 (1981) 50. CC. Schenk. RX. Blankenship and W-W. Parson, Biochim. Biophys. Acta 680 (1982) 44. CC. Schenk, W-W. Parson, D. Holten and hi-W_ Windsor, Biochim. Biophys. Acta 635 (I 981) 383. R. Haberkom and WE. Michel-Beyerle, FEBS Letters 75 (1977) 5. A. Oprodnik, H-W_ Kruger, H. Orthuber, R. Haberkorn, ME. Michel-Bcyerle and H. Scheer. Biophys. J. (1982). to be published. AH. Hoff, P. Cast and J.C. Romihn. FEBS Letters 73 (1977) 18.5. M.K. Bowman. D.E. Budil, G.L. Gloss. A.C. Kosta. CA. Wraight and J.R. Norris, Proc. NatI. Acad. Sci. US 78 (1981) 3305. WM. Vredenberg and L.N.M_ Duysens, Nature 197 (1963) 355. W.G. van Dorp, T.J. Schaafsma. W. Soma and J.H. van der Waals. Chem. Phgs. Letters 21 (1973) 221. A. Angerhofer. Diplomarbeit, Universitit Stuttgart (1982). J. Beck, J.U. van Schiitr and H.C. Wolf, Photosynthese. Rundgespr%zh der Deurschcn Forschungsgemcinschaft Whirburg, November 1981; DPG-Friihjshrstagung 1982, Verhandl. DPG 4 (I 982) 394. H.J. den Blanken, G.P. van der Zwct and A.H. Hoff, Bio-
chim. Biophys. Acta (1982). to be published. 1351 TS. Kuan, D.S. Tinti and MA. El-Sayed. Chem. Phys. Letters 4 (1970)
507.
I271 J. Beck, G.H. Kaiser, J.U. von Schiitz and H.C. Wolf,
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