Photothemsty and Photobiology Vol. 40. No. 2, pp. 277 - 281, 1984 Printed in Great Britain. Ail rights rcscrved

0031-8655184 $03.@@+@ W Copyright 0 1984 Pergamon Press Ltd

RESEARCH NOTE

SPECTRAL EVIDENCE FOR PHOTO-INDUCED TSOMERIZATION OF CAROTENOIDS IN BACTERIAL

PHOTOREACTION CENTER

FRAN~OIS BOUCHER and GABRIEL GINGRAS~

Quebec, Canada and tDCpartement de Biochimie, UniversitC de Montreal, Montreal, Quebec, Canada

(Received 22 July 1983; accepted 14 March 1984)

*Centre de Recherche en Photobiophysique, Universitt du QuCbec Trois-Rivikres, Trois-Rivieres,

Abstract-The aim of this work was to determine whether spirilloxanthin and sphaeroidene bound to the same site of the photoreaction center isolated from Rhodospirillurn rubrum can display cis-trans isomerization under conditions that lead to formation of triplet state PR of the primary electron donor. To this end, we monitored changes in the absorption spectrum of these bound carotenoids as induced by red light at low redox potentials. This experiment was performed both with the intact photoreaction center isolated from strain S1 and with the photoreaction center isolated from carotenoidless strain G9 reconstituted with either spirilloxanthin or sphaeroidene. In both preparations, spirilloxanthin exhibited light-induced absorption changes that can be interpreted as a cis-trans isomerization. Under our cxperimental conditions, the absorption changes attained their full extent in about 1 min and were not or wcre only partially reversed when the light was switched off. Under our experimental conditions, the extent ot these changes indicate that about 15% of the bound spirilloxanthin undergoes isomerization. Sphaeroidene artificially attached to the G9 photoreaction center also undergoes light-induced absorbance changes, but these cannot easily be interpreted as a cis-trans isomerization.

INTRODUCTION

All wild type bacterial photoreaction centers appear to contain a single carotenoid molecule as an intrinsic constituent (van der Rest and Gingras, 1974; Cogdell et al., 1976). This carotenoid plays the double role of an tenna and protector against photodynamic damage. The bacteriochlorophyll of the photo- reaction centers lacking carotenoids is rapidly destroyed under bright illumination in the presence of oxygen. This photodynamic damage is caused by singlet oxygen (Boucher et a l . , 1977) presumably produced by triplet-triplet energy transfer from state PK of the primary electron donor. Reconstitution studies (Boucher et al., 1977) showed that the photoreaction centre from Rhodospirillum (Rs.)$ rubrum G9, a carotenoidless strain, contains a site to which a single carotenoid molecule can be tightly b o u n d . A m o n g t h e c a r o t e n o i d s tes ted f o r reconstitution, spirilloxanthin and sphaeroidene conferred photoprotection to the same extent as does spirilloxanthin in the wild type photoreaction center.

These reconstitution studies have allowed precise measurements of the in situ spectral properties of the bound carotenoids. Bound spirilloxanthin displays a spectral flattening of its visible absorption bands ('B t 'A transition) accompanied by a large increase of the cis bands ('C t 'A transition) while bound sphaeroidene shows similar features but to a lesser

tTo whom reprint requests should be addressed. $Abbrev ia t ions: C D , circular dichroism; Rp,

Rhodopseudornonas; Rs, Rhodospirillurn.

extent. Both carotenoids become optically active upon binding. O n the basis of these spectra, we have proposed that spirilloxanthin is bound to the photo- reaction centre as a hindered central cis isomer. The absorption and C D bands in the ultraviolet were explained by a strong exciton coupling between two identical segments of the molecule (Boucher et a/. , 1977). On the basis of resonance Raman spectro- scopy data, Lutz et al. (1978) and Agalidis et al. (1980) have questioned the validity of the central cis model for sphaeroidene and sphaeroidenone in the photoreaction center from Rp. sphaeroides 2.4.1 and G a . They have proposed instead that these carotenoids are in a di-cis configuration. More recently, Koyama et al. (1982) systematically compared the resonance Raman spectra of Rp. sphaeroides G1C with those of various cis-trans isomers of beta-carotene in solution. They concluded that the carotenoid in the photoreaction center takes the 15-cis configuration but that it probably is not twisted in a protohelix as proposed by Boucher et al. (1977).

Photoprotection of bacteriochlorophyll probably results from a quenching of triplet state PR inducing a T t S transition in the carotenoid (Cogdell et a l . , 1975; Parson and Monger, 1977; Frank et al . , 1980). Once in the triplet state, certain carotenoids are known to readily isomerize (Claes and Nakayama, 1959; Claes, 1961; Foote et al . , 1970b; Jensen et al., 1982). One might, therefore, expect the carotenoids of the photoreaction center to isomerize under conditions where state PR is formed. Recently, Lutz et al. (1982) have measured time resolved resonance

217

278 FRANCOIS BOUCHER and GABRIEL GINGRAS

Raman spectra of triplet cis-sphaeroidene and methoxyneurosporene bound to the photoreaction center of Rp. sphaeroides, they concluded that isomerization may occur in bound carotenoids in the triplet state but that it does not lead to an all-trans isomer. The aim of the present work was to determine whether bound triplet cis-spirilloxanthin and sphaeroidene could indeed decay into all-trans isomers in the Rs. rubrum photoreaction center. To detect such isomers, we relied on absorption spectroscopy of wild type and of carotenoidless and reconstituted photoreaction center from Rs. rubrum. Our results suggest that, in these preparations, isomerization of spirilloxanthin yields an all-trans isomer as the final product.

MATERIALS AND METHODS

Most experimental procedures have been previously described. Photoreaction centers from Rs. rubrurn strains S1 and G9 were prepared according to Vadeboncoeur et al. (1979). Reconstitution of the carotenoidless photoreaction center with spirilloxanthin and sphaeroidene was performed according to Boucher et al. (1977).

Spectra were measured with a Cary 14R spectro- photometer equipped with a side illumination attachment consisting of a 650 W halogen-tungsten lamp (Sylvania DVY) whose light was filtered through an 870 nm interference filter (Baird Atomic, 10 nm half-bandwidth) (or a cut-off filter (Schotf RG9 A> 690 nm) and finally focused on the cell of the sample compartment. The corresponding light intensities at the cell surface were 2.0 X lo5 and 1.5 X lo7 ergs cm-’s-’ for the interference and cut-off filters, respectively. Both were sufficient to completely oxidize P 870 in the absence of secondary electron donor. State PR, and consequently the T t S transition in the carotenoid (Cogdell etal . , 1975; Parson and Monger, 1977), was induced by illuminating the preparation with these light intensities in the presence of sodium dithionite, added as a solid, to a final Concentration of 1 mg/me.

For in vitro photo-isomerization experiments, spiril- loxanthin was extracted fro Rs. rubrurn S1 and purified according to van der Rest and Gingras (1975). Purified spirilloxanthin was solubilized in n-hexane and racemized by iodine catalysis following the procedure described by Polgar et al. (1944) and photo-isomerized by irradiation in the presence of methylene blue (Foote et al., 1970b). The experimental set-up was essentially the same as just described, except that the actinic light was provided through a cut-off interference filter (Optics Technology, A > 650 nm).

RESULTS

Primary photochemistry in the isolated photo- reaction center results in a separation of charges between the primary electron donor, bacterio- chlorophyll, and the primary ubiquinone acceptor. Prolonged illumination in the absence of a secondary electron donor results in the formation of state P+ Q- the steady state concentration of which depends on light intensity and on the rate of charge recombination. In the photoreaction center from Rp. sphaeroides 2.4.1., the carotenoid absorption spectrum is known to undergo small changes associated with the oxidation of the primary electron

donor (Cogdell et al., 1977). We were able to confirm this observation in the photoreaction center from Rs. rubrum S1 containing spirilloxanthin as the carotenoid. The light minus dark difference spectrum between 450 and 650 nm (Fig. 1.-) shows the expected bleaching at 600 nm due to the oxidation of the primary donor. This spectrum also reveals a hyperchromism and a bathochromic shift (from 505 and 525 nm to 507 and 545 nm) in the absorption spectrum of spirilloxanthin. These changes are made more evident by comparison with the absorption spectrum of spirilloxanthin bound to the photoreaction center (Fig. 1 ,----). Identical changes in the spectrum of spirilloxanthin are obtained by oxidation with ferricyanide (not shown) indicating that they are essentially related to the oxidation of the primary donor. In addition to a red shift of the carotenoid bands upon oxidation of the primary donor, Cogdell et al. (1977) also observed a blue shift upon placement of a negative charge on the primary acceptor by reduction with dithionite in the dark. We were unable to reproduce this result with spirilloxanthin in the Rs. rubrum photoreaction center.

Besides this small electrochromic shift induced by oxidation of the primary electron donor, consider- ably larger spectral changes of spirilloxanthin can be observed under conditions which favor formation of state PR. Figure 2 (-) shows the light minus dark difference spectrum recorded with Rs. rubrum photoreaction center illuminated in the presence of sodium dithionite. Identical results are obtained with photoreaction centers from strain S1 or from strain G9 reconstituted with spirilloxanthin. This spectrum shows negative bands at 370 and 395 nm and positive bands at 480,510 and 548 nm. Comparison with the in situ absorption spectrum of spirilloxanthin (----)

I I I I I

n

I I I 1- 400 SO0 600

WAVELENQTH , nm

Figure 1. Light minus dark difference absorption spectrum in the photoreaction center from Rs. rubrurn S1 (-). Also shown, the in situ absorption spectrum of spirilloxanthin (----) taken as the difference between reconstituted and native Rs. rubrum G9 photoreaction center. Photoreaction center (2.8 pM) wasin 10mMTris-HCI (pH8.0)containing

0.1% (wtivol) Triton X-100.

Isomerization of carotenoids in photoreaction center 219

W 0

a i: I I I I I I

400 500 600 WAVELENGTH, nm

Figure 2 . Light minus dark difference spectrum in Rs. rubrum G9 photoreaction center reconstituted with spiril- loxanthin and kept at a low redox potential (-). Photo- reaction center (1.5 pM) was in 10 mM Tris-HCI (pH 8.0) containing 0.1% (wthol) Triton X-100 and 1 m g h t sodium dithionite. Also shown (cf. Fig. l), the in situ

absorption spectrum of spirilloxanthin (----).

shows that this effect is accompanied by a red shift of the 510 and 548 nm bands.

Since this difference spectrum is typical of cis-trans isomerization, we show, for comparison, a difference spectrum obtained with spirilloxanthin in n-hexane (Fig. 3). In this experiment, chromatographically pure spirilloxanthin was submitted to iodine catalysis which yields a racemic mixture of different cis isomers in addition to the all-trans isomer (Polgar et al . , 1944). The absorption spectrum of this racemic mixture is shown in Fig. 3 (----). When irradiated with red light in the presence of methylene blue, a significant amount of cis spirilloxanthin undergoes isomerization. The light minus dark difference

W 0 z

LI1 U

-0,02 2 0.3-

-0.01 : 0,oo f

P -0.01 2

z 4

W 0 - 0

a

400 500 600 WAVELENGTH , nm

Figure 3. Light minus dark (trans minus cis) difference absorption spectrum oE spirilloxanthin in n-hexane (-). This spectrum was recorded after 30 s. illumination (84 > 650 nm) of the racemic mixture of spirilloxanthin in the presence of methylene lue (Ah5" = 0.8). Also shown, the absorption spectrum of this racemic mixture of spiril-

loxanthin in n-hexane (----).

spec t rum (Fig. 3,-) shows the expected hypochromism of the cis bands with a hyper- chromism at 485,497 and 500 nm. Comparison with the absolute spectrum also shows a red shift of the latter two bands.

We next attempted to verify whether similar effects could be observed with the Rs. rubrum G9 p h o t o r e a c t i o n c e n t e r r econs t i t u t ed with sphaeroidene. Under the same conditions used for the preceding experiment, the photoreaction center reconsituted with sphaeroidene behaved very differently from that reconstituted with spiril- loxanthin. The light minus dark difference spectrum of Fig. 4 (-) shows hyperchromism at 430, 456 and 489 nm. Comparison with the in situ absorption spectrum of sphaeroidene on this preparation (----) shows that this effect is accompanied by a blue shift of these bands. Remarkably, this difference spectrum has no negative components under the cis bands. A similar result was reported by Cogdell et al. (1977) for the same carotenoid in the photoreaction center of Rp. sphaeroides 2.4.1.

Since according to the preceding results, only spirilloxanthin seems to undergo the expected cis-trans isomerization, we next sought to measure its extent and rise time in the photoreaction center from Rs. rubrum S1. The time course of the absorption changes at different wavelengths was recorded after illumination in the presence of sodium dithionite. When plotted against wavelength with their proper sign and amplitude, these results reproduced the difference spectrum of Fig. 2 (-). Figure 5 shows that the absorbance changes recorded at 380 nm reached a plateau within the first minute of illumination and decayed to another plateau upon switching off the actinic illumination. This dark

w 0

K 0,02 z

e k a

m a m I 600 a

W 0

0,Ol 4

%

WAVELENGTH, nm

Figure 4. Light minus dark difference spectrum of Rs. rubrum G9 photoreaction center reconstituted with sphaeroidene and kept at low redox potential (-). Photoreaction center (4.2 KM) was in Tris-HCl (pH 8.0) containing 0.1% (wthol) Triton X-100 and 1 mgime of sodium dithionite. Also shown, the in situ absorption spectrum of sphaeroidene taken as the difference between reconstituted and native Rs. rubrum G9 photoreaction (-).

280 FRANCOW BOUCHER and GABRIEL GINGRAS

t 1

I T

10,Ol A 1

I I I I

0 2 4 6

Time , min

Figure 5. Time course of light-induced spectral changes in Rs. rubrum S1 photoreaction center at low redox potential. Photoreaction center (2.1 p M ) was in 20 mMTris-HC1 (pH 8.0) containing 0.2% (wtlvol) Triton X-100 and 1 mg/mt of sodium dithionite. Absorbance decrease observed at 380 nrn elicited by two successive illuminations through a 690 nrn high pass cut-off filter. Light intensity was 1.5 X lo7 ergs ern-' s- ' . The onset and extinction of the actinic light are indicated respectively by upward and downward pointing arrows. An upward deflection of the signal

corresponds to a decrease in absorbance.

reversal was not complete. Subsequent illumination induced much smaller changes. The amplitude of the signal was found to depend on light intensity; a 100-fold decrease in actinic light intensity produced a signal about six times smaller (not shown). On the basis of the absorbance decrease of the cis peaks at 370 nm and at 395 nm, we calculated that approximately 15% of the spirilloxanthin was isomerized within the first 5 s of the most intense illumination at our disposal. Higher light intensities presumably would yield higher values.

DISCUSSION

In this work, we were able to show that in the Rs. rubrum photoreaction center, spirilloxanthin under- goes electrochromic spectroscopic changes associated with the placement of a positive charge on the primary electron donor. This confirms similar observations previously made with sphaeroidene bound to the Rp. sphaeroides 2.4.1 photoreaction center (Cogdell et al., 1977).

Our primary aim, however, was to determine whether the bound carotenoid can undergo an isomerization reaction associated with the formation of state PR of the primary electron donor. Carotenoids in solution have been shown to readily undergo cis-trans isomerization upon quenching singlet oxygen or an excited triplet state (Claes and Nakayama, 1959; Foote et al., 1970a,b; Jensen et al . , 1982). The light-induced spectral changes observed here with spirilloxanthin in solution and bound to the Rs. rubrum photoreaction center are typical of

cis-trans isomerization. Figures 2 and 3 show hypochromicity of the 'C + 'A transition accompanied by hyperchromicity and a batho- chromic shift of the 'B t 'A transition. Although these spectra are similar, they are not expected to be identical, since the isomeric compositions of bound and free spirilloxanthin are different. Although the photosensitized isomerization procedure is known to yield mainly trans isomers (Foote et al., 1970b), free spirilloxanthin in hexane is a racemic mixture of unknown isomeric composition both before and after the reaction, with the result that the difference spectrum of Fig. 3 corresponds to the conversion of different cis configurations of trans spirilloxanthin. In contrast, spirilloxanthin bound to the photo- reaction center is in only one specific form, probably the 1 5 4 s (monocis central) configuration (Boucher et al . , 1977; Koyama et al., 1982). According to the electronic spectra measured for different isomers of beta-carotene by Jensen etal. (1982) and Koyama et al. (1982), the reaction amplitude of the cis peaks is higher for the 154s isomer. Therefore, the cis peaks should show a different extent of bleaching according to whether the cis-trans isomerization is from a 15-cis carotenoid or from a racemic mixture. This is essentially what we observe in the difference spectra of Figs. 2 and 3; the relative intensities of the negative 'C c 'A to the positive 'B +- 'A band is 1:2 for spirilloxanthin in solution and 3:2 for spirilloxanthin bound to the photoreaction center,

The isomerization product of the bound 15-cis isomer is probably all trans spirilloxanthin, since another isomer should be blue-shifted (Koyama er al., 1982) rather than red-shifted as observed here. Hence, we feel that the most likely interpretation of the light-induced spectra of Figs. 2 and 5 is that bound spirilloxanthin undergoes a cis-trans isomerization around its central 15-15' double bond.

Unlike spirilloxanthin, sphaeroidene bound to the photoreaction center of Rs. rubrum shows no clear sign of cis-trans isomerization under our experimental conditions. In particular, there was no hypochromicity of the 'C t 'A transition with a shift to the blue rather than to the red (Fig. 4). In light of the electronic spectra of defined cis-trans isomers of beta-carotene (Jensen et al., 1982; Koyama et af., 1982), this cannot be interpreted as a cis-trans isomerization. Moreover, when expressed on a molar basis, the hyperchromicity was about four-fold less intense in bound sphaeroidene than in bound spirilloxanthin. This difference spectrum is akin to that obtained by Cogdell et al. (1977) upon place- ment of a negative charge on the intermediary acceptor and may perhaps be interpreted in the same manner. An explanation for the very different behavior of spirilloxanthin and of sphaeroidene bound to the Rs. rubrum photoreaction center must be sought in their molecular structures and in their interaction with their binding site. In spirilloxanthin but not in sphaeroidene, a plane of symmetry passes

Isomerization of carotenoids in photoreaction center 281

through the central 15-15’ bond of the molecule; sphaeroidene lacks a methoxyl group on carbon 1’. While these considerations seem to explain their different CD spectra in their photoreaction center binding site, they provide by themselves no simple interpretation for the apparent lack of cis-trans isomerization of bound sphaeroidene. It may be hypothesized that the spirilloxanthin binding site imposes the selection of a cis isomer of sphaeroidene, thus preventing the formation of detectable amounts of the trans isomer.

A question of fundamental importance is what is the physiological significance of the alleged isomerization of the photoreaction center caro- tenoid? More limited but more easily assessed subquestions are (1) whether it is the spirilloxanthin bound to its natural site that undergoes the isomerization reaction, (2) what is the extent of the reaction and (3) whether it is reversible? To all these questions, we can offer as yet only partial answers. That it is spirilloxanthin bound to its native site is indicated by the fact that the very same difference spectra were obtained with the fresh photoreaction center from strain S1 as with preparations from strain G9 reconstituted with isolated spirilloxanthin; more- over, as we found earlier (Boucher et al . , 1977), spirilloxanthin offered to both preparations the same photoprotection against photodynamic damage. From the amplitude of the light-induced hypo- chromicity of the cis peak (Fig. 5 ) , we estimate that at least 15% of the bound spirilloxanthin can be isomerized under our experimental conditions. Foote et al. (1970b) have observed that photo- sensitized isomerization of dissolved carotenoids can yield more than 50% of the all-trans isomer. This is in line with the quantum yield of triplet sensitized isomerization of beta-carotene in the cis-trans direction being higher by at least an order of magnitude than that in the trans-cis direction (Jensen et al . , 1982). The maximal yield of trans isomer that we obtained with spirilloxanthin bound to the photoreaction center is clearly smaller than this. While better experimental conditions probably would lead to larger spectral changes, bound spiril- loxanthin may never show the same extent of isomerization as free spirilloxanthin. This prediction is based not only on the apparent partial reversibility of the phenomenon in the dark (Fig. 5) but also on

the observation that the carotenoid binding site probably does not accommodate a trans isomer. The latter circumstance is expected to lead to selection of the cis isomer during the lifetime of triplet state spirilloxanthin, thus decreasing the yield of the trans isomer. Although we have no evidence for this, it is conceivable that the trans isomer is then forced out of the carotenoid binding site. What effect this would have on the photoprotection capacity of the caro- tenoid remains to be verified.

Despite the problems left open by this Note, the behavior of spirilloxanthin is intriguing since it appears to undergo trans-cis isomerization upon binding to the photoreaction center and ci.y-truns isomerization when playing its role of photo- protector.

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