[28] Electron paramagnetic resonance studies of carotenoids

[28] Electron paramagnetic resonance studies of carotenoids

[28] EPR OF CAROTENOIDS 305 method and a Raman spectrometer equipped with a multichannel detector is absolutely necessary for excited-state Raman m...

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[28]

EPR OF CAROTENOIDS

305

method and a Raman spectrometer equipped with a multichannel detector is absolutely necessary for excited-state Raman measurements, 1° which are not covered in this chapter.) Spectral comparison of an isomer isolated from a pigment-protein complex, the configuration of which has been determined by IH N M R spectroscopy, with the original isomer bound to the pigment-protein complex ensures that no artificial isomerization has taken place during the isolation procedures and spectroscopic measurements. It leads to a straightforward and definitive configurational assignment. Some distortion of the polyene backbone, due to the binding of the carotenoid, can be detected in this step as a difference in relative Raman intensitiesJ ,H Further, Raman spectroscopy can predict at least the all-trans and some mono-cis configurations. Figure 1 shows the spectral patterns in the 13001000 cm -~ region for isomeric fl-carotene. A strong single peak around 1160 cm -~ is the key Raman line of an all-trans isomer. The relative intensity of the peak around 1140 cm -1 vs the one around 1160 cm -~ increases in the order 7-cis ( 9-cis ( 13-cis, and they are comparable for the 13-cis isomerJ 2 A medium peak around 1240 cm -1 is the key Raman line of a 15-cis isomerJ ,2,13 ~o H. Hashimoto and Y. Koyama, J. Phys. Chem. 92, 2101 (1988). H K. Iwata, H. Hayashi, and M. Tasumi, Biochim. Biophys. Acta 810, 269 (1985). 12 y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988). 13 y. Koyama, T. Takii, K. Saiki, and K. Tsukida, Photobiochem. Photobiophys. 5, 139 (1983).

[28] Electron

Paramagnetic Resonance Carotenoids

Studies of

B y H A R R Y A. F R A N K

Introduction It is well known that carotenoids act as protective devices against the irreversible photodestruction of the cells of bacteria, plants, and animalsJ It is generally accepted that the triplet states of carotenoids are involved in the photoprotective mechanism. 2 The triplet states of carotenoid molecules N. I. Krinsky, in "Photophysiology" (A. C. Giese, ed.), Vol. III, pp. 123-195. Academic Press, New York, 1968. 2 R. J. Cogdell and H. A. Frank, Biochim. Biophys. Acta 895, 63 (1987).

METHODS IN ENZYMOLOGY, VOL. 213

Copyright© 1992by Academic~ inc. All rightsof reproductionin any formreserved.

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SEPARATION AND QUANTITATION

[28]

are poorly understood, however, owing to the fact that the direct population of triplet states of isolated carotenoids via singlet-triplet intersystem crossing is not a very efficient process. The low yields of intersystem crossing make investigations of carotenoid triplets feasible only through the use of energy donors that sensitize the formation of the carotenoid triplet state. 3 The triplets are then detected using pulse radiolysis, 3 flash photolysis, 3,4 or electron paramagnetic resonance5 (EPR) spectroscopic techniques. Pulse radiolysis and flash photolysis optical spectroscopic experiments have been used to determine triplet-triplet absorption spectra of carotenoids, quantum yields of carotenoid triplet formation, and relative triplet state energies (e.g., by a judicious choice of donors of known triplet energies).3,4 EPR detection of carotenoid triplet states provides information about three other important factors. 1. The mechanism of carotenoid triplet state formation: This information is derived from the polarization pattern of the triplet state EPR signals. Because essentially all carotenoid triplets are formed via triplet energy transfer from a suitable triplet energy donor, the polarization pattern of the carotenoid triplet state signals reveals whether the donor triplet originated via the intersystem crossing mechanism or via the radical-pair mechanism of triplet energy formation. 5 2. The structure of the carotenoid: The zero-field splitting parameters, IOl and IEL provide a sensitive probe of the molecular structure of the carotenoid by assessing the extent of dipolar interaction between the two electrons that comprise the triplet state. 6 3. The geometry of the carotenoid molecule relative to other chromophores: Advantage can be taken of the inherent anisotropy of the dipolar interaction that exists in molecular triplet states to map the orientation of the carotenoid relative to other chromophores whose orientations have also been determined (e.g., bacteriochlorophyll).7,s This requires the use of an oriented sample (e.g., membranes dried onto Mylar strips or single crystals 7'9) or polarized light (i.e., magnetophotoselectionS). This chapter illustrates how each of these three factors can be addressed 3 R. Bensasson, E. J. Land, and B. Maudinas, Photochem. Photobiol. 23, 189 (1976). 4 p. Mathis and J. Kleo, Photochem. Photobiol. 18, 343 (1973). 5 H. A. Frank, J. D. Bolt, S. M. De B. Costa, and K. Sauer, J. Am. Chem. Soc. 102, 4893 (1980). 6 H. A. Frank, B. W. Chadwick, J. J. Oh, D. Gust, T. A. Moore, P. A. Liddell, A. L. Moore, L. R. Makings, and R. J. CogdeU, Biochim. Biophys. Acta 892, 253 (1987). 7 H. A. Frank, J. Machnicki, and P. Toppo, Photochem. Photobiol. 39, 429 (1984). a W. J. McGann and H. A. Frank, Biochim. Biophys. ActaS07, 101 (1985). 9 D. E. Budil, S. S. Taremi, P. Gast, J. R. Norris, and H. A. Frank, lsr. J. Chem. 28, 59 (1988).

[28]

EPR OF CAROTENOIDS

i

EPR

307

] Lock in I Amplifier

Chopper

~ \

Computer

i

Light 0

Reference

[email protected] FIG. 1. Experimental diagram of the double-modulation EPR spectrometer used for detecting carotenoid triplet states. The output of the EPR is routed to a lock-in amplifier referenced to the frequency of the light modulation. The output of the lock-in is fed to a computer, which serves as the recorder and also sweeps the magnetic field. The light source is a 1000-W xenon arc lamp filtered by - 5 cm of water in a Pyrex bottle. Typically the magnetic field modulation frequency is 100 kHz, and the light modulation frequency is 10-100 Hz.

and uses examples from the photosynthetic literature, which is now replete with carotenoid triplet state EPR spectroscopic observations. 2 Materials and M e t h o d s The optimal method for the EPR detection of carotenoid triplet states is schematically represented in Fig. 1. The unfiltered DC output of the EPR magnetic field modulation amplifier is fed directly to an external lock-in amplifier that is referenced to the frequency of a modulated excitation light source. This experimental arrangement offers a substantial improvement in the sensitivity of triplet state detection over the traditional "light-minusdark" spectral approach to observing light-induced signals and is necessary for the detection of carotenoid triplets, probably because of two factors: (1) The steady state triplet population of carotenoids is low owing to their typically very rapid ( - 10 psec) decay back to the ground state; ~° and (2) carotenoid triplets may exhibit substantial spin-lattice relaxation between triplet spin sublevels, which may diminish the EPR signal amplitudes, t~ Thus, a "double-modulation" (field and light modulation) technique, although not obligatory, facilitates detection of carotenoid triplet states. to S. S. Taremi, C. A. Violette, and H. A. Frank, Biochim. Biophys. Acta 973, 86 (1989). " W. J. McGann and H. A. Frank, Chem. Phys. Lett. 121, 253 (1985).

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Results and Discussion The first EPR observation of a carotenoid triplet state was made on the B800-850 light-harvesting complex purified from the photosynthetic bacterium, Rhodobacter (previously Rhodopseudomonas) sphaeroides, wildtype strain 2.4. I. 5 Subsequently, carotenoid triplet state signals were detected in whole cells, chromatophores (photoactive membrane vesicles), several different light-harvesting and reaction center pigment-protein complexes prepared from a wide variety of photosynthetic organisms, and in synthetic, covalently linked carotenoporphyrin molecules.6,~2The initial unambiguous assignment of the triplet state EPR signals to carotenoids was made in the photosynthetic bacterial reaction center and deduced from the following observations: ( 1) The triplet state EPR signals displayed the so-called "radical-pair" triplet state spin polarization pattern (eaa eea, where e denotes a signal in emission and a denotes a signal in absorption). This pattern (or its inverse, aee aae) is observed only when triplets are formed via a charge separation-recombination (i.e., radical-pair) mechanism or when they coherently quench triplets formed via this mechanism. 5 The reaction center-bound carotenoid had been shown from previous optical triplet-triplet absorption experiments to be quenching the primary donor triplet) 3 It is well known that the primary donor triplet state is born via the radical pair mechanism; ~4 (2) no triplet state signals displaying zero-field splitting parameters similar to those observed in the R. sphaeroides wild-type strain 2.4. l preparations were observed in samples from the carotenoidless mutant R. sphaeroides R-26; 5 (3) the temperature dependence of the triplet state EPR signals from the carotenoid-containing R. sphaeroides wild-type strain 2.4.1 was precisely the same as that observed for the optically detected, triplet-triplet absorption signals belonging to the reaction center-bound carotenoid and reported by Cogdell et al. ~3 It was observed that the carotenoid triplet state signal amplitudes increased relative to the primary donor triplet signals as the temperature was raised from l0 to 100 K; ~3 (4) the final piece of evidence that confirmed that the observed triplet state EPR signals were arising from carotenoids came from experiments carried out on reaction centers from the carotenoidless mutant R. sphaeroides R-26 that had been reconstituted with the carotenoid, spheroidene. 15 This sample displayed an EPR spectrum (see Fig. 2) identical to that of R. sphaeroides wild-type strain 2.4. l, which naturally con-

12 H. A. Frank, J. Machnicki, and M. Felber, Photochem. PhotobioL 35, 713 (1982). 13 R. J. Cogdell, T. G. Monger, and W. W. Parson, Biochim. Biophys. Acta 408, 189 (1975). 14 A. J. Hoff, Phys. Rep. 54, 75 (1979). 15 B. W. Chadwick and H. A. Frank, Biochim. Biophys. Acta 851,257 (1986).

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>, ,u c (b +_,

2700

2900

5100

3300

3500

3700

Magnetic Field Strength (Gauss)

FIG. 2. Triplet state EPR signals from the carotenoid, spheroidene, reconstituted into reaction centers isolated from R. sphaeroides R-26. Note this spectrum displays the radicalpair polarization pattern, eaa eea. The experimental conditions were as follows: Magnetic field modulation frequency, 100 kHz; modulation amplitude, 25 G; microwave frequency, 9.054 GHz, microwave power, 20 mW; field modulation lock-in amplifier sensitivity, 2.5 mV; light modulation lock-in amplifier sensitivity, 10 mV; time constant, 3 sec light modulation frequency, 33 Hz; temperature, 90 K. tains spheroidene, thus confirming that the EPR signal was due to the bound carotenoid. M e c h a n i s m o f Carotenoid Triplet State Formation

With few exceptions 2 all carotenoid triplet states are formed via energy transfer from a triplet energy donor. The reaction is 3Donor* + ~Car-~ ~Donor+ 3Car* where ~Car and 1Donor are the ground state singlets, and 3Car* and 3Donor* are the excited triplet states o f the carotenoid and the energy donor, respectively. Because, the reaction involves a change in the spin multiplicity o f each molecular state, the exchange (or Dexter) mechanismS6 is usually invoked to account for the process. Moreover, the polarization pattern observed for the carotenoid triplet state signals reveals whether the d o n o r triplet was born via the intersystem crossing mechanism or via the radical-pair mechanism of triplet energy formation. Figure 2 shows the triplet state signals from spheroidene in the reaction center of R. sphaeroides wild-type strain 2.4.1. Note the polarization pattern is eaa eea, ,6 D. L. Dexter, J. Chem. Phys. 21,836 (1953).

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SEPARATIONAND QUANTITATION

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indicating that it was transferred from the primary donor, whose triplet was formed via the radical-pair mechanism. Figure 3 shows the triplet state spectrum of the same molecule, spheroidene, in the B800-850 lightharvesting complex from R. sphaeroides wild-type strain 2.4.1. Note the polarization pattern in this triplet is eae aea, indicating that it was transferred from a triplet energy donor, presumably bacteriochlorophyll, whose triplet was formed by intersystem crossing. The polarization patterns offer an unambiguous assignment of the mechanism of formation of the triplet state that ultimately resides on the carotenoid. In whole cells of the photosynthetic bacteria, this provides a convenient method for deciding whether the observed carotenoid triplet signal originates in the reaction center or in the light-harvesting pigment-protein complexes. Structure of Carotenoid

The zero-field splitting parameters of carotenoid triplet states correlate with the extent of It electron conjugation in their structures. For example, Table I gives the ]DI and lED values for the B800-850 light-harvesting complexes obtained from a series of photosynthetic bacterial preparations. ~ The data can be understood in terms of variations in the extent of dipole-

>,, d) C

@

+_, mc

i

2700

2900 Magnetic

i

3100

i

3300

Field Strength

i

3500

3700

(Gauss)

FIG. 3. Triplet state EPR signals from the carotenoid, spheroidene, in the B800-850 light-harvesting complex from R. sphaeroides wild-type strain 2.4.1. Note this spectrum displays an intersystem crossing mechanism polarization pattern, eae aea. The experimental conditions were as follows: Magnetic field modulation frequency, 100 kHz; modulation amplitude, 29 G; microwave frequency, 9.050 GHz; microwave power, 16 mW; field modulation lock-in amplifier sensitivity, 2.5 rnV; light modulation lock-in amplifier sensitivity, 2.5 mV; time constant, 30 sec; light modulation frequency, 100 Hz; temperature, 100 K.

[28]

EPR OF CAROTENOIDS

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TABLE I ZERO-FIELD SPLITTING PARAMETERS OF B800-850 COMPLEXES FROM DIFFERENT PREPARATIONS OF PHOTOSYNTHETIC BACTERIAa Number of conjugated double bonds

IDI (cm -2)

IEI (cm -2)

Triplet assignment

0.0365 + 0.0002 0.0324 _+ 0.0002

0.0035 + 0.0002 0.0036 + 0.0002

Neurosporene Spheroidene

9 10

0.0318 _ 0.0002

0.0032 + 0.0002

Spheroidenone

11

Rhodopseudomonas acidophila 0.0279 _ 0.0003

0.0029 __. 0.0003

Rhodopin

13

Sample

Rhodobacter sphaeroides GA Rhodobacter sphaeroides wild type (anaerobically grown)

Rhodobacter sphaeroides wild type (aerobically grown) 7750 a From Ref. 6.

dipole interaction arising from different amounts of n electron conjugation in the various carotenoids. The least conjugated carotenoid, neurosporene (nine conjugated double bonds), was found to have the largest ID[ value (largest dipole interaction). The [DI values decrease in order of increasing n electron conjugation, with rhodopin (13 conjugated double bonds) having the smallest value. This demonstrates the utility of the zero-field splitting parameters in deducing the structure of the EPR-detected carotenoid.

Geometry of Carotenoid Molecule Relative to Other Chromophores Owing to the inherent anisotropy of the dipolar interaction that exists in the triplet state, EPR spectra of carotenoids can be used to determine the orientations of carotenoids relative to other chromophores (e.g., bacteriochlorophylls). These experiments require the use of an oriented sample or polarized light. In the former case, it was shown that large orientation effects on the EPR spectra of spheroidene were observed from chromatophores of R. sphaeroides wild-type strain 2.4.1 dried onto Mylar strips. 7 The data yielded the projections of the carotenoid triplet state magnetic axes with respect to the normal to the membrane plane. 7 In the latter case, magnetophotoselection was used to determine the orientations of the primary donor and carotenoid magnetic axes with respect to each other) These data are particularly important because the triplet state EPR spectrum of crystalline r-carotene has been reported, and the assignment of the triplet state magnetic axes with respect to the molecular symmetry of the

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SEPARATION AND QUANTITATION

carotenoid has been made. 17 Thus, it is now possible to deduce carotenoid molecular geometries from the orientation effects observed in their triplet state EPR spectra. Conclusions EPR detection of carotenoid triplet states continues to be an important spectroscopic tool for the elucidation of the mechanism of triplet state formation, the determination of carotenoid structures in vivo, and the specification of carotenoid molecular geometries. All of these factors are central to the issue of how carotenoids protect biological systems from photodestruction. 2 This brief chapter has not dealt with such questions as concern (1) the specific molecular features of carotenoids contributing to triplet energy transfer; (2) how the zero-field splitting parameters of carotenoids are modulated by the various stereochemical isomeric forms they can adopt; and (3) how different environments (i.e., different biological systems such as the photosynthetic pigment-protein complexes) affect the spectroscopic and functional properties of carotenoids. These questions remain for future investigations. Acknowledgments I thank Dr. John Bolt for sharing in the initial discovery of the triplet state EPR spectra of carotenoids, which was made in the laboratory of Professor Kenneth Sauer at the University of California, Berkeley. This work is currently supported by grants from the National Institutes of Health (GM-30353), the Competitive Research Grants Office of the U.S. Department of Agriculture (88-37130-3938), the NATO Scientific Affairs Division (880107), and the University of Connecticut Research Foundation. 17 j. Frick, J. U. Von Schiitz, H. C. Wolf, and G. Kothe, Mol. Cryst. Liq. Cryst. 183, 269 (1990).

[29] Identification of Carotenoid

Pigments

in Birds

By JOCELYN HUOON and ALAN H. BRUSH

One extraordinary feature of avian carotenoids is their packaging. Feathers are unique to birds and form the interface between a remarkable metabolic machine and an unforgiving environment. The colors and patterns of their display are exceedingly important in individual and species recognition and in myriad aspects of communication. Historically, there has been an interest in both the molecules themselves and the nature of METHODS IN ENZYMOLOGY, VOL. 213

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