Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

Absorbance changes of carotenoids and bacteriochlorophylls responding to the change of local electrical field induced by hydrophobic anion,tetraphenylborate in membranes of photosynthetic bacteria

Large blue-shifts of carotenoid absorption bands were induced by dark addition of a hydrophobic anion, tetraphenylborate, in chromatophores and cell membranes of photosynthetic bacteria, Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata. Tetraphenylborate also induced a red-shift of the 850 nm absorption band and a blue-shift and broadening of the 800 nm band of bacteriochlorophyll. From the analysis of the relation between the magnitude and isosbestic wavelength of the absorbance changes the tetraphenylborate-induced carotenoid band shift were assumed to reflect the change of local electrical field close to each carotenoid molecule which exists as a minor pool on the light-harvesting pigment-protein complex II (LHC II). Absorbance changes of carotenoid and chlorophylls were also induced by tetraphenylborate in membranes of spinach chloroplasts.     

Fluorescence emission spectra of cells and subcellular preparations of a green photosynthetic bacterium. Effects of dithionite on the intensity of the emission bands

Fluorescence emission spectra were measured of intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii in the presence and in the absence of dithionite. A 3–5-fold increase in bacteriochlorophyll a fluorescence at 816 nm occurred upon addition of dithionite in a membrane vesicle preparation (Complex I), in a photochemically active pigment-protein complex and in a bacteriochlorophyll a protein complex free from reaction centers. The pigment-protein complex showed a relatively strong long-wave emission band (835 nm) of bacteriochlorophyll a, which was preferentially excited by light absorbed at 670 nm and was not stimulated by dithionite. With Complex I, which contains some bacteriochlorophyll c in addition to bacteriochlorophyll a, a 3–4-fold stimulation of bacteriochlorophyll c emission was also observed. Emission bands at shorter wavelengths, probably due to artefacts, were quenched by dithionite. With intact cells, the effect of dithionite was smaller, and consisted mainly of an increase of bacteriochlorophyll a emission.

The results indicate that the strong increase in the yield of bacteriochlorophyll emission that occurred upon generating reducing conditions is, at least mainly, due to a direct effect on the light-harvesting systems, and does not involve the reaction center as had been earlier postulated.     

Fluorescence emission by wild-type- and mutant-strains of Rhodopseudomonas capsulata

Absorption and fluorescence emission spectra of Rhodopseudomonas capsulata, strains 37b4 (wild type), A1a⁺ (blue-green mutant strain), Y5 (phototroph negative, having only B-800–850 bacteriochlorophyll-carotenoid-protein complex) at 4 K, 77 K and 300 K were measured. The fluorescence emission at 890 nm of the B-870 bacteriochlorophyll band dominates the emission of other spectral forms of the strains 37b4 and A1a⁺, while in strain Y5 a fluorescence emission band at 865 nm of the B-850 bacteriochlorophyll dominates. Very little fluorescence was observed at 805 nm. A linear relation between relative fluorescence intensity and the exciting light intensity was observed. The integrated fluorescence yield increased as the temperature was lowered from 300 K to 4 K. The results are discussed in the light of the arrangement of pigment molecules in the membrane and the process of energy migration within the photosynthetic apparatus.     

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360–410 mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores.

The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C₃₄₀) which has an oxidation-reduction midpoint potential close to 340 mV (n = 1) at pH 6.0–7.6. The E_m of this component varies by 60 mV/pH unit between pH 7.6 and 9.2.

The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C₃₄₀.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Orientation of the bacteriochlorophyll triplet and the primary ubiquinone acceptor of Rhodospirillum rubrum in membrane multilayers determined by ESR spectroscopy (I)

Chromatophores from Rhodospirillum rubrum were oriented as multilayers on quartz slides under reducing conditions. Irradiation of these multilayers in the resonance cavity of an ESR spectrometer at 6 K yielded the spectrum of the bacteriochlorophyll dimer triplet. The relative intensities of the main six lines of the triplet were dependent on the angle subtended by the direction of the external magnetic field with plane of the multilayers. The angular dependence of the intensities of these transitions can best be interpreted in terms of one of the principal axes of the triplet lying along the plane of the membrane while the other two axes are tilted 10–20° away from the parallel to and normal to the membrane directions. If we assume the porphyrin planes of the dimer to be parallel and the largest splitting of the triplet transitions to correspond to those transitions in a direction normal to this plane, then these data imply that the dimer planes are nearly perpendicular to the membrane plane.Purified iron-depleted phototrap complexes were similarly oriented in reconstituted phosphatidylcholine multilayers and the angular dependence of the light-induced spectrum recorded at room temperature. A computer analysis of this angular dependence suggests that the plane of the primary ubiquinone acceptor molecule is parallel to the plane of the membrane and therefore, perpendicular to the donor.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Purification and characterization of the photochemical reaction center of the thermophilic purple sulfur bacterium Chromatium tepidum

Pure reaction center preparations from the thermophilic species Chromatium tepidum have been obtained by lauryldimethylamine N-oxide treatment of chromatophores. The light-induced difference spectrum in presence of 10 mM sodium ascorbate revealed the presence of two high-potential cytochrome c hemes (-band, 555 nm; γ-band, 422 nm). The dithionite-minus-oxidized difference spectrum in the -band suggests the presence of additional hemes of low potential. These hemes are associated with a single polypeptide (M_r = 36 000). The reaction center pigments, probably four bacteriochorophyll a and two bacteriopheophytin a molecules, are associated with three polypeptides of apparent molecular weights equal to 33 000, 30 000 and 22 000. A carotenoid molecule is also bound to the reaction center. The three main absorption bands of this molecule are located at 480, 510 and 530 nm at liquid helium temperature. Photochemical activity is found to be stable, even after heating for 10 min at temperatures higher than 60 °C in intact chromatophore membranes. On the other hand, isolated reaction centers or chromatophores treated with 1% lauryldimethylamine N-oxide are fully inactivated after heating at temperatures higher than 50 °C. From these results, we propose that lipid-protein interactions are of prime importance in the thermal stabilization of Chromatium tepidum reaction centers.     

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Linear dichroism and the orientation of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films

Aqueous mixtures of reaction centers of Rhodopseudomonas sphaeroides and gelatin were dried to form thin films. Following hydration, these films were stretched as much as two to three times their original length. Polarized absorption spectra showing linear dichroism were obtained for both unstretched and stretched films, with the planes and stretching axes of the films mounted in various geometries relative to the electric vector of the measuring beam. These data were analyzed in terms of the following model: Reaction centers possess an axis of symmetry that is fixed in relation to the reaction center structure. In unstretched films this axis is confined to the film plane and oriented at random within the plane. In stretched films the symmetry axis is aligned with the direction of stretching. In both preparations reaction centers are distributed randomly with respect to rotation about the axis of symmetry. The data are consistent with this model when the analysis acknowledges less than perfect orientation. For perfect orientation in a stretched film the model predicts uniaxial symmetry about the axis of stretching. The approach to this condition was examined with films stretched to different extents. Extrapolation yielded dichroic ratios for the ideal case of perfect orientation, and allowed calculation of the angles between the axis of symmetry and the various optical transition dipoles in the reaction center. This treatment included the two absorption bands of the bacteriochlorophyll ‘special pair’ (photochemical electron donor) in the Q_x region, at 600 and 630 nm, which we were able to resolve in light minus dark difference spectra.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

The primary charge separation,cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations.Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex.The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840⁺ was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

The triplet exciton of chlorophyll a and carotenoid in solution and in photosynthetic antenna proteins

We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll $\text{a}\text{b}$ protein complex by high-intensity laser excitation. The carotenoid triplet (³Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of ³Car in CP-II decreased 11-fold as intensity was increased from 10¹⁶ to 2 · 10¹⁷ photons/cm² per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 10¹⁶ photons/cm² per s, the ³Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between ³Car and chlorophyll triplets which is compatible with the observed increased rate of ³Car decay. Indirect evidence suggests similar effects occur in LHC.