Stoichiometrically Bound beta -Carotene in the Cytochrome b6f Complex of Oxygenic Photosynthesis Protects against Oxygen Damage*

Huamin Zhang, Deru Huang, and William A. CramerDagger

From the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The cytochrome b6f complex of oxygenic photosynthesis carries out "dark reactions" of electron transfer that link the light-driven reactions of the reaction centers, and coupled proton transfer that generates part of the electrochemical potential utilized for ATP synthesis. In contrast to the bc1 complex of the respiratory chain, with which there are many structural and functional homologies, the b6f complex contains bound pigment molecules. Along with the specifically bound chlorophyll a previously found to be bound stoichiometrically in the dimeric b6f complex, it was found in the present study that beta -carotene is also present in the b6f complex at stoichiometric levels or nearly so. Chlorophyll and carotenoid pigments were quantitatively extracted from b6f complex purified from (i) the thermophilic cyanobacterium, Mastigocladus laminosus, (ii) spinach chloroplasts, and (iii) the green alga, Chlamydomonas reinhardtii. Visible and mass spectra showed the carotenoid to be a beta -carotene of molecular weight = 536, with a stoichiometry of 1.0:1 relative to cytochrome f in the highly active M. laminosus complex but somewhat lower stoichiometries, 0.77 and 0.55, in the b6f complex obtained from spinach chloroplasts and C. reinhardtii. A photoprotective function for the beta -carotene was inferred from the findings that the rate of photobleaching of the chlorophyll a bound in the complex was found to vary inversely with beta -carotene content and to decrease markedly in the presence of ambient N2 instead of air. The presence of beta -carotene in the b6f complex, and not in the related bc1 complexes of the mitochondrial respiratory chain and photosynthetic bacteria, suggests that an additional function is to protect the protein complexes in oxygenic photosynthetic membranes against toxic effects of intramembrane singlet O2.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Elucidation of the structural basis of energy transduction in the cytochrome bc complexes is proceeding rapidly. High resolution structure data from x-ray diffraction have been obtained for the 11 subunits of the integral cytochrome bc1 complex from the bovine respiratory chain (1-3). For the cytochrome b6f complex, atomic structures (<2.0-Å resolution) have been obtained for the lumen side domains of cytochrome f (4-5) and the Rieske iron-sulfur protein (6). It is clear that along with many similarities between the b6f and bc1 complexes (7), there are significant differences in structure, now most obvious in the comparison of the structures of cytochromes f and c1 (Refs. 2 and 3 versus 4). The presence of stoichiometrically bound pigment molecules in the cytochrome b6f complex, discussed in the present work, is another contrasting aspect.

The electronic connection between the reducing and oxidizing sides of photosystems II and I, provided by the cytochrome b6f complex, through which the proton electrochemical potential is generated, is traditionally considered part of the "dark" reactions of oxygenic photosynthesis (8). Thus, the finding of a molecule of the light-absorbing pigment, chlorophyll a, to be present at an approximate 1:1 stoichiometry in active cytochrome b6f complex after exhaustive chromatography, was unexpected (9). In addition to the unit stoichiometry, a specific binding site for the chlorophyll molecule in the complex was implied by the dichroism of the bound chlorophyll a in oriented monomeric b6f complex isolated without the Rieske protein from the cyanobacterium, Synechocystis sp. PCC 6803 (10). A specific binding site of a chlorophyll a in the complex from the green alga, Chlamydomonas reinhardtii, was implied by spectroscopic identification of a specific hydrogen-bonding mode of the chlorophyll and slow rates of exchange of the b6f-bound Chl1 with 3[H]Chl in detergent micelles (11).

The conclusion that the chlorophyll a molecule is bound at a specific site in the b6f complex raises the questions of where the two chlorophyll molecules per dimer are located and the functional role of the pigment molecules in the complex. It has been proposed that the function of the chlorophyll a may be to stabilize the dimeric form of the b6f complex (12). The presence of a chlorophyll molecule also implies that there should be a neighboring carotenoid to quench the chlorophyll triplet state that, otherwise, will inevitably lead to photodamage through formation of singlet oxygen (13). It is reported in the present study that beta -carotene is present in the cytochrome b6f complex of the cyanobacterium, Mastigocladus laminosus, at a unit stoichiometry relative to cytochrome f, approximately equivalent to that of the bound chlorophyll a. The Chl a in the M. laminosus b6f complex, compared with the complex isolated from C. reinhardtii with half the beta -carotene content, is twice as resistant to bleaching by actinic light, which was found to be O2-dependent.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and Purification of Cytochrome b6f Complex

The b6f complex was purified from spinach chloroplasts and C. reinhardtii according to the procedures described previously (9) and from cyanobacteria as described (14). The differences in the latter procedure are the use of a propyl-agarose column before the sucrose gradient and of dodecylmaltoside in the sucrose gradient step.

Determination of Chlorophyll and Carotenoid Concentration

Pigment Extraction-- Pigments were extracted with 80% acetone, 20% methanol at 4 °C under dim light (15), vortexed (1 min), sonicated (2 min), and centrifuged at 5,000 × g (10 min). The supernatant was decanted and filtered through a Millex SLCR L04 NS (Millipore) 0.5-µm filter. A second extraction was carried out with 80% acetone, 20% methanol to ensure complete extraction. Because of the prominence of the spectrum of the bound chlorophyll a, epsilon mM = 79 for the absorbance peak at 669 nm (16), the efficiency of extraction based on the absorbance of first and second extracts could be determined to be > 98%. The second extract was colorless by visual inspection.

HPLC Separation---The pigments were resolved on HPLC following procedures in (17), using a Microsorb-MV C-18 column (Rainin Instruments ODS (4.6 mm, inner diameter × 25 cm)), and eluted isocratically (0-- 4 min) with acetonitrile/Tris-HCl (0.1 M, pH 8.0), 80:3 (v/v), and then by a 4.5-min linear gradient to methanol:hexane, 4:1 (v/v). All solvents were filtered through GH-Polypro 0.2-µm filters (Gelman Sciences). Sample injections were 20 µl, the flow-rate for all separations was 2 ml/min, and the eluent was monitored at 440 nm.

Pigment Identification and Calibration-- Pigments were identified by UV-visible spectrophotometry and mass spectrometry. For visible spectra, chlorophyll a and beta -carotene fractions were collected from the HPLC column, dried under an N2 stream, the fractions resolubilized in methanol and hexane, respectively, and their absorption spectra recorded on a Cary 3 UV-visible spectrophotometer. The pigment concentrations were calculated from the visible spectra using the extinction coefficients for chlorophyll a, epsilon mM (A665-A750) = 71.4 in methanol (16), and for beta -carotene, epsilon mM (445 nm) = 134 (18). The pigment content was quantitated by measurement of the area under the peaks in reverse-phase HPLC corresponding to addition of known amounts of pigment standards, and the standard samples were diluted into 80-20% acetone-methanol. The two separated components from the HPLC column were collected, dried under a nitrogen stream, redissolved in methanol (Chl a) and hexane (carotenoids), and their concentrations determined from their visible spectra. The two methods agreed within approximately 3%.

Mass Spectroscopy

Mass spectra were obtained at the Nebraska Center for Mass Spectrometry by high resolution fast atom bombardment using Micromass AutoSpec double focusing-liquid secondary ion mass spectrometry. Neat samples were dissolved in 1-2 µl of matrix (3-nitrobenzyl alcohol doped with LiI) on the probe tip. Mass calibration was achieved by using ions of known mass present in a saturated solution of CsI in glycerol. The putative chlorophyll a sample in the 3-nitrobenzyl alcohol matrix produced a mixture of M+ and (M-H)+ ions with a mass/charge ratio = 892 and 893, respectively. The mass of M+, determined with high precision, was 892.5421 Da and that for the putative beta -carotene sample was determined to be 536.4371.

Determination of Cytochrome Concentration

The cytochrome content was determined from difference spectra of the cytochrome b6f complex, ~0.1 mg/ml in 30 mM Tris-HCl, pH 7.5, 20 mM NaCl, 30 mM beta -D-octyl glucoside, using a Cary 3 UV-visible spectrophotometer with a measuring beam half-bandwidth of 2.0 nm. Ferricyanide, ascorbate, and dithionite were used as redox agents. A reduced minus oxidized Delta epsilon mM = 26 mM-1 cm-1 (19) of cytochrome f at 554 nm, relative to the isosbestic wavelengths at 534, 544, and 561 nm, was used to determine cytochrome concentrations.

Fluorescence Excitation and Energy Transfer Spectra

Room temperature fluorescence excitation and emission spectra of the chlorophyll a in the M. laminosus cytochrome b6f complex, at a cytochrome f concentration of 0.3-1 µM in 30 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 25 mM beta -D-octyl glucoside, were measured with an SLM-Aminco 8000 spectrofluorimeter thermostatted at 20 °C. The excitation and emission wavelengths, for measurements of emission and excitation spectra, respectively, were 440 and 676 nm, with bandwidths of 4 and 8 nm, respectively.

Photobleaching of Chl a in the cyt b6f Complex

A stirred 1-ml cyt b6f sample, ~1 µM cyt f in 25 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.02% beta -D-dodecyl maltoside, was illuminated with 1.6 × 106 µE/m2 of white light filtered through a 12.5-cm 1% CuSO4 solution as a heat filter. 0.2 ml of sample were taken at different time intervals and diluted 5-fold into the same buffer. The chlorophyll a visible spectrum was measured with a Cary 3 UV-visible spectrophotometer. The photobleaching was measured in the presence and relative absence of air, in the latter case by de-aerating the stirred sample on ice for 2 h with N2 gas, adding the b6f complex to the degassed buffer with a syringe, and then flushing the air space above the sample with N2 gas before and during illumination.

Circular Dichroism Spectra

Circular dichroism (CD) spectra were measured on a JASCO J600 spectropolarimeter. Spectral parameters were as follows: sampling, 0.2-nm wavelength increments; time constant, 2 s; spectral half-bandwidth, 2 nm; optical path length, 1 cm; spectra, average of 4 scans after subtraction of the buffer blank. Cyt b6f (6 µM) from spinach thylakoid membranes was suspended in 20 mM MOPS, pH 7.2, 50 mM NaCl, and 20 mM n-octyl-beta -D-glucoside.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characterization of the Cytochrome b6f Complex-- The cytochrome b6f complex contains the four "large" petA-D gene products, cytochrome f, cyt b6, the Rieske protein, and subunit IV, seen in the first dimension SDS-PAGE (9, 20) and three "small" polypeptides, the petG, M, and L hydrophobic polypeptides (21-24), separated on SDS-PAGE in the second dimension after blue native gel electrophoresis (8). The turnover of the chloroplast b6f complex used in the present work was approximately 100, 150, and 450 electrons transferred from decyl-plastoquinol to plastocyanin-ferricyanide per cyt f per second at 25 and 28 °C, respectively, for the complex from spinach chloroplasts (9), C. reinhardtii and the thermophilic cyanobacterium, M. laminosus.

The ratio of f to b heme in the complex from spinach chloroplasts (Fig. 1A) C. reinhardtii or M. laminosus (not shown) was determined from the amplitudes in the cytochrome alpha -band region of the chemical difference spectra, ascorbate minus ferricyanide for cyt f (Fig. 1A, a) and dithionite minus ascorbate for cytochrome b6 (Fig. 1A, b), whose ratio was 1:2. The concentration of cytochrome f and the complex were determined using the extinction coefficient, epsilon mM = 26 (19). Although it has not been separately determined, we infer from the ratio of amplitudes and from the known structural requirement for two b hemes in bc complexes (1-3) that their average extinction coefficient is also approximately 24 mM-1 cm-1 (19).


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Fig. 1.   Room temperature absorbance spectra of cytochrome b6f from spinach chloroplasts. A, difference spectra of cytochrome b6f complex (~1 µM in 30 mM Tris-HCl, pH 7.5, 20 mM NaCl, 30 mM octyl glucoside) showing amplitude of cyt f (a) and b6 (b) obtained as described under "Materials and Methods." Measuring beam half-bandwidth, 2.0 nm. Ferricyanide (0.1 mM) and excess dry ascorbate and dithionite were used, respectively, as oxidant and reductants. B, a, absolute visible absorbance spectrum of the cyt b6f complex, 1 µM cyt f. The spectral bands associated with the presence of carotenoid and chlorophyll a are marked. B, b, inset, room temperature fluorescence spectra of M. laminosus cytochrome b6f complex. The concentration of b6f complex was 0.3 µM in 30 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 0.2% beta -D-dodecyl maltoside. The excitation wavelength was 440 nm with half-bandwidths 4 and 8 nm, respectively, for excitation and emission. The putative excitation band in the region of beta -carotene absorbance is marked (down-arrow ).

Pigment Content of the Cytochrome b6f Complex-- The presence of carotenoid, as well as the chlorophyll a already noted and characterized (9-11, 25), can be seen in the absolute absorbance spectrum of the complex from spinach chloroplasts (Fig. 1B). An essentially identical spectrum was obtained for the complex isolated from M. laminosus (not shown). Extraction of all pigments from the b6f complex was carried out using 80% acetone, 20% methanol at 4 °C under dim light. Total extraction (>98%) was determined as described under "Materials and Methods." The pigments from the chloroplast (Fig. 2A), M. laminosus (Fig. 2B), and C. reinhardtii (not shown) b6f complex were separated into two major components by reverse phase HPLC in hexane:methanol, 4:1, detected by their absorbance at 440 nm, whose visible and mass spectra are diagnostic of chlorophyll a and beta -carotene. The visible absorbance spectra of the two HPLC peaks from the chloroplast b6f complex (Fig. 3) are characteristic, respectively, of beta -carotene (Fig. 3A; Ref. 26) and chlorophyll a (Fig. 3B). Essentially identical spectra were obtained for the HPLC-separated (Fig. 2B) pigment components of M. laminosus (not shown). Through the features of the major and minor peak positions at 434 and 473 nm, the spectrum for beta -carotene (Fig. 3A) most closely resembles a 9-cis or 15-cis beta -carotene (26). The mass of the isolated pigments was determined by fast atom bombardment ionization mass spectrometry to be 892.5421 and 536.4371, respectively (cf. "Materials and Methods"), for the two pigment components shown to be separated by reverse phase HPLC in Fig. 2A. The calculated molecular masses of chlorophyll a (C55H72N4O5Mg) and beta -carotene (C40H56) are 892.96 and 536.85, leading to an absolute assignment of chlorophyll a and beta -carotene for the two isolated pigments.


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Fig. 2.   HPLC separation of pigments from cyt b6f complex as described under "Materials and Methods." A, from spinach chloroplasts; B, from M. laminosus. The ratios of the areas of peak 1 to peak 2 in panels A and B are 0.64:1 and 0.68:1, respectively.


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Fig. 3.   Room temperature absorption spectra of HPLC-purified (A) beta -carotene dissolved in 100% hexane and (B) chlorophyll a dissolved in 100% methanol, both pigments obtained from spinach cytochrome b6f complex. The spectrum (A) is characteristic of 9-cis or 15-cis-beta -carotene (26).

The concentration of the pigment components separated by HPLC was determined using the areas under the peaks, from addition of known concentrations of pigment standards, and by collecting and drying each of the two components, redissolving them in methanol (Chl) and hexane (beta -carotene), and determining their spectra, checked through the extinction coefficients (epsilon mM (A665-A750)) of 71.4 mM-1 for chlorophyll a in methanol and 134 mM-1 cm-1 at 445 nm for beta -carotene in 100% hexane. Using the differential extinction coefficient, Delta epsilon mM = 26 mM-1 cm-1, for cytochrome f, the stoichiometry of the chlorophyll a and beta -carotene relative to cytochrome f is 0.97 ± 0.13 and 0.77 ± 0.07, respectively, in the chloroplast complex (Table 1, spinach chloroplasts), 1.65 ± 0.3 and 1.02 ± 0.1 in that from M. laminosus (Table 1), and 1.37 ± 0.3 and 0.55 ± 0.08 in C. reinhardtii. Recalculation of the data of Huang et al. (9) and Pierre et al. (11), using the recently redetermined Delta epsilon mM = 26 mM-1 cm-1 for cyt f yields values of the Chl a/cyt f ratio of 1-1.2 and 1.1-1.6, respectively. This eliminates any problem of the significance of a stoichiometry smaller than 1:1 (25), but raises the possibility of either a second bound chlorophyll a or a small amount of nonspecifically bound chlorophyll. The latter is a crucial issue in studies of the fluorescence properties of the specifically bound chlorophyll a. Recalculation of the beta -carotene/cyt f stoichiometry previously reported (11) using the cyt f extinction coefficient determined previously (19) yields a value of 0.38-0.55, close to the value found in the present study.

Possible Inter-pigment Interactions-- Because the fluorescence yield and lifetime of beta -carotene are small (27), these two pigments have to be located within a small distance of each other for energy transfer from beta -carotene to the chlorophyll a to occur. The excitation spectrum for chlorophyll a fluorescence at 676 nm from the b6f complex shows a broad band of small amplitude centered near 490 nm (Fig. 1B, inset, spectrum (b)). This band is absent in a solution mixture of chlorophyll a and beta -carotene (not shown). Such a band was absent in the excitation spectrum for chlorophyll a fluorescence at low temperature (77 K) for the complex from the green alga C. reinhardtii (11). In the latter work, the issue of a carotenoid excitation band was not specifically addressed because the carotenoid content was found to be less than stoichiometric.

Photobleaching of the Chlorophyll a, Dependence on beta -Carotene Content-- The existence of three preparations from different sources that have different contents and stoichiometries of beta -carotene to cytochrome f creates the possibility of testing the hypothesis that one of the functions of the beta -carotene, as in the photosynthetic antenna proteins, is to protect the chlorophyll a from photodamage. Such damage can arise from production of chemically reactive singlet oxygen because of the interaction of ground state triplet oxygen with the excited state (*) chlorophyll triplet (13) shown.
<SUP> 3</SUP>Chl*+<SUP>3</SUP>O<SUB>2</SUB> →<SUP> 1</SUP>Chl+<SUP>1</SUP>O<SUP>*</SUP><SUB>2</SUB>
<UP><SC>Reaction</SC> 1</UP>
beta -carotene (Car) can serve as an alternative acceptor of the excited state chlorophyll triplet, preventing the production of excited singlet oxygen, 1O2*.
<SUP> 3</SUP>Chl*+<SUP>1</SUP>Car →<SUP> 1</SUP>Chl+<SUP>3</SUP>Car*
<UP><SC>Reaction</SC> 2</UP>
Significant photobleaching of the Chl a in the C. reinhardtii b6f complex could be observed after a 10-min exposure to heat-filtered white light with an intensity of 2.7 × 103 µE/m2 s (Fig. 4A, b, dashed spectrum, compared with the spectrum of unilluminated sample, Fig. 4A, a, solid curve). The residual spectrum in the 400-450 nm region (Fig. 4A, b, dashed spectrum) arises mostly from the Soret bands of cytochrome f and the two hemes of cytochrome b6 and explains why the amplitude of the beta -carotene near 500 nm appears relatively small compared with that in the Soret region arising from one-two chlorophylls and three hemes from cytochromes b6 and f (Fig. 1B).


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Fig. 4.   Bleaching of Chl a in the cytochrome b6f complex as a function of incident light energy. A, visible absorption spectra of Chl a in the cytochrome b6f complex of C. reinhardtii, solid line, before (a) and after (b) illumination for 10 min with 1.6 × 106 µE/m2 of white light filtered through 12.5 cm of 1% CaSO4 solution as a heat filter. B, stability of the Chl a molecule in the cytochrome b6f complex as a function of illumination energy with heat-filtered white light at an intensity of 2.7 × 103 µE/m2 s, after which the residual absorbance of the Chl 676-nm band was 92, 70, and 46%. b6f complex from M. laminosus (a), spinach chloroplasts (b), and C. reinhardtii (c).

The time course of the bleaching of the Chl a in C. reinhardtii is shown (Fig. 4B, curve c). Also shown in Fig. 4B is the time course of the bleaching of the Chl a in the b6f complex of M. laminosus (curve a) and spinach chloroplasts (curve b). It can be seen that the bleaching of Chl a in the M. laminosus complex is very small. The residual absorbance of the 676 nm Chl a band, after the 10-min illumination (1.7 × 106 µE/m2) was 92 ± 2%, 70 ± 5%, and 46 ± 10% (n = 3), respectively, for the complex from M. laminosus, spinach chloroplasts, and C. reinhardtii (Fig. 4B). The relative magnitude of this residual absorbance for the Chl a from the b6f complex from these three sources was 1.0, 0.76, 0.50, almost proportional to the respective stoichiometries of beta -carotene to cytochrome f (Table I). The bleaching of the Chl a band in the C. reinhardtii complex was reduced substantially, the residual absorbance increasing from 47 ± 8 to 74 ± 4% (n = 3) when the sample was equilibrated with N2 instead of air before exposure to the actinic light (Fig. 4B, black-square, linked by arrow, showing the change in photobleaching of the b6f complex from C. reinhardtii).

                              
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Table I
Pigment content (mol/mol) of purified cytochrome b6f complex from spinach chloroplasts, M. laminosus, and Chlamydomonas reinhardtii

Circular Dichroism Spectra Show beta -Carotene Is Bound in an Asymmetric Environment-- Carotenoids in a symmetric environment in organic solvent show no optical activity (26). CD spectra of beta -carotene in the spinach chloroplast b6f complex have minima at 430, 457, and 485 nm (Fig. 5), reasonably close to the absorbance peaks at 422, 451, and 478 nm for beta -carotene in 100% hexane (Fig. 3A). In a somewhat more polar solvent, 80% acetone, these peaks are red-shifted to 429, 456, and 483 nm, very close to the extrema in the CD spectrum. The CD spectra are indicative of an asymmetric binding environment of the beta -carotene in the complex. The ratio of the ellipticity peak of the beta -carotene at 457 nm to that of the Chl a at 670 nm is 4.6:1. This ratio in the CP43 light-harvesting complex, which contains chlorophyll a and beta -carotene in a ratio of 4:1, is 1:1 (28). It is concluded that all of the bound beta -carotene is bound in the complex in an asymmetric environment.


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Fig. 5.   Circular dichroism spectrum of the cyt b6f complex at room temperature. Cyt b6f complex from spinach chloroplasts was in 20 mM MOPS, pH 7.2, 50 mM NaCl, 20 mM octyl glucoside corresponding to an absorbance of 0.7 for Chl a at 672 nm.


    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Function of b6f-bound Pigments-- Carotenoids can have multiple roles in light-harvesting and oxygen-related reactions of photosynthesis (29, 30): (a) light harvesting via energy transfer to chlorophyll, (b) photoprotection via quenching of chlorophyll excited triplet states, (c) excess excited state energy dissipation, (d) singlet oxygen scavenging, and (e) structure stabilization (31). A possible function of beta -carotene as a radical-trapping antioxidant that can limit the damage induced by oxy-radical generating systems (32) seems unlikely for a stoichiometrically bound beta -carotene because it would involve chemical modification of the carotene. For chlorophylls, the range of possibilities for function is smaller. They function, most obviously (i), in light harvesting, (ii) they could conceivably influence pathways of electron transfer because of their aromaticity, and (iii), in addition, they could participate in structure stabilization. The latter function would involve the long hydrophobic phytyl chain as well as the bulky ring moiety.

Function related to light-harvesting seems unlikely for the pigments in the b6f complex. Although this possibility has been proposed (33) in the context of "super-complexes" that could be formed with the PSI reaction center (34), (a) the fractional contribution of the b6f-bound pigment to the total antenna function of such a super-complex is small; (b) the lack of inhibition of electron transfer through the b6f complex caused by photobleaching of the chlorophyll in the C. reinhardtii b6f complex implies that the chlorophyll does not facilitate a rate-limiting step of electron transfer (11); and (c) a light energy transfer function of beta -carotene to chlorophyll is either small, as shown in the present work (Fig. 1B, inset), or absent (11).

Thus, one is led to think of a function for the b6f-bound beta -carotene in quenching of the chlorophyll excited triplet state, and protection against the formation of single O2, and in structure stabilization. The latter function for the long chain beta -carotene and the Chl a with its long phytyl chain is suggested by the role for beta -carotene in stabilization of the assembly of the D1 protein into a functional photosystem II complex (31) and of the LHCI polypeptides (35) and, as well, the proposal that bound chlorophyll a prevents dissociation of the cytochrome b6f dimer into monomers (12).

Evidence for a role of the beta -carotene in protection against oxygen-mediated damage presumably involving formation of singlet O2 from the chlorophyll-excited triplet state is provided in the present work. The extent of the residual absorbance of the chlorophyll a molecule in the cytochrome b6f complex after photobleaching (Figs. 4, A and B) correlates well with the stoichiometry of beta -carotene relative to cytochrome f (Fig. 4B, a-c, beta -carotene:cyt f = 1.0, 0.77, 0.55), and is markedly dependent upon the ambient O2 concentration (Fig. 4B). One beta -carotene per monomer of b6f complex confers virtually complete protection against photodamage to the 1-2 Chl a molecules bound per monomer. It is inferred that the lower content of beta -carotene in the complexes isolated from spinach chloroplast and C. reinhardtii is an artifact of preparation.

The mechanism of protection by beta -carotene is inferred to be triplet transfer from the excited state of the chlorophyll to the beta -carotene. However, triplet transfer to carotenoid was not found in the b6f complex of the cyanobacterium, Synechocystis sp. PCC 6803 (36). From the precedents of loss of chlorophyll upon monomerization or dissociation of the Rieske protein (12) and of diminished content of beta -carotene in the C. reinhardtii complex (Table I), it is possible that the measurements of triplet transfer to beta -carotene in Peterman et al. (36) were made difficult by a lowered content of beta -carotene and chlorophyll a that is a consequence of the loss of the Rieske protein in this preparation, which results in complete loss of activity. Preliminary experiments designed to detect by EPR the presence of a beta -carotene triplet in the b6f complex following laser excitation of the chlorophyll a did not reveal any component that could be unambiguously assigned to a carotenoid triplet with a decay time in the expected 0.5-10 µs time range.2

Location of b6f-bound Pigments-- The bound chlorophyll a molecule is inferred to be located close to the pathway of electron and/or proton transfer to cytochrome f, from the correspondence of the rates of reduction of cytochrome f and the rise time of an electrochromic band shift of a chlorophyll a (37). This is consistent with the tendency of the chlorophyll molecules in the LHCII protein structure to be localized near the membrane interfacial domain (38). The LHCII structure also provides a precedent for glutamate residues serving as chlorophyll ligands. The carboxylate residue on the lumen side of the small hydrophobic petG, L, and M polypeptides could provide such a residue as a ligand for the b6f-bound chlorophyll. These polypeptides are uniquely present, along with the bound pigments, in the cytochrome b6f compared with the bc1 complex. It has been proposed that the Chl-specific binding site is associated with the cytochrome b6 polypeptide (39). However, because the stoichiometry of this association was approximately only 10%, the association with cyt b6 might be a consequence of preferential and adventitious binding of the chlorophyll with the most hydrophobic polypeptide of the complex. From the present studies on the role of beta -carotene in protecting the Chl a in the b6f complex from O2-dependent photobleaching, it is inferred that the beta -carotene is positioned close to the chlorophyll a and the membrane polar interface (Fig. 6).


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Fig. 6.   Schematic of cytochrome b6f complex, assuming a total of 12 transmembrane helices/monomer (4, cyt b6 (pink); 3, suIV (yellow); 1, cyt f (green); 1, Rieske iron-sulfur protein (red); pet G, M, and L (brown)). Approximate shapes and sizes of lumen-side extrinsic domains of cyt f and Rieske [2Fe-2S] protein are from solved structures (4, 6). A quinone, chlorophyll, and carotenoid are shown in the bilayer region. The position of the chlorophyll a shown in the excited triplet state that is believed to be quenched by beta -carotene and the beta -carotene ring near the interface is based on the precedent for the position of Chl a molecules in LHCII (38) and kinetic correlation of an electrochromic shift of the Chl a with cyt f reduction (37).

Differences between b6f and bc1 Complex, Significance of Absence of Pigments in the Photosynthetic bc1 Complex-- The cytochrome b6f complex is similar in many respects to the bc1 complex of the mitochondrial respiratory chain and photosynthetic bacteria, to which it is known to be related in many structure-function aspects. The possibility of major differences between the complexes has recently become clear with the discovery that cytochromes f and c1 are completely dissimilar proteins, and provide an excellent example of divergent functional evolution in membrane proteins.

A major role for oxygen scavenging in oxygenic photosynthetic membranes would explain the apparent absence of pigments in the cytochrome bc1 complex of mitochondria and photosynthetic bacteria. Singlet oxygen, which readily causes oxidative damage at conjugated bonds, e.g. unsaturated fatty acids and aromatic amino acids (40), can be readily produced by the reaction of ground state triplet oxygen with the excited triplet state of many dyes and pigments. It is produced in PSII, presumably by oxygen quenching of the triplet-excited state of the reaction center, 3P680* (41). beta -carotene is known to be an extremely efficient quencher of singlet oxygen, able to quench at a rate that is diffusion-controlled (40). Two molecules of beta -carotene per monomer can quench singlet oxygen in isolated photosystem II reaction centers (41).

We infer that the oxygen produced in the PSII region of the membrane will readily diffuse because it is highly soluble in the non-polar membrane and must be present, perhaps at a somewhat lower concentration, in the region of other thylakoid membrane-bound protein complexes (Fig. 6). Here it can also inflict similar molecular damage to lipids and protein. The range of action of the 1O2 depends upon its lifetime and diffusion rate in the membrane. The lifetime of 1O2 in dioxane, whose dielectric constant (epsilon  = 2.2) is similar to that of the lipid bilayer core, is 30 µs and is not very dependent on solvent polarity (40). If the viscosity of the relatively fluid photosynthetic membrane is assumed to be at the lower end of the range of viscosities, 0.5-5 poise, of biological membranes (42), the effective diffusion constant of O2 in the membrane would be ~10-7 cm2 s-1. The root mean square distance of 1O2 diffusion from its site of generation in PSII, where the O2 concentration is highest, is then ~350 Å. This distance is larger than the average distance between the PSII reaction center and the b6f complex, which has been estimated to be 200 Å (43).

The data in the present study imply that one function of beta -carotene in the b6f complex is to prevent generation of singlet O2 from the photoexcited Chl a in the complex. The role of the chlorophyll itself, presumably structural, is still unclear. It is suggested that it is also needed to exert a protective role against the toxicity of photosynthetically produced O2 toward the cytochrome b6f complex. One may suggest that all protein complexes within an effective diffusion distance of PSII in the oxygenic photosynthetic membrane may require such protection.

The bound chlorophyll and beta -carotene may be "evolutionary relics" that resulted from the appearance of reaction centers prior to that of quinol oxidoreductases (11). However, there is no report of bound bacteriochlorophyll or carotenoid in the cyt bc1 complex isolated from photosynthetic bacteria. Therefore, it seems likely that these "relics" are utilized functionally in the oxygenic photosynthetic membrane. In the case of carotenoids, these pigment molecules are synthesized not only to assist in light harvesting but also as a "bonus" (44) to cope with the problem of oxygen toxicity and structure stabilization in both the light-dependent and "dark" integral membrane protein complexes in the membranes of oxygenic photosynthesis.

    ACKNOWLEDGEMENTS

We thank L. Mets for posing a question that led to these studies; H. Frank, P. Laible, M. V. Ponamarev, G. M. Soriano, and M. Thurnauer for helpful discussions; D. L. Smith and R. Cerny at the Nebraska Center for mass spectrometric determination of pigment masses; and J. Hollister for skilled assistance in the writing of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-38323 (to W. A. C.) and United States Department of Agriculture Grant 95-37306-2045 (to J.  L. Smith and W. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 765-494-4956; Fax: 765-496-1189; E-mail: wac{at}bilbo.bio.purdue.edu.

The abbreviations used are: Chl, chlorophyll; Car, carotenoid; 1Car* or 3Car*, first excited singlet or triplet state of carotenoid; 3Chl*, first excited triplet state of chlorophyll; cyt, cytochrome; epsilon , dielectric constant; epsilon mM, millimolar extinction coefficient; HPLC, high performance liquid chromatography; LHC, light-harvesting chlorophyll pigment protein; PAGE, polyacrylamide gel electrophoresis; PC, plastocyanin; PS, photosystem; µE, microeinstein; MOPS, 4-morpholinepropanesulfonic acid; 1O2, singlet O2.

2 P. D. Laible, H. Zhang, M. C. Thurnauer, and W. A. Cramer, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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