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• W2091936935 abstract "Highly purified preparations of cytochromeb6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6 f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed. Highly purified preparations of cytochromeb6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6 f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed. Cytochrome b6 f, the central complex in the photosynthetic electron transfer chain, receives from plastoquinol (PQH2) 1The abbreviations and trivial names used are: PQH2, plastoquinol; HG, Hecameg (6-O-(N-heptylcarbamoyl)-methyl-α-d-glycopyranoside); AP-HP buffer, ammonium phosphate/Hecameg/phosphatidylcholine buffer; Chl, chlorophyll; Chla, chlorophyll a; Cytf, cytochrome f; HA, hydroxylapatite; HPLC, high-pressure liquid chromatography; LM, laurylmaltoside (dodecyl-β-d-maltoside); PC,l-α-phosphatidylcholine; PMSF, phenylmethylsulfonyl fluoride; TAP, Tris acetate/phosphate growth medium; TMK, Tris/magnesium/potassium buffer; TMK-HP, TMK buffer supplemented with HG and egg PC; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type.1The abbreviations and trivial names used are: PQH2, plastoquinol; HG, Hecameg (6-O-(N-heptylcarbamoyl)-methyl-α-d-glycopyranoside); AP-HP buffer, ammonium phosphate/Hecameg/phosphatidylcholine buffer; Chl, chlorophyll; Chla, chlorophyll a; Cytf, cytochrome f; HA, hydroxylapatite; HPLC, high-pressure liquid chromatography; LM, laurylmaltoside (dodecyl-β-d-maltoside); PC,l-α-phosphatidylcholine; PMSF, phenylmethylsulfonyl fluoride; TAP, Tris acetate/phosphate growth medium; TMK, Tris/magnesium/potassium buffer; TMK-HP, TMK buffer supplemented with HG and egg PC; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type. electrons stripped from water by photosystem II reaction centers and transfers them to plastocyanin, the electron donor to photosystem I. In the process, part of the electron free energy drop is transduced into a proton electrochemical potential gradient (1Malkin R. Photosynth. Res. 1992; 33: 121-136Crossref PubMed Scopus (39) Google Scholar, 2Hope A.B. Biochim. Biophys. Acta. 1993; 1143: 1-22Crossref PubMed Scopus (132) Google Scholar, 3Cramer W.A. Martinez S.E. Furbacher P.N. Huang D. Smith J.L. Curr. Opin. Struct. Biol. 1994; 4: 536-544Crossref Scopus (38) Google Scholar, 4Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Crossref PubMed Scopus (161) Google Scholar, 5Hauska G. Schütz M. Büttner M. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht1996: 377-398Google Scholar). A homologous complex, cytochrome bc1, carries out an equivalent function in the respiratory chains of mitochondria and many prokaryotes (6Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (467) Google Scholar, 7Nitschke W. Mühlenhoff U. Liebl U. Photosynthesis: A Comprehensive Treatise.in: Raghavendra A.S. Cambridge University Press, Cambridge1997Google Scholar). Chlorophyll molecules are part of the two photosystem reaction centers and the associated antenna complexes. In the photosynthetic electron transfer chain, there are 500–1000 chlorophyll (Chl) molecules/b6 f complex (see Refs. 8Kok B. Biochim. Biophys. Acta. 1956; 22: 399-400Crossref PubMed Scopus (90) Google Scholarand 9Joliot P. Joliot A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1034-1038Crossref PubMed Scopus (57) Google Scholar, and Table I). It was no surprise, therefore, that traces of Chl should be found in purified b6 fpreparations (see e.g. Ref. 10Hurt C. Hauska G. Eur. J. Biochem. 1981; 117: 591-599Crossref PubMed Scopus (297) Google Scholar), and they have long been regarded as contaminants. In recent years, however, several reports have noted that, in very pure preparations, the amount of Chl tends to be close to 1/cytochrome f (Cytf) (11Bald D. Kruip J. Boekema E.J. Rögner M. Murata N. Research in Photosynthesis. Proceedings of the IXth International Congress on Photosynthesis. Kluwer Academic Publishers, Nagoya1992: 629-632Google Scholar, 12Huang D. Everly R.M. Cheng R.H. Heymann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (102) Google Scholar, 13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 14Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. Kluwer Academic Publishers, Montpellier1995: 507-512Google Scholar), raising the intriguing possibility that its presence might not be adventitious (12Huang D. Everly R.M. Cheng R.H. Heymann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (102) Google Scholar, 14Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. Kluwer Academic Publishers, Montpellier1995: 507-512Google Scholar).Table IProsthetic group composition (mol/mol ratios) of thylakoid membranes and cytochrome b6 f complexes purified from either wild-type or LDS C. reinhardtii strainsWTLDSMembranesCytb6 fMembranesCyt b6 fChla/Cytf∼9001-aMixture of Chlb and Chla.0.78 –1.1ND1-bConcentration of Cyt f not determined.0.33β-carotene/Cytf21 –330.26 –0.38ND1-bConcentration of Cyt f not determined.0.036PQ/Cytf130.05 –0.07ND1-bConcentration of Cyt f not determined.0.14β-carotene/Chla0.035 –0.050.23 –0.481.010.16Chlb/Chla0.37 –0.480.09 –0.100.350.45Lutein/Chla0.09 –0.110.06 –0.0811.10Neoxanthin/Chla0.09 –0.110.010.360.50Violaxanthin/Chla0.08 –0.090.020.060.71Most determinations were performed on two different wild-type preparations. All other pigments (antheraxanthin, lutein-5,6-epoxide, zeaxanthin, α-carotene, β-carotene-5,6-epoxide) were either absent or present in trace amounts (≤1% of total pigment mass).1-a Mixture of Chlb and Chla.1-b Concentration of Cyt f not determined. Open table in a new tab Most determinations were performed on two different wild-type preparations. All other pigments (antheraxanthin, lutein-5,6-epoxide, zeaxanthin, α-carotene, β-carotene-5,6-epoxide) were either absent or present in trace amounts (≤1% of total pigment mass). In the present article, we present further evidence in support of our earlier conclusion that the nativeb6 f complex fromChlamydomonas reinhardtii comprises 1 molecule of chlorophyll a (Chla) per monomer as an authentic component (14Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. Kluwer Academic Publishers, Montpellier1995: 507-512Google Scholar). Namely: (i) free [3H]Chlaadded to C. reinhardtii thylakoid membranes at the time of solubilization does not associate with theb6 fcomplex; 2This observation has been reported in preliminary form in Ref. 14Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. Kluwer Academic Publishers, Montpellier1995: 507-512Google Scholar.2This observation has been reported in preliminary form in Ref. 14Popot J.-L. Pierre Y. Breyton C. Lemoine Y. Takahashi Y. Rochaix J.-D. Mathis P. Photosynthesis: From Light to Biosphere. Proceedings of the Xth International Congress on Photosynthesis. Kluwer Academic Publishers, Montpellier1995: 507-512Google Scholar. (ii) the rate of exchange of b6 f-associated Chla for free [3H]Chla is extremely slow; and (iii) Chla is bound to theb6 f complex at a single, specific site. Putative functional roles for a chlorophyll in theb6 f complex are examined and some of them are experimentally ruled out. Decylplastoquinone (C10-PQ), Tricine, egg yolk l-α-phosphatidylcholine (PC), phenylmethylsulfonyl fluoride (PMSF), ε-aminocaproic acid, benzamidine, and sucrose were purchased from Sigma; acetone Uvasol was from Merck; sodium dodecyl sulfate (SDS) was from Pierce; Hecameg (HG) was from Vegatec (Villejuif, France); hydroxylapatite (HA) was from Bio-Rad; dithiothreitol was from Boehringer Mannheim; 3,3′,5,5′-tetramethylbenzidine was from Fluka Chemie AG; urea was from Tebu; [3H]acetic acid was from ICN; and Aqualuma was from Packard Instruments. TMK buffer contained 20 mm Tricine-NaOH, pH 8.0, 3 mm MgCl2, 3 mm KCl. TMK-HP buffer contained TMK buffer supplemented with 20 mmHG and 0.1 g/liter egg PC. AP-HP buffer contained 400 mmammonium phosphate, pH 8.0, 20 mm HG, 0.1 g/liter egg PC, protease inhibitors (200 μm PMSF, 1 mmbenzamidine, 5 mm ε-aminocaproic acid). Wild-type strain (WT) and mutant strain LDS (lacking chlorophyll synthesis when grown in the dark) were kindly provided by J. Girard-Bascou and P. Bennoun (CNRS UPR 9072, Institut de Biologie Physico-Chimique). C. reinhardtiiwas grown in Tris acetate-phosphate medium (TAP) (15Gorman D.S. Levine R.P. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1665-1669Crossref PubMed Scopus (1246) Google Scholar) at 25 °C under an illumination of 300–400 lux (WT) or in the dark (LDS) on a rotary shaker until stationary phase (∼107 cells/ml). Cells were harvested at 5,000 × g for 10 min. Thylakoid membranes were prepared as described previously (WT, Ref. 16Atteia A. de Vitry C. Pierre Y. Popot J.-L. J. Biol. Chem. 1992; 267: 226-234Abstract Full Text PDF PubMed Google Scholar; LDS, Ref. 17Bennoun P. Atteia A. Pierre Y. Delosme M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10202-10206Crossref PubMed Scopus (11) Google Scholar), resuspended in 10 mm Tricine-NaOH, pH 8.0, containing protease inhibitors (200 μm PMSF, 1 mmbenzamidine, 5 mm ε-aminocaproic acid), and stored at −80 °C. The final concentration of WT membranes was adjusted at 3 mg of Chl/ml. The concentration of LDS etioplast membranes was estimated from their optical density at 460 nm. Cytochromeb6 f complex was purified and analyzed, and its PQH2-plastocyanin oxidoreductase activity was determined as described previously (13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). UV-visible absorbance spectra were recorded either on a Kontron Uvikon 930, a Varian Cary 2300, or a Joliot-type homemade spectrophotometer (18Joliot P. Joliot A. Biochim. Biophys. Acta. 1984; 765: 210-218Crossref Scopus (94) Google Scholar), as specified in the legends to Figs. 2, 3, and 6. Cytf concentrations were determined from the ascorbate-reduced minus ferricyanide-oxidized spectra, using ε554 = 18,000m−1·cm−1 (19Rich P.R. Heathcote P. Moss D.A. Biochim. Biophys. Acta. 1987; 892: 138-151Crossref Scopus (70) Google Scholar, 20Dawson R.M.C. Elliot D.C. Elliot W.H. Jones K.M. Data for Biochemical Research. Clarendon Press, Oxford1986: 232-233Google Scholar);A554 was measured by reference to a line joining isosbestic points at 545 and 575 nm. Chl concentrations were determined from the absorption at 668 nm, using ε668 = 75,000m−1·cm−1 (20; we checked that the extinction coefficient of Chla bound to the complex is identical to that in acetone).Figure 3Fluorescence excitation and emission spectra of b6 f-bound chlorophyll a. Panel A, absorption spectra of free Chla in AP-HP buffer (a) and of purifiedb6 f complex in the same buffer without addition (b) and after addition of dithionite (c), using Varian Cary 2300 spectrophotometer. Panel B, fluorescence emission spectra at 77 K of purifiedb6 f complex in AP-HP buffer without addition (a) and after 1 h incubation in 100 mm HG in the same buffer (b), and emission spectrum of free Chla in AP-HP buffer (c); excitation light at 440 nm. Panel C, fluorescence excitation spectra at 77 K of purified b6 fcomplex without addition (○) and following addition of dithionite (+); emission was measured at 673 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Visible absorption spectra ofb6 f complexes purified from wild-type and LDSC. reinhardtii strains. Cytochromeb6 f was purified from WT and LDS strains as described under “Experimental Procedures,” and the preparations were analyzed for Cytf (left), following addition of ascorbate, and for Chl (right). Spectra were recorded on a Kontron Uvikon 930 spectrophotometer normalized to the same absorbance at 554 nm and vertically displaced.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pigments were extracted from thylakoid membranes or from cytochrome b6 fpreparations by 10 volumes of ice-cold 100% acetone under vigorous stirring. Precipitated proteins were spun down at 5,000 ×g for 10 min. The supernatant was collected, evaporated to dryness in a glass flask under a flow of N2 and stored at −80 °C. Pigments were first separated by chromatography on thin-layer silica gel plates according to Eichenberger and Grob (21Eichenberger W. Grob E.C. Helv. Chim. Acta. 1962; 45: 974-981Crossref Scopus (14) Google Scholar). After methanol extraction from the silica powder, each fraction was further purified by reversed-phase HPLC on a Zorbax-ODS column (Rockland Technologies, Inc.; 4.6 × 250 mm, 5 μm granulometry). Elution proceeded in the following three phases: (i) during 8 min, 0.1% methylene chloride in acetonitrile/methanol (70:30 v/v); (ii) during 4 min, a 0.1–40% (v/v) gradient of methylene chloride in the same solvent mixture; and (iii) a constant concentration of 40% methylene chloride in the same mixture. The absorption spectrum of the eluted fractions was continuously monitored with a Hewlett-Packard 1040 A diode array detector (wavelength range 230–600 nm). The detector response was calibrated with standards, using extinction coefficients given by Lichtenthaler (22Lichtenthaler H.K. Methods Enzymol. 1987; 148: 350-382Crossref Scopus (8701) Google Scholar) for pheophytins and chlorophylls, by Britton (23Britton G. Methods Enzymol. 1985; 111: 113-149Crossref PubMed Scopus (236) Google Scholar) for carotenoids, and by Barr and Crane (24Barr R. Crane F.L. Methods Enzymol. 1971; 23: 372-408Crossref Scopus (92) Google Scholar) for quinones. Wild-typeC. reinhardtii cells were grown in TAP medium under standard conditions until stationary phase, diluted 10 times into 200 ml of TAP medium containing 3.7 GBq of sodium [3H]acetate, and further grown under about 1000 lux until stationary phase. Cells were harvested, thylakoid membranes were prepared, and3H-labeled pigments were separated as described above (Fig. 1). Their specific activity was determined by liquid scintillation counting in Aqualuma in a LS1801 counter (Beckman) and spectrometry. A 500-μl sample of C. reinhardtii thylakoid membranes (containing 1.5 mg Chl) in 10 mm Tricine-NaOH buffer, pH 8.0, plus protease inhibitors, was solubilized by addition of an equal volume of HG 50 mmin 2× concentrated TMK buffer supplemented with 37 μg of [3H]Chla (3.25 × 107 cpm). After 15 min of incubation at 4 °C in the dark and 10 min of centrifugation at 80,000 rpm (160,000 × g) in the TLA 100.3 rotor of a TL100 ultracentrifuge (Beckman), the supernatant was layered on top of an 11-ml 10–30% (w/w) sucrose gradient in TMK-HP buffer and centrifuged for 24 h at 40,000 rpm (270,000 ×g) in the SW41 rotor of an L8 ultracentrifuge (Beckman). Fractions of 400 μl were collected. The top fractions were analyzed for Chl and radioactivity. The fractions containing theb6 f complex were pooled, and the complex was purified by HA chromatography as described (13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The specific activity of free Chl was taken to be that of the Chl present in the uppermost three fractions of the gradient (Fig. 5). An acetone solution containing 2 nmol of [3H]Chla (∼480,000 cpm) was evaporated to dryness under N2. The [3H]Chla was redissolved in 0.5 ml of a 5 μm solution of purifiedb6 f complex in AP-HP buffer and incubated in the dark under N2 at 4 °C. At time intervals, 0.1-ml aliquots were layered onto 2-ml 10–30% (w/w) sucrose gradients in TMK-HP buffer and centrifuged for 3 h at 55,000 rpm (260,000 × g) in the TLS 55 rotor of a TL100 ultracentrifuge (Beckman) to separate free fromb6 f-bound Chl. The brownish band containing the complex was collected, and the specific radioactivity ofb6 f-bound Chl was determined and compared with that of the free Chl present at the top of the gradient. Low-temperature (77 K) fluorescence spectra were recorded on a homemade instrument (see Ref.25Ajlani G. Vernotte C. DiMagno L. Haselkorn R. Biochim. Biophys. Acta. 1995; 1231: 189-196Crossref Scopus (75) Google Scholar). The exciting beam and the fluorescence emission were passed through a Y-shaped light guide. The sample (∼5 μm in AP-HP buffer) was placed in a flat quartz cuvette (0.1-mm light path), immersed in liquid nitrogen, and held at the common end of the guide. The exciting beam wavelength was selected by a monochromator, with a bandwidth set at 3 nm for excitation spectra and 12 nm for emission spectra. The fluorescence emitted by the sample was monitored by a photomultiplier through a monochromator with a bandwidth of 12 nm for excitation spectra and 3 nm for emission spectra. Spectra were recorded as uncorrected responses of the photomultiplier. Resonance Raman spectra were recorded with a U1000 Raman spectrometer equipped with a charge-coupled device camera (Jobin-Yvon, France) on pellets of oxidized cytochromeb6 f obtained by ultracentrifugation after exposure to 1.5 mm ferricyanide and dilution under the critical micellar concentration of HG. The 441.6 nm excitation light (less than 15 milliwatts on the sample) was provided by a HeCd continuous laser (Model 4270N, Liconix, CA). To prevent photodegradation of Chla during the experiments, samples were cooled at 77 K in a gas flow cryostat (TBT, France). Purified b6 f complex in AP-HP buffer was diluted 10 times into 20 mm HG, 0.1 g/liter PC, to a final concentration of 0.5 μmCytf, 40 mm AP. A 300-μl sample was irradiated at 4 °C under gentle stirring by the two light beams produced by a KL 1500 lamp (Schott; power set at 3). The white light was filtered by a red Wratten low-pass filter No. 92 (Kodak; cut-off wavelength 620 nm) and by 1-cm plastic cuvettes filled with water. The b6 fcomplex from C. reinhardtii contains seven subunits in stoichiometric ratio and four identified redox carriers, onec-type heme, two b-type hemes, and a [2Fe-2S] cluster (13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In addition to the three cytochromes, UV-visible spectra of even the most highly purified preparations reveal the presence of carotenoids (absorbance peaks at ∼460 and 483 nm) and of Chla (peak at 667–668 nm) (13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Within experimental accuracy, the visible spectrum of the Chl does not depend on the redox state of the complex (Ref. 13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar; see Fig. 3 A). Using thein situ extinction coefficient ofb6 f-associated Chla(ε668 = 75,000m−1 · cm−1; cf.“Experimental Procedures”) and an extinction coefficient ε554 = 18,000m−1 · cm−1 for Cytf (19Rich P.R. Heathcote P. Moss D.A. Biochim. Biophys. Acta. 1987; 892: 138-151Crossref Scopus (70) Google Scholar), the Chla/Cytf ratio was found to be 0.93 ± 0.18 (mean ± S.D. over 26 preparations). Chemical analysis confirmed thatb6 f preparations contain essentially pure Chla; Chlb, which makes up to ∼30% of Chl in thylakoid membranes from WT C. reinhardtii, represents less than 10% of Chl in purified b6 f (Table I). Carotenoids are present in substoichiometric ratio with respect to Chla, while other pigments and quinones either are totally absent or are present in trace amounts (Table I). The approximate 1:1 molar ratio of Chla to Cytf, the excess of Chla over Chlb, as compared with thylakoid membranes, and the retention of Chla throughout the purification procedure suggest that there exists, on theb6 f complex, one binding site with high affinity and specificity for Chla. However, the average stoichiometry is somewhat smaller than 1:1, and its variation from one preparation to the next tends to be larger than the uncertainty on the measurements would lead one to expect. Several factors may explain the dispersion of the data. (i) Traces of Chl collected from the sucrose gradient and incompletely washed from the hydroxylapatite column may contaminate some preparations; (ii) theb6 f-associated Chl is easily bleached (see below); and (iii) exposure of the complex to detergent micelles tends to release the Chl (26Breyton C. Tribet C. Olive J. Dubaq J.-P. Popot J.-L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Exposing the complex to an excess of laurylmaltoside (LM) micelles induced a bathochromic shift of the Chla peak by ∼2 nm, from 667–668 to 669–670 nm (Fig. 2 A). Similar shifts were observed following denaturing treatments, such as heating the preparation at 50 °C or adding 8m urea, and occurred whether theb6 f complex was in its detergent-solubilized state or reconstituted into lipid vesicles (Fig. 2 B). The spectrum of Chla inb6 f preparations treated with an excess of detergent resembles that of pure Chla dissolved in LM micelles (Fig. 2 A), suggesting that this treatment releases Chl from the complex. Delipidation by detergents indeed induces dissociation of the complex into chlorophyll-free monomers (26Breyton C. Tribet C. Olive J. Dubaq J.-P. Popot J.-L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, closer examination reveals that the spectral shift actually precedes Chl dissociation. We show elsewhere that mild treatment of the complex with detergent first generates a dimeric form that has lost the Rieske protein and retains the Chl, while a harsher treatment is required for the complex to release the Chl and break down into monomers (26Breyton C. Tribet C. Olive J. Dubaq J.-P. Popot J.-L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Analysis of the visible spectrum of the Chl bound to purified, Rieske-depleted b6 fdimer revealed a red-shifted (and broadened) absorption peak (Fig. 2 C), indicating that the environment of the Chl has been affected even though it is still bound to the complex and co-purifies with it (26Breyton C. Tribet C. Olive J. Dubaq J.-P. Popot J.-L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Interactions of the Chl with its environment in theb6 f complex were further examined by low temperature fluorescence measurements. Cytochromeb6 f exhibits, in the Soret region, several absorption bands due to the Chl, the carotenoids, and cytochromes f and b6, the latter bands being modulated by the redox potential (Ref. 13Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar and Fig. 3 A). The possible occurrence of energy transfer between hemes and Chla was examined by analyzing the fluorescence characteristics of the Chl under various redox conditions. At 77 K, excitation at 440 nm in the Chl band of cytochrome b6 f produced an emission of fluorescence with a maximum at 673 nm (Fig. 3 B). Excitation spectra of the fluorescence emitted at 673 nm and emission spectra of the fluorescence excited at 440 nm were recorded in the presence or absence of 5 mm ascorbate or ∼5 mm sodium dithionite. The effects of these additions on the redox state of the cytochromes were checked by absorption spectroscopy (Fig. 3 A). Regardless of the addition, there was no significant change in the fluorescence excitation and emission spectra (Fig. 3 C, and data not shown). Incubation at room temperature for 1 h with 100 mm HG, which is known to induce partial dissociation of the cytochromeb6 f complex (26Breyton C. Tribet C. Olive J. Dubaq J.-P. Popot J.-L. J. Biol. Chem. 1997; 272: 21892-21900Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), did not modify the shape of the fluorescence spectra, but enhanced fluorescence intensity by a factor of ∼2 (Fig. 3 B). A more limited increase in fluorescence intensity was observed after freezing and thawing the b6 f solution (Table II). A comparison of these fluorescence intensities with that of free Chla in the same buffer is shown in Fig. 3 B and Table II. These observations indicate that association of Chla with theb6 f complex results in a redox-independent quenching of its fluorescence (by a factor of ∼4), which is partially relieved following detergent treatment.Table IICompared fluorescence intensities of free and b6 f-bound ChlaChlorophyll aCytochromeb6 fFreshAfter freeze/thaw+100 mm HecamegRelative fluorescence intensity at 77 K10025 ± 339 ± 451 ± 5 Open table in a new tab The mode of binding of Chla to the protein was further investigated using resonance Raman spectroscopy. To detect selective contributions from the Chl molecules present in the samples, experiments were performed at 441.6 nm excitation wavelength on oxidized cytochrome b6 f. This laser line is located on the red side of the Soret electronic transition of Chla, more than 1500 cm−1 away from the Soret band of the oxidized cytochromes (∼413 nm). As expected, resonance Raman spectra recorded under these conditions led to barely detectable signals from cytochromeb6. Nevertheless, under these conditions of excitation, intense contributions typical of carotenoid molecules partially masked the middle frequency modes of Chla (data not shown). Analysis, therefore, was focused on the high-frequency region of the spectrum (Fig. 4). Below 1600 cm−1, resonance Raman spectra of Chlamolecules typically feature an intense band at ∼1550 cm−1, which has been attributed to complex vibrational modes of the chlorin ring (27Lutz M. Mäntele W. Scheer H. The Chlorophylls. CRC Press Inc., Boca Raton, FL1991: 855-902Google Scholar). Between ∼1600 and 1710 cm−1, two or three bands may be observed: (i) a band between 1595 and 1615 cm−1, arising from the stretching modes of the methine bridges of the molecule (27Lutz M. Mäntele W. Scheer H. The Chlorophylls. CRC Press Inc., Boca Raton, FL1991: 855-902Google Scholar), the frequency of which depends on the conformation of the chlorin ring (28Fujiwara M. Tasumi M. J. Phys. Chem. 1986; 90: 5646-5650Crossref Scopus (90) Google Scholar) and is thus sensitive to the coordination state of the central Mg2+ ion (27Lutz M. Mäntele W. Scheer H. The Chlorophylls. CRC Press Inc., Boca Raton, FL1991: 855-902Google Scholar); (ii) a band at ∼1620 cm−1, arising from the stretching mode of the conjugated vinyl group in position C2 (29Feiler U. Mattioli T.A. Katheder I. Scheer H. Lutz M. Robert B. J. Raman Spectrosc. 1994; 25: 365-370Crossref Scopus (32) Google Scholar), and often just appearing as a weak shoulder on the high frequency side of the methine stretching band (29Feiler U. Mattioli T.A. Katheder I. Scheer H. Lutz M. Robert B. J. Raman Spectrosc. 1994; 25: 365-370Crossref Scopus (32) Google Scholar); and (iii) between 1640 and 1710 cm−1, bands arising from the stretching modes of the conjugated 9-keto carbonyl group, the frequency of which is extremely sensitive to the H-bonding state and to the environment of this group (27Lutz M. Mäntele W. Scheer H. The Chlorophylls. CRC Press Inc., Boca R" @default.
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