Báo cáo khoa học: Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts doc

Cytochrome b 6 f is a dimeric protochlorophyll a binding complex in etioplasts Veronika Reisinger, Alexander P. Hertle, Matthias Plo ¨ scher and Lutz A. Eichacker Department Biology I, University Munich, Germany In respiration and photosynthesis, cytochrome binding protein complexes (Cyt) of the bc 1 (Cyt bc 1 ) and b 6 f type (Cyt b 6 f) couple hydrogen and electron transfer across a membrane phase [1]. In the Cyt b 6 f complex, two protons per electron are translocated across the membrane to build up an electrochemical gradient for the generation of ATP [2]. Seven prosthetic groups per monomer Cyt b 6 f com- plex have been identified. One 2 Fe-2 S-cluster, four hemes (one c-, two b- and one x-type), one chloro- phyll a (Chl) and one b-carotene were described per monomer [3]. The participation of hemes in the elec- tron transport process is indisputable. Chl was found in Cyt b 6 f preparations of both pro- and eukaryotic origin [3–6], and b-carotene was shown to be echine- none in the prokaryote Synechocystis sp. PCC 6803 [7], indicating a structural or a functional role for both pigments. A structural role was indicated in a Chl-less mutant that was reported to lack accumulation of the Cyt b 6 f complex in Clamydomonas [4]. We therefore set out to characterize the protein complex in etioplast isolated from angiosperm seedlings grown under dark- ness. At this developmental phase, no accumulation of Chl and of Chl binding photosystem proteins is found; however, protochlorophyllide a (Pchlide) and the light dependent enzyme NADPH: protochlorophyllide oxi- doreductase (POR) accumulate [8]. We show in etio- plasts that a Cyt b 6 f complex can be isolated with a molecular mass and subunit composition indistinguish- able from dimeric Cyt b 6 f isolated from chloroplasts. Analysis of the pigment composition identified phyty- lated protochlorophyll a (Pchl) bound to the b 6 sub- unit of the dimeric Cyt b 6 f protein complex in the absence of Chl. We conclude that binding of a phyty- lated tetrapyrrol is essential for assembly and accumu- lation of the Cyt b 6 f complex. Results Chloroplasts and etioplasts share protein complexe ATP synthase, Cyt b 6 f and ribulose- 1,5-bisphosphate carboxylase For direct comparison of subunit composition of protein complexes in etioplasts and chloroplasts, we Keywords chlorophyll; cytochrome b 6 f; etioplast; protochlorophyll Correspondence L. A. Eichacker, Department Biology I, University Munich, Menzingerstrasse 67, 80638 Munich, Germany Fax: +49 89 17861 209 Tel: +49 89 17861 272 E-mail: lutz.eichacker@lrz.uni-muenchen.de (Received 30 October 2007, revised 20 December 2007, accepted 2 January 2008) doi:10.1111/j.1742-4658.2008.06268.x The cytochrome b 6 f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to asso- ciate per cytochrome b 6 f monomer and the structural or functional impor- tance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b 6 f. However, the phyty- lated chlorophyll precursor protochlorophyll a, and not chlorophyll a,is associated with subunit b 6 . The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b 6 f. Abbreviations BN, blue native; Chl, chlorophyll; Cyt, cytochrome; DIGE, 2D fluorescence difference gel electrophoresis; LN, lithium dodecylsulfate native; Pchl, protochlorophyll; Pchlide, protochlorophyllide; POR, NADPH: protochlorophyllide oxidoreductase. 1018 FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS employed 2D fluorescence difference gel electrophore- sis (DIGE) technology [9]. After labelling of the proteins in the membrane fractions from both devel- opmental stages with Cy5 and Cy3, the two samples were mixed and subunits of protein complexes were analyzed by blue native (BN)-DIGE (Fig. 1). Protein subunits corresponding to the Pchlide-binding protein subunits of the POR complex that accumulated only in etioplasts were characterized by the red Cy5 fluo- rescence emission in the fluorescent image (Fig. 1). Protein subunits of Chl-binding photosynthetic com- plexes from photosystem I, photosystem II and the light harvesting complex family that accumulated only in chloroplasts were visualized as green Cy3 fluores- cence emissions in the fluorescent image. In addition, Chl released from Chl-binding photosynthetic com- plexes was recorded as a red autofluorescence signal in the low molecular mass region of the gel. Protein subunits corresponding to the dimeric Cyt b 6 f com- plex, ATP synthase CF1 complex and a complex con- taining the ribosomal protein L12 revealed identical electrophoretic mobilities in etioplasts and chloro- plasts. These proteins were visualized as yellow spots in the fluorescence overlay image of the proteins (Fig. 1). Since the dimeric Cyt b 6 f complex was the only Chl- binding complex identified in chloroplasts and present in its fully assembled state in etioplasts, and since no Chl could be isolated from etioplasts, we were inter- ested to discover how the dimeric assembly state of the Cyt b 6 f complex is achieved. The dimeric Cyt b 6 f complex contains a chlorophyll derivative in etioplasts To identify whether a chromophore is bound to the Cyt b 6 f complex in etioplasts, we set up a noncol- oured lithium dodecylsulfate native electrophoretic system (LN-PAGE) for isolation of the dimeric Cyt b 6 f complex. In comparison to BN-PAGE, LN- PAGE is compatible with spectroscopic methods enabling analysis of fluorescent protein complexes after electrophoresis. After excitation at 633 nm, an autofluorescent image could be recorded from two membrane protein complexes in etioplasts. Identifica- tion of the corresponding proteins by MS identified subunits from the Cyt b 6 f complex. A molecular weight determination of approximately 270 and 140 kDa for the two protein complexes further indicated that the Cyt b 6 f complex was present in a dimeric and monomeric form (Fig. 2A). To our surprise, two proteins were released from the dimeric and the monomeric Cyt b 6 f complex, respectively. These proteins still exhibited autofluorescence proper- ties after second dimension SDS-PAGE. In order to identify the corresponding protein subunits, we combined a Cy2 labeling and readout of the native etioplast membrane protein complexes with autofluo- rescence detection in the Cy5 channel. Clearly, Cyt b 6 emitted a Cy2 signal and the strongest autofluores- cent from the identical molecular mass position. This overlay signal indicated that Cyt b 6 retained the majority of the autofluorescent pigment (Fig. 2B). In addition, a weaker overlay signal could be recorded from the Cyt f protein subunit, indicating that Cyt f also retained pigment bound to the protein despite the solubilization of the protein complex by SDS. Thus, we concluded that the autofluorescent emissions corresponded to subunits Cyt b 6 and Cyt f from the mono- and dimeric Cyt b 6 f complexes, respectively. Fig. 1. DIGE of subunits from etioplast and chloroplast protein complexes in a mass range of 100–300 kDa (BN ⁄ PAGE). After iso- lation of inner membranes from either 5 · 10 7 etioplasts or chloro- plasts, the membrane proteins were labelled by Cy5 (etioplast) or Cy3 (chloroplast). After mixing of the two samples, they were sepa- rated by BN- ⁄ SDS-PAGE and the gel was read out in a Typhoon imager 9400. Proteins originating from the etioplast are shown in red; proteins originating from the chloroplast are shown in green; proteins present in both membranes in equal amounts are shown in yellow. Complex subunits are labelled according to Granvogl et al. [23]. The dimeric Cyt b 6 f complex is boxed (white lines). V. Reisinger et al. Protochlorophyll in the Cyt b 6 f dimer FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS 1019 For identification of the autofluorescent pigment, we recorded an absorption spectrum and analysed an organic extract from the dimeric Cyt b 6 f complex after LN-PAGE (Fig. 3). In etioplasts, absorption spectroscopy of dimeric Cyt b 6 f revealed four differ- ent maxima that could be compared with the absorption spectrum of the dimeric Cyt b 6 f complex reported for chloroplasts [10]. Direct correlation was found at k = 420 nm for the Soret bands, at approximately k = 490 nm for the carotinoid and ferredoxin-NADP + -reductase bands, and at k = 554 nm for the Cyt f a-band (Fig. 3). However, the absorbance maximum at k = 668 nm characteristic for Chl was lacking in etioplasts, whereas a peak at k = 635 nm indicated the presence of Pchl(ide) (Fig. 3). Besides the Chl precursor Pchlide, which is bound to the POR complex [11], etioplasts also syn- thesize a small fraction of approximately 4.3% Pchl with unknown function [12]. Since both Chl derivates feature the same spectral properties, we performed TLC analysis of chromophore standards against an organic extract isolated from the dimeric Cyt b 6 f complex for chromophore identification (Fig. 4). In parallel, the standards and pigment extracts were analysed by MS (Fig. 5). Identification of the chlorophyll derivative in the Cyt b 6 f complex in etioplasts It was evident from TLC and autofluorescence visuali- zation of the pigments that the Pchl standard and the pigment extracted from Cyt b 6 f dimers revealed the AB Fig. 2. (A) Autofluorescence emission of protein complexes after LN-PAGE. Inner membranes from 2 · 10 8 etioplasts were sepa- rated by LN-PAGE. The gel was scanned for autofluorescence. The dimeric (2) and monomeric (1) assembly stage of the Cyt b 6 f com- plex are labelled. (B) Overlay of Cy2 labelled etioplast membranes with autofluorescene signals after LN- ⁄ SDS-PAGE. After isolation of inner membranes from 1 · 10 8 etioplasts the membrane pro- teins were labelled by Cy2 and separated by LN- ⁄ SDS-PAGE. After electrophoresis, the gel was read out in a Typhoon Trio. Signals originating from Cy2 are shown in blue, signals originating from autofluorescence are shown in yellow. Proteins are labelled accord- ing to Fig. 1. Pchl Cytf 550 600 650 700 0.000 0.005 0.010 0.015 0.020 0.025 Absorption Wavelength (nm) 400 450 500 550 600 650 700 0.25 0.20 0.15 0.10 0.05 0.00 421 553 631 Absorption Wavelen g th (nm) 484 Fig. 3. Absorbance spectrum of dimeric Cyt b 6 f complexes from etioplasts. 2 · 10 8 etioplasts were separated by LN-PAGE and the dimeric Cyt b 6 f complex was cut after fluorescent excitation. Five bands were combined and an absorption spectrum from 400– 700 nm was recorded. The wavelength region in the range 540– 700 nm is enlarged (insert). Fig. 4. Identification of Pchl as component of the dimeric Cyt b 6 f complex by TLC. Pigments of the dimeric Cyt b 6 f complex were extracted from LN-PAGE gels. After extraction, pigment extracts of the dimeric Cyt b 6 f complex (Cytb 6 f) and pigment standards of Pchl and Pchlide were separated by TLC. Protochlorophyll in the Cyt b 6 f dimer V. Reisinger et al. 1020 FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS Fig. 5. Mass spectrometry of the pigments bound to the dimeric Cyt b 6 f complex. Mass spectrometric characterization of Pchl in the dimeric Cyt b 6 f complex was carried out by comparison of pigment standards protopheophytin (Pchl standard ) and protopheophorbide (Pchlide standard ), and of cofactors isolated from the dimeric Cyt b 6 f complex (869 cytochrome and 591 cytochrome ). V. Reisinger et al. Protochlorophyll in the Cyt b 6 f dimer FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS 1021 same low chromatographic mobility, whereas Pchlide was characterized by a high mobility. This indicated a binding of Pchl to dimeric Cyt b 6 f in etioplasts (Fig. 4). For identification of the alcohol esterified to the tetrapyrrol, MS was employed (Fig. 5). Fragmenta- tion of protopheophorbide a standard (originating from Pchlide) at 591.15 m ⁄ z and quadrupole mass selection of the Cyt b 6 f extract at 591.15 m ⁄ z did not yield overlapping fragmentation signals, whereas frag- mentation of protopheophytin a standard (originating from Pchl) at 869.319 m ⁄ z matched the quadrupole mass selection at 869.319 m ⁄ z (Fig. 5). This result con- firmed the conclusion proposed after TLC that Pchl is a component of the dimeric Cyt b 6 f complex in etiop- lasts. The mass difference of 278.169 m ⁄ z between the Pchl and Pchlide mass signals selected from the dimeric complex further revealed that Pchl bound to the Cyt b 6 f was esterified with phytol. Discussion The Cyt b 6 f complex assembles as a dimer in etioplasts In chloroplasts, the dimeric complex is characterized by an increased electron transport rate compared to the monomer and is therefore assumed to be the func- tional assembly state [13,14]. In the crystal structure of the dimeric Cyt b 6 f complex, at least eight different transmembrane subunits have been identified [15,16]. Our finding that the complexes in etioplasts and chlo- roplast exhibited an identical molecular mass in native PAGE studies was corroborated further by mass spec- trometric de novo sequence analysis of the four large subunits Cyt f (PetA), Cyt b 6 (PetB), the iron sulfur protein (PetC), and subunit IV (PetD), which were isolated from the dimeric complex of both organelles (Fig. 1). We therefore conclude that the dimeric Cyt b 6 f complex potentially may be an already enzy- matically active complex in etioplasts. Our localization of Cyt b 6 f dimer in etioplasts therefore fosters the dis- cussion concerning the components proposed to oper- ate in an alternative electron transfer chain. The NAD(P)H dehydrogenase complex, a peroxidase act- ing on reduced plastoquinone, a superoxide dismutase and an iron sulfur protein have been proposed [17,18]. Protochlorophyll a replaces Chl in the Cyt b 6 f complex in etioplasts Both published crystal structures of the Cyt b 6 f complex show the presence of one Chl molecule per monomeric complex. These reports confirmed previous component analyses of dimeric Cyt b 6 f complexes from photosynthetic pro- and eukaryotic organisms [19,20] and spectra showing an absorbance maximum at 670 nm [4,5,7,10]. By contrast, the dimeric Cyt b 6 f complex of etioplasts exhibited an absorbance maxi- mum at 631 nm (Fig. 3). These findings argue for a replacement of Chl against Pchl in etioplasts. Replace- ment of a cofactor in the dimeric Cyt b 6 f complex has been reported also in Synecochystis mutants deficient in echinenone synthesis. In the present study, the cofactor was replaced by a mixture of b-carotene, zeaxanthine and mono-hydroxy-b-carotene [7]. The role of Pchl remains open Our finding that Chl is selectively replaced by Pchl in etioplasts indicates an essential role of the pigment for the assembly of the Cyt b 6 f complex. It remains unknown, however, whether Pchl fulfils a functional or structural role in the complex. For Chl in chloroplasts, a distance of 16.7 A ˚ to the b-type hemes was interpreted to indicate a func- tional participation of Chl in electronic interactions [21]. Alternatively, Chl and Pchl may be required for stable assembly of the Cyt b 6 f subunits into a func- tional protein complex. In the present study, the data indicate that the phytyl chain in Chl is of central importance. Bleaching of the tetrayrrol moiety in Chl maintained the Cyt b 6 f complex in a functional state [4]; however, a chlorophyll-less Clamydomonas mutant lacked accumulation of the Cyt b 6 f complex [4]. It is therefore concluded that the phytyl chain in Chl and Pchl causes the co-isolation of the pigment with Cyt b 6 in the etioplast (Fig. 2B) and chloroplast (data not shown) [21]. Our finding demonstrates that the Cyt b 6 f complex in etioplast selectively binds the phytylated minority component Pchl (4.3%) over the nonphytylated principal component Pchlide that con- stitutes 95.7% of the Chl precursor molecules in the organelle. We therefore conclude that the phytyl chain in Pchl and Chl may be essential for assembly of a functional Cyt b 6 f complex in the two develop- mental states of the organelles in etiolated and light grown tissue. Experimental procedures Isolation of membrane protein complexes Barley (Hordeum vulgare, L. var. Steffi) seeds were grown for 4.5 days and intact plastids were isolated from the primary leaves as described by Eichacker et al. [22]. After Protochlorophyll in the Cyt b 6 f dimer V. Reisinger et al. 1022 FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS isolation of intact plastids, native membrane protein com- plexes were prepared for BN-PAGE as described previously [23]. For LN-PAGE, protein complexes were solubilized by a detergent mixture with a final concentration of 0.38% n-dodecyl b-d-maltoside (w ⁄ v), 0.64% (w ⁄ v) digitonin, and 0.006% (w ⁄ v) lithium dodecyl sulfate. 2D native ⁄ SDS gel electrophoresis Solubilized membrane proteins were separated either by BN- ⁄ SDS-PAGE [23,24] or by LN- ⁄ SDS-PAGE. LN- PAGE was based on BN-PAGE with a modified cathode buffer composed of 80 mm tricine, 15 mm Bis-Tris and 0.002% (w ⁄ v) lithium dodecyl sulfate. CyDye labeling and protein identification For direct comparison of complex composition from chloroplasts and etioplasts, membrane samples were labelled with Cy3 and Cy5 [25], mixed, and separated by BN- ⁄ SDS-PAGE. The overlay of exogenous and endogenous fluorescence was obtained by labelling of etioplast membranes with Cy2 [25] and separation by LN- ⁄ SDS-PAGE. In some cases, the gel was scanned for fluorescence after SDS-PAGE and fluorescent spots were cut. After two washing steps, in-gel digestion and peptide identification was carried out as described previously [26]. Proteins were digested by trypsin and analysed after ESI-MS⁄ MS frag- mentation by a Q-TOF premier (Waters Corporation, Mil- ford, MA, USA). The peptide sequences, obtained by manual interpretation from the fragment spectra, were used for protein database searches using the frame ‘fasta3’ from the European Bioinformatics Institute (EBI; http://www. ebi.ac.uk/fasta33) [27]. Characterization of the chlorophyll derivatives in the Cyt b 6 f complex Pigments and autofluorescent protein complexes were detected by a Typhoon Trio scanner (633 nm laser excita- tion ⁄ 670 BP30 emission filter; GE Healthcare UK Ltd, Bucks, UK). For absorption spectroscopy, fluorescent bands were cut from the LN-PAGE. An absorption spec- trum from 400–700 nm was recorded from five combined bands. Cofactor extraction was carried out by cutting fluorescent bands from the LN-PAGE. Pottered gel pieces were incu- bated in dimethylformamide at 4 °C for 1 h. The cofactor containing solution was separated from the extracted gel by centrifugation and cofactors were dried by SpeedVac (Eppendorf, Hamburg, Germany). Reference pigments and extracted cofactors were dissolved in the mobile phase solution (acetone : methanol : H 2 O in a ratio of 20 : 30 : 1) and spotted on the HPTLC RP-8 F 254 plate (Merck, Darmstadt, Germany). Dry samples were dissolved in 25% formic acid, 62.5% acetonitrile, 7.5% isopropanol and cleaned up by a C-18 ZipTip column (Millipore Corporation, Billerica, MA, USA). After elution in in 25% formic acid, 62.5% aceto- nitrile, 7.5% isopropanol, 5% H 2 O samples were measured by ESI-MS ⁄ MS. 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