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

Analysis of the pigment composition identified phyty-lated protochlorophyll a Pchl bound to the b6 sub-unit of the dimeric Cyt b6f protein complex in the absence of Chl.. Results Chloropl

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 bc1 (Cyt bc1) and b6f

type (Cyt b6f) couple hydrogen and electron transfer

across a membrane phase [1] In the Cyt b6f 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 b6f

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 b6f 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 b6f 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 b6f complex can be isolated with a molecular mass and subunit composition indistinguish-able from dimeric Cyt b6f isolated from chloroplasts Analysis of the pigment composition identified phyty-lated protochlorophyll a (Pchl) bound to the b6 sub-unit of the dimeric Cyt b6f 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 b6f complex

Results

Chloroplasts and etioplasts share protein complexe ATP synthase, Cyt b6f 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 b6f 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 b6f 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 b6f However, the phyty-lated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6 The data imply that a phytylated tetrapyrrol is

an essential structural requirement for assembly of cytochrome b6f

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.

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 b6f 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 b6f 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 b6f complex is achieved

The dimeric Cyt b6f complex contains a chlorophyll derivative in etioplasts

To identify whether a chromophore is bound to the Cyt b6f complex in etioplasts, we set up a noncol-oured lithium dodecylsulfate native electrophoretic system (LN-PAGE) for isolation of the dimeric Cyt b6f 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 b6f complex A molecular weight determination of approximately 270 and

140 kDa for the two protein complexes further indicated that the Cyt b6f 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 b6f 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 b6 emitted a Cy2 signal and the strongest autofluores-cent from the identical molecular mass position This overlay signal indicated that Cyt b6 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 b6 and Cyt f from the mono- and dimeric Cyt b6f 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 b6f complex is boxed (white lines).

For identification of the autofluorescent pigment,

we recorded an absorption spectrum and analysed an

organic extract from the dimeric Cyt b6f complex

after LN-PAGE (Fig 3) In etioplasts, absorption

spectroscopy of dimeric Cyt b6f revealed four

differ-ent maxima that could be compared with the

absorption spectrum of the dimeric Cyt b6f 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 b6f

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 b6f 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 b6f dimers revealed the

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

Wavelength (nm)

0.25

0.20

0.15

0.10

0.05

0.00

421

Wavelength (nm) 484

Fig 3 Absorbance spectrum of dimeric Cyt b6f 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 b6f 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.

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 b6f complex was carried out by comparison of pigment standards protopheophytin (Pchlstandard) and protopheophorbide (Pchlidestandard), and of cofactors isolated from the dimeric Cyt b6f complex (869cytochromeand 591cytochrome).

same low chromatographic mobility, whereas Pchlide

was characterized by a high mobility This indicated a

binding of Pchl to dimeric Cyt b6f 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 b6f 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 b6f 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 b6f was esterified with phytol

Discussion

The Cyt b6f 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 b6f 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 b6 (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 b6f complex potentially may be an already

enzy-matically active complex in etioplasts Our localization

of Cyt b6f 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 b6f

complex in etioplasts

Both published crystal structures of the Cyt b6f

complex show the presence of one Chl molecule per

monomeric complex These reports confirmed previous component analyses of dimeric Cyt b6f 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 b6f 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 b6f 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 b6f 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 b6f 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 b6f complex in a functional state [4]; however, a chlorophyll-less Clamydomonas mutant lacked accumulation of the Cyt b6f complex [4] It is therefore concluded that the phytyl chain in Chl and Pchl causes the co-isolation of the pigment with Cyt b6 in the etioplast (Fig 2B) and chloroplast (data not shown) [21] Our finding demonstrates that the Cyt b6f 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 b6f 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

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%

2D native⁄ SDS gel electrophoresis

by BN-⁄ SDS-PAGE [23,24] or by ⁄ SDS-PAGE

LN-PAGE was based on BN-LN-PAGE with a modified cathode

buffer composed of 80 mm tricine, 15 mm Bis-Tris and

CyDye labeling and protein identification

For direct comparison of complex composition from

labelled with Cy3 and Cy5 [25], mixed, and separated

endogenous fluorescence was obtained by labelling of

etioplast membranes with Cy2 [25] and separation by

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

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 b6f complex

Pigments and autofluorescent protein complexes were

detected by a Typhoon Trio scanner (633 nm laser

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

containing solution was separated from the extracted gel by

centrifugation and cofactors were dried by SpeedVac

(Eppendorf, Hamburg, Germany)

dissolved in the mobile phase solution (acetone : methanol :

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%

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