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:

(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.
References
1 Crofts AR (2004) The cytochrome bc
1
complex: func-
tion in the context of structure. Annu Rev Physiol 66,
689–733.
2 Mitchell P (1966) Chemiosmotic coupling in oxidative
and photosynthetic phosphorylation. Biol Rev Camb
Philos Soc 41, 445–502.
3 Stroebel D, Choquet Y, Popot JL & Picot D (2003) An
atypical haem in the cytochrome b
(6)
f complex. Nature
426, 413–418.
4 Pierre Y, Breyton C, Lemoine Y, Robert B, Vernotte C
& Popot JL (1997) On the presence and role of a mole-
cule of chlorophyll a in the cytochrome b
6
f complex.
J Biol Chem 272, 21901–21908.
5 Poggese C, Polverino de Laureto P, Giacometti GM,
Rigoni F & Barbato R (1997) Cytochrome b
6
⁄ f

complex from the cyanobacterium Synechocystis 6803:
evidence of dimeric organization and identification of
chlorophyll-binding subunit. FEBS Lett 414, 585–589.
6 Zhang H & Cramer WA (2004) Purification and crystal-
lization of the cytochrome b6f complex in oxygenic
photosynthesis. Methods Mol Biol 274, 67–78.
7 Boronowsky U, Wenk S, Schneider D, Jager C & Rogner
M (2001) Isolation of membrane protein subunits in their
native state: evidence for selective binding of chlorophyll
and carotenoid to the b
(6)
subunit of the cytochrome b
(6)
f
complex. Biochim Biophys Acta 1506, 55–66.
8 Eichacker LA, Soll J, Lauterbach P, Rudiger W, Klein
RR & Mullet JE (1990) In vitro synthesis of chlorophyll
a in the dark triggers accumulation of chlorophyll a
apoproteins in barley etioplasts. J Biol Chem 265,
13566–13571.
9 Lilley KS & Friedman DB (2004) All about DIGE:
quantification technology for differential-display 2D-gel
proteomics. Expert Rev Proteomics 1, 401–409.
10 Zhang H, Whitelegge JP & Cramer WA (2001) Ferre-
doxin:NADP+ oxidoreductase is a subunit of the chlo-
roplast cytochrome b
6
f complex. J Biol Chem 276,
38159–38165.
11 Armstrong GA, Runge S, Frick G, Sperling U & Apel

K (1995) Identification of NADPH:protochlorophyllide
oxidoreductases A and B: a branched pathway for
light-dependent chlorophyll biosynthesis in Arabidopsis
thaliana. Plant Physiol 108, 1505–1517.
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 1023
12 Schoch S, Lempert U & Ruediger W (1977) The last
steps of chlorophyll biosynthesis. Intermediates between
chlorophyllide and phytol-containing chlorophyll.
Z Pflanzenphysiol 83, 427–436.
13 Huang D, Everly RM, Cheng RH, Heymann JB, Schag-
ger H, Sled V, Ohnishi T, Baker TS & Cramer WA
(1994) Characterization of the chloroplast cytochrome
b6f complex as a structural and functional dimer.
Biochemistry 33, 4401–4409.
14 Breyton C, Tribet C, Olive J, Dubacq JP & Popot JL
(1997) Dimer to monomer conversion of the cyto-
chrome b
6
f complex. Causes and consequences. J Biol
Chem 272, 21892–21900.
15 Hurt E & Hauska G (1982) Identification of the poly-
peptides in the cytochrome b
6
⁄ f complex from spinach
chloroplasts with redox-center-carrying subunits.
J Bioenerg Biomembr 14, 405–424.
16 Widger WR, Cramer WA, Herrmann RG & Trebst A

(1984) Sequence homology and structural similarity
between cytochrome b of mitochondrial complex III
and the chloroplast b6-f complex: position of the cyto-
chrome b hemes in the membrane. Proc Natl Acad Sci
USA 81, 674–678.
17 Casano LM, Zapata JM, Martin M & Sabater B (2000)
Chlororespiration and poising of cyclic electron trans-
port. Plastoquinone as electron transporter between thy-
lakoid NADH dehydrogenase and peroxidase. J Biol
Chem 275, 942–948.
18 Guera A, de Nova PG & Sabater B (2000) Identifica-
tion of the Ndh (NAD(P)H-plastoquinone-oxidoreduc-
tase) complex in etioplast membranes of barley: changes
during photomorphogenesis of chloroplasts. Plant Cell
Physiol 41, 49–59.
19 Pierre Y, Breyton C, Kramer D & Popot JL (1995)
Purification and characterization of the cytochrome b
6
f
complex from Chlamydomonas reinhardtii. J Biol Chem
270, 29342–29349.
20 Zhang H, Huang D & Cramer WA (1999) Stoichiomet-
rically bound beta-carotene in the cytochrome b
6
f com-
plex of oxygenic photosynthesis protects against oxygen
damage. J Biol Chem 274, 1581–1587.
21 Wenk SO, Schneider D, Boronowsky U, Jager C, Klug-
hammer C, de Weerd FL, van Roon H, Vermaas WF,
Dekker JP & Rogner M (2005) Functional implications

of pigments bound to a cyanobacterial cytochrome b
6
f
complex. FEBS J 272, 582–592.
22 Eichacker LA, Muller B & Helfrich M (1996) Stabiliza-
tion of the chlorophyll binding apoproteins, P700,
CP47, CP43, D2, and D1, by synthesis of Zn-pheophy-
tin a in intact etioplasts from barley. FEBS Lett 395,
251–256.
23 Granvogl B, Reisinger V & Eichacker LA (2006) Map-
ping the proteome of thylakoid membranes by de novo
sequencing of intermembrane peptide domains. Proteo-
mics 6, 3681–3695.
24 Schagger H & von Jagow G (1991) Blue native electro-
phoresis for isolation of membrane protein complexes
in enzymatically active form. Anal Biochem 199, 223–
231.
25 Reisinger V & Eichacker LA (2006) Analysis of
membrane protein complexes by blue native PAGE.
Proteomics 6(Suppl. 2), 6–15.
26 Granvogl B, Gruber P & Eichacker LA (2007) Stan-
dardisation of rapid in-gel digestion by mass spectrome-
try. Proteomics 7, 642–654.
27 Pearson WR (1990) Rapid and sensitive sequence com-
parison with FASTP and FASTA. Methods Enzymol
183, 63–98.
Protochlorophyll in the Cyt b
6
f dimer V. Reisinger et al.
1024 FEBS Journal 275 (2008) 1018–1024 ª 2008 The Authors Journal compilation ª 2008 FEBS