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

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:

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

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.

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

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).

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

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-

[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

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

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