(Received for publication, July 21, 1995; and in revised form, September 21, 1995)
From the
A protocol has been developed for the purification of the
cytochrome bf complex from the
unicellular alga Chlamydomonas reinhardtii. It is based on the
use of the neutral detergent Hecameg
(6-O-(N-heptylcarbamoyl)-methyl-
-D-glycopyranoside)
and comprises only three steps: selective solubilization from thylakoid
membranes, sucrose gradient sedimentation, and hydroxylapatite
chromatography. The purified complex contains two b hemes
(
bands, 564 nm; E
=
-84 and -158 mV) and one chlorophyll a (
= 667-668 nm) per cytochrome f (
band, 554 nm; E
= +330 mV). It is highly active in transferring
electrons from decylplastoquinol to oxidized plastocyanin (turnover
number 250-300 s
). The purified complex
contains seven subunits, whose identity has been established by
N-terminal sequencing and/or peptide-specific immunolabeling, namely
four high molecular weight subunits (cytochrome f, Rieske
iron-sulfur protein, cytochrome b
, and subunit IV)
and three
4-kDa miniproteins (PetG, PetL, and PetX). Stoichiometry
measurements are consistent with every subunit being present as two
copies per b
f dimer.
The photosynthetic electron transfer chain, the device that
provides essentially all of the free energy dissipated by living
beings, comprises two membrane protein complexes that are homologous to
complexes of the respiratory chain, the CFCF
(respectively, F
F
) proton ATP-synthase
and the b
f (respectively, bc
) oxidoreductase. The bf/bc complexes
transfer electrons from a liposoluble quinol to a hydrosoluble protein,
plastocyanin or cytochrome c, and couple the resulting
electron free energy drop to setting up a transmembrane proton
electrochemical potential. Neither the structure of the b
f nor that of the bc
complex is known in any detail, and the existence of an unique versus alternative mechanism(s) of electron transfer remains a
matter of discussion (see, e.g., (1, 2, 3, 4) and references
therein).
Most structural studies of the bf complex to date have been carried out either on higher
plants or on cyanobacteria (for reviews, see Refs. 1, 2, and
5-8). Unicellular algae, such as Chlorella sorokiniana and Chlamydomonas reinhardtii, have long been recognized
as convenient organisms for combined spectroscopic, biochemical and
genetic studies of the b
f complex
structure and function (9, 10) . In the present
article, we describe the purification of the b
f complex from C. reinhardtii. From the point
of view of the experimentalist, C. reinhardtii combines a
number of advantages: (i) cell suspensions are amenable to in vivo studies of the electron transfer and proton pumping processes
using spectroscopic and other biophysical approaches; (ii) C.
reinhardtii being a facultative phototroph, mutant strains with
defective photosynthetic complexes can be generated and maintained;
(iii) large fractions of its chloroplast genome have been sequenced;
(iv) techniques for modification of its chloroplast and nuclear genes
are being developed; (v) it is amenable to in vivo isotopic
labeling; (vi) large scale growth, although not currently resorted to,
is not an unrealistic objective (for a general review of C.
reinhardtii laboratory use, see (10) ).
Previous
studies have shown that the bf complex
from C. reinhardtii is similar to that of higher plants in its
complement of high molecular mass subunits(11, 12) .
It comprises two heme-bearing subunits (cytochrome f and
cytochrome b
), a protein carrying a
[2Fe-2S] cluster (the so-called Rieske protein), and an
integral protein devoid of prosthetic group (``subunit IV'')
that is homologous to the C-terminal region of mitochondrial cytochrome b(13) . More recent studies have shown that
additional, very small subunits (
4 kDa) are also present, namely:
1) the product of the petG chloroplast gene, initially
identified in higher plants (14, 15) and subsequently
shown to be present in C. reinhardtii b
f(15, 16) ; 2) the PetX protein,
identified both in C. reinhardtii(15, 16) and in spinach (15) complexes,
which is the product of a nuclear gene(16, 17) ; and
3) the product of the chloroplast gene ycf 7 (petL), (
)a gene that is also present in higher plant chloroplast
genomes.
Obtaining large scale preparations of active integral
membrane proteins is a major stumbling block in understanding the
function of these proteins at the structural level. Two limitations are
the amount of protein produced by laboratory organisms and scale limits
on purification procedures. Until now, preparations of bf complex from C. reinhardtii(11, 12, 15) have suffered from limited
purity, low yields, and low or unknown enzymatic activity. In the
present article, we describe a rapid (three-step) protocol for the
preparation of highly purified C. reinhardtii b
f complex that is enzymatically fully active. The
neutral detergent used throughout the
purification,6-O-(N-heptylcarbamoyl)-methyl-
-D-glycopyranoside
(Hecameg (
)(19) ), is found to be an interesting
substitute to the widely used but much more expensive
octyl-
-D-glucopyranoside (OG). At variance with previous b
f purification protocols, the new
procedure does not resort at any step to an electrically charged
detergent, and it is easily amenable to scaling up: two favorable
factors for growing crystals. The purified complex has been
characterized with respect to its subunit composition and its
spectroscopic and redox properties.
Spectral changes recorded from -250 to
+450 mV were analyzed by taking the peak wavelengths
(approximately 564 nm for cytochrome b and 554 nm
for cytochrome f) and fitting the absorbance values to Nernst
curves. Component spectra were obtained by redox cut analysis; the
spectrum of cytochrome f was obtained by taking the average of
three spectra recorded at 400 ± 10 mV and subtracting the
average of four spectra taken at 230 ± 30 mV; the spectrum of
cytochrome b
was obtained by subtracting from a
spectrum recorded at -80 mV a spectrum recorded at 0 mV; the
spectrum of cytochrome b
was obtained by
subtracting from a spectrum taken at -250 mV a spectrum recorded
at -132 mV. The ratio of cytochrome b
to
cytochrome f was estimated from the redox titration curves,
assuming extinction coefficients of 18,000 M
cm
(cytochrome f) and 20,000 M
cm
(average of
the two b-type cytochromes) (peak absorbance minus absorbance
at isosbestic point; (40) ).
For selective
solubilization, thylakoid membranes of wild type cells were resuspended
in ice-cold TMK buffer containing 25 mM HG at a chlorophyll
concentration of 1.5 g/liter and incubated at 4 °C for 15 min. with
occasional agitation. The suspension was centrifuged either at 160,000
g (80,000 rpm) for 10 min in the TLA 100.3 rotor of a
Beckman TL100 ultracentrifuge, or at 200,000
g (43,000
rpm) for 30 min in the 70 Ti rotor of a Beckman L8 ultracentrifuge,
depending on the volume (6 or 30 ml).
The solubilization supernatant
was fractionated by centrifugation in 10-30% (w/w) sucrose
density gradients in TMK buffer containing 20 mM HG and 0.1
g/liter egg PC. The presence of egg PC during this and the following
step of purification is necessary to prevent the dissociation of the
Rieske protein from the complex(43) . The gradients, onto which
were layered 600 µl (Beckman SW41 Ti rotor) or
3 ml (45
Ti rotor) of supernatant, were centrifuged at 270,000
g (40,000 rpm) for 24 h (SW41 Ti rotor), or at 180,000
g (38,000 rpm) for 16 h (45 Ti rotor). The yellow-brown band
containing the cytochrome b
f complex was
collected with a syringe.
The band collected from the gradient was
layered onto a HA column (0.7 cm 2 cm, or 1 cm
3 cm)
pre-equilibrated with 20 mM Tricine-NaOH, pH 8.0, 20 mM HG, 0.1 g/liter egg PC. The column was washed with 3 column
volumes of 100-200 mM ammonium phosphate (depending on
HA batches), pH 8.0, containing 20 mM HG and 0.1 g/liter egg
PC. Pure cytochrome b
f complex was
eluted with 400 mM ammonium phosphate, pH 8.0, containing 20
mM HG and 0.1 g/liter egg PC. Typical yields are
600
µl of pure b
f complex at
6
µM cytochrome f for one SW41 rotor or
3 ml
at
10 µM for one 45 Ti rotor, with larger
preparations resulting in better overall yields.
Our initial
experiments with HG were made somewhat frustrating by irreproducible
batch-to-batch results. These appeared to be due to the presence of
diheptylurea, a side product from the synthesis. ()In the
presence of traces of diheptylurea, abnormal migration of the b
f complex on sucrose gradient was
observed, and electron transfer activity was inhibited. The presence of
diheptylurea is easily detected by leaving a 20 mM solution of
HG in water overnight in the cold room. Diheptylurea precipitates as a
fine white powder. This problem has been solved by the manufacturer,
and recent HG batches (batches 2001, 2101, WK1-20, and
CP1-57) have been free from diheptylurea. From a practical point
of view, we note that the CMC of HG is similar at 4 °C and at room
temperature (
)(see also Refs. 44 and 45), but appears to be
sensitive to high salt concentrations, since the CMC at 4 °C
dropped from
19.5 mM to
15 mM when moving
from the low ionic strength TMK buffer to 0.4 M ammonium
phosphate (see also (45) ).
Figure 1:
Urea/SDS-PAGE and immunoblot analysis
of C. reinhardtii bf complex
preparations. Lanes 1-4, polypeptide composition of
thylakoid membranes (lane 1), solubilization supernatant (lane 2), b
f-enriched fraction
from sucrose gradient (lane 3), and purified b
f complex eluted from the hydroxylapatite column (lane 4). Lanes 5-8", Immunoblot analysis of
wild type (lanes 5-8) and FuD4 (5`-8`)
thylakoid membranes and of the purified b
f complex (5"-8"), using antisera raised against the
N-terminal peptides of mature cytochrome f (5 -5"), cytochrome b
(6-6"), mature Rieske protein (7-7") and subunit IV (8-8"). Immunodetection was carried out with
I-protein A.
Handling the
complex under delipidating conditions, e.g. sedimenting it in
a sucrose gradient containing 25 mM HG in the absence of
lipids and salts, resulted in the loss of the Rieske protein and of the
oxidoreductase activity(43) . Identified stabilizing factors
are (i) working close to the CMC of HG and in the presence of lipids
(0.01-0.1 g/liter egg PC) and (ii) adding low concentrations of
salt (3 mM KCl, 3 mM MgCl).
Figure 2:
Absorbance spectra of purified bf preparations. Oxidized ( . .
. ), ascorbate-reduced(- - -) and dithionite-reduced (-)
UV-visible spectra of a purified preparation. The spectrum of oxidized b
f was calculated by subtracting from
the spectrum of air-equilibrated b
f the
contribution of reduced cytochrome f (see ``Materials and
Methods''). Inset A, UV-visible difference spectra:
ascorbate-reduced minus air-oxidized ( . . . ) and dithionite-reduced
minus ascorbate-reduced (-). Inset B, visible
difference spectra (from a different b
f preparation): ascorbate-reduced minus ferricyanide-oxidized(- - -)
and dithionite-reduced minus ascorbate-reduced
(-).
Upon addition of ascorbate, the
contribution of reduced cytochrome f to the spectrum increases
more or less strongly, indicating that, in air-equilibrated
preparations, cytochrome f is partially to totally reduced.
Further reduction with dithionite or borohydride reveals the presence
of cytochrome b . Difference spectra (Fig. 2, insets A and B) show absorption
maxima at 523 and 554 nm for cytochrome f and 534 and 564 nm
for cytochrome b
, and isosbestic points in the
Soret region at 414 and 420 nm, respectively.
Visible absorbance
spectra were recorded over the redox potential range from -250 to
450 mV. They were analyzed by fitting the absorbance values at peak
wavelengths (approximately 564 nm for cytochrome b and 554 nm for cytochrome f) to Nernst curves. A single
curve with m = 1 and E
=
+ 330 mV was sufficient to fit the cytochrome f data (Fig. 3A). Two titration curves with n = 1 and E
= -84 and
-158 mV were necessary to fit the cytochrome b
data (Fig. 3B). These values are similar to those
obtained at pH 8.75 in spinach thylakoids for cytochromes b
and b
,
respectively(4) . The visible spectra of the two b-type cytochromes at room temperature, calculated from the
redox titration data, were undistinguishable (Fig. 3C).
The concentrations of hemes b
and b
were comparable (Fig. 3C) and the b
/f ratio determined from the redox
titration curves was
1.9:1, indicating that none of the two b-type hemes had been lost during the purification.
Figure 3:
Redox titration curves of cytochromes f (A) and b (B) and absorbance spectra of the three
cytochromes in the
band region (C). Redox potential
values are given against the standard hydrogen electrode (SHE).
Figure 4:
Electron transfer catalysis by purified C. reinhardtii bf complex.
Reduction of oxidized spinach plastocyanin (5 µM) was
initiated by addition of decylplastoquinol (15 µM) and
monitored as the absorbance change at 600 nm. Data are plotted as the
logarithm of A
(absorbance at time t)
minus A
(final absorbance). +, no b
f present;
, in the presence of
0.3 nM purified b
f;
,
same conditions, but 1 µM C
-stigmatellin was
added 20 s after the beginning of the reaction. Inset, initial
rate of electron transfer to spinach plastocyanin (SP)
measured at different concentrations of purified b
f complex.
After 2-week storage at 0 °C in
the dark, purified preparations retained 75% of their activity and
exhibited no obvious change of their polypeptide pattern. They could be
frozen without loss of activity.
Identification of the bands was based on their apparent M, heme content as revealed by TMBZ staining,
N-terminal sequencing, and cross-reaction with antipeptide antisera (Table 2). These approaches confirmed that the four high M
bands correspond, in this order, to cytochrome f, the Rieske iron-sulfur protein, cytochrome b
, and subunit IV(11, 12) . 1) N-terminal
sequencing of the TMBZ-stainable 39.5-kDa band yielded a sequence (Table 2) that is internal to that of the cytochrome f precursor, as predicted from the sequence of the petA gene(51, 52) . The band was labeled by antisera
raised either against this N-terminal peptide or against a peptide with
the predicted C-terminal sequence of cytochrome f (Fig. 1, lane 5", and data not shown). 2)
Sequencing of the 18.5-kDa band yielded a sequence (Table 2) that
is homologous with N-terminal sequences of other Rieske proteins. The
18.5-kDa band was labeled by antisera directed against this N-terminal
sequence (Fig. 1, lane 6"). Oligonucleotides coding for
this sequence have been used to clone and sequence C. reinhardtii
petC nuclear gene (53) . 3) The diffuse TMBZ-stainable
18-kDa band did not yield any phenylthiohydantoin derivatives upon
amino acid sequencing. It was labeled by antisera directed against
peptides corresponding to the predicted N- and C-terminal sequences of
cytochrome b
deduced from that of the petB gene (52) (Fig. 1, lane 7", and data not
shown). 4) The 12.5-kDa band did not yield any N-terminal sequence
either. This band was labeled by two antisera (Fig. 1, lane
8", and data not shown), raised respectively against the
N-terminal and C-terminal sequences predicted from the petD gene, which encodes subunit IV(52) .
We have shown
previously that the 4-kDa band(s) represent(s) more than one
polypeptide ((16) ; see also (15) ). The presence of
the product of the petG gene was inferred from labeling with
an antipeptide directed against the C-terminal part of the predicted
PetG protein. The presence of a novel subunit, product of an
unidentified nuclear gene provisionally named petX, was
established by N-terminal sequencing and pulse labeling in the presence
of various inhibitors of protein synthesis ( (15) and (16) ; Table 2). Immunolabeling shows that PetX and PetG
comigrate during SDS/urea-PAGE and migrate close one to another in
three-step gels in the absence of urea(16) . The complete
sequence of PetX has recently been established by further protein
sequencing and by cloning and sequencing of the petX gene(17) . Finally, the presence of a third
4-kDa
subunit has been recently established by deletion of the chloroplast ycf7 (petL) gene and immunoblot analysis of purified b
f preparations obtained from wild-type
and ycf7- strains(47) .
Immunoblotting of
SDS-PAGE gels shows that all of these seven proteins are absent in
thylakoid membranes prepared from mutant FuD4, that lacks the bf complex (Fig. 1, lanes
5`-8`; (16) and Footnote 1).
Figure 5:
Subunit stoichiometry in C-labeled b
f complex. A, autoradiogram of purified
C-labeled b
f complex
following urea/SDS-PAGE. B, relative radioactivity associated
with each subunit or group of subunits; the radioactivity in each band
was quantified using PhosphorImager plates, divided by the number of
carbon atoms contained in the subunit(s) present in the band and
normalized to the average activity per carbon atom in the complex (see
``Materials and Methods''). Panel shows average ± S.D.
of 10 measurements on two different preparations. C, a scan
through the middle of the lane shown in panel A; note that
because of the variable width and broadness of the bands, peak heights
and areas are not proportional to relative
activities.
In this latter respect,
Hecameg (HG) appears as an economically interesting substitute to the
much more expensive octyl--D-glucopyranoside (OG) for
membrane protein purification. HG has been developed by Plusquellec and
co-workers(19) . Its chemical structure, molecular mass,
aggregation number, and CMC are similar to those of
OG(19, 44, 45) , and it is likely to be able
to substitute for it under many circumstances. HG has been hitherto
applied to the purification of mycoplasma surface
antigens(19, 59) , to the solubilization of the
sarcoplasmic Ca
-ATPase(45) , and to the
crystallization of beef heart cytochrome bc
(60) . Proteins that are sensitive to OG
are not good candidates for purification with HG, as indicated by
preliminary experiments with Ca
-ATPase (45) (
)and with the nicotinic acetylcholine
receptor. (
)C. reinhardtii b
f complex, on the other hand, is solubilized by HG somewhat more
specifically than by OG, and purified preparations in HG are stable for
weeks provided lipids are also present.
Anaerobic redox titrations were performed on isolated
cytochrome bf complex in order to
determine the redox midpoint potential of the components. Cytochrome b
and cytochrome b
were found
to have E
values of -158 and
-80 mV, respectively, similar to those found in spinach
cytochrome b
f in thylakoids at pH
8.3(65) . Cytochrome f was found to have a slightly
lower E
(approximately +340 mV) than reported
for other systems (+350 to +370 mV; (1) and (65) ).
We
show elsewhere that the purified complex is a dimer, whose
experimentally determined mass matches well that expected from its
protein and detergent composition(43) . Equilibrium C labeling is compatible with each subunit being present
as two copies per b
f dimer. The accuracy
of the data, however, does not permit exclusion of the possibility that
one or the other of the 4-kDa subunits is present as a single copy per
dimer.
Lemaire et al.(11) noted that a 19.5-kDa
polypeptide, which was present in partially purified preparations of C. reinhardtii bf complex, was missing
from the thylakoid membranes of b
f-less
strains. They considered it an additional b
f subunit, which they called ``subunit V.'' Other authors (1, 8, 14) subsequently have called
``subunit 5'' or ``subunit V'' the product of the petG gene (initially called petE) identified in maize b
f preparations(14) . In order
to avoid confusion while retaining some similarity with the former
denomination, we will designate the 19.5-kDa protein hereafter by the
italic letter v. Our results show that protein v is
absent in purified b
f preparations that
are highly active in electron transfer. This absence was confirmed by
immunodetection using an antiserum raised against a synthetic peptide
with the sequence of the N terminus of protein v. (
)From this point of view, protein v does not seem
to be an essential component of C. reinhardtii b
f complex. On the other hand, the earlier observations
indicate that the accumulation of protein v in thylakoid
membranes somehow depends on the presence of cytochrome b
f. One simple explanation would be
that, in situ, protein v physically associates with
the b
f complex. The absence of protein v in b
f-less mutants then might
result from its more rapid degradation when not bound to its proper
site. While protein v clearly is not required for the b
f efficiently to transfer electrons and
is not strongly bound to the complex, it cannot be excluded that it
carries out an unidentified function associated with it. Further
characterization of protein v is in progress in our
laboratory.