Báo cáo khoa học: Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides pptx

Role of the N- and C-terminal regions of the PufX protein
in the structural organization of the photosynthetic core
complex of
Rhodobacter sphaeroides
Francesco Francia
1,2
, Jun Wang
1,
*, Hans Zischka
1,
†, Giovanni Venturoli
2
and Dieter Oesterhelt
1
1
Department of Membrane Biochemistry Max-Planck-Institute for Biochemistry, Martinsried, Germany;
2
Department of Biology,
Laboratory of Biochemistry and Biophysics, University of Bologna, Italy
The core complex of Rhod obacter sphaeroides is formed by
the a ssociation o f the light-harvesting antenna 1 (LH1) and
the reaction center (RC). The P ufX protein is essential for
photosynthetic gr owth; i t i s located within the core in a 1 : 1
stoichiometry with the RC. PufX is required for a fast
ubiquinol exchange between the Q
B
site of the RC and the
Qo site of the cytochrome bc
1
complex. In vivo the LH1–
PufX–RC complex is assembled in a dimeric form, where

PufX is involved as a structural organizer. We have modified
the PufX protein at the N and the C-terminus with pro-
gressive deletions. T he nine mutants obtained have b een
characterized for th eir ability f or photosynthetic growth, the
insertion of PufX in the core LH1–RC complex, the stability
of the dimers and the kinetics of flash-induced reduction of
cytochrome b
561
of the cytochrome bc
1
complex. Deletion
of 18 residues a t t he N-terminus destabilizes the dimer in vitro
without preventing photo synthetic growth. The dimer (or a
stable dimer) does not seem to be a necessary requisite for
the photosynthetic phenotype. Partial C-terminal d eletions
impede the insertion of PufX, while the complete absence of
the C -terminus leads to t he insertion of a PufX protein
composed of only its first 53 residues and does not affect the
photosynthetic growth of the bacterium. Overall, the results
point to a complex role of the N and C domains in the
structural organization of the core complex; the N-terminus
is suggested to be responsible mainly for d imerization, while
the C-terminus is thought to be involved mainly in PufX
assembly.
Keywords: LH1-RC; photosynthesis; PufX; Rhodobacter
sphaeroides.
The purple b acterium Rhodobacter (Rb.) sphaeroides can
grow photosynthetically or heterotrophically via aerobic or
anaerobic respiration. When growing photosynthetically, it
uses light energy as a driving force to form ATP via a cyclic

electron transfer. Photons are captured from the light-
harvesting (LH) complex(es) and the excitation energy
funnelled towards a bacteriochlorophyll (BChl) special pair
(P), located in the reaction centre (RC). The excited P
delivers an electron via an accessory BChl and a bacterio-
pheophytin molec ule to a primary ubiquinone acceptor
(Q
A
). In a m uch slower reaction the electron is transferred to
a second ubiquinone acceptor (Q
B
). The full reduction of
the quinone m olecule at Q
B
to quinol requires a second
photoexcitation of the RC and is coupled to the uptake of
two protons from the cytoplasmic space. The formed
ubiquinol dissociates from the RC and is released into the
membrane lipid phase [1]. Ubiquinol molecules are oxidized
at the Qo site of the cytochrome bc
1
complex (cyt bc
1
). Here
the electron pathway branches into a high and a low
potential chain. The first electron reduces in series an iron
cluster centre and a cytochrome c
1
in the high potential
chain, while the second electron reduces the low poten tial

chain, composed of cytochrome b
566
, cytochrome b
561
and
a ubiquinone molecu le located at the Qi site. A second
ubiquinol oxidized at the Qo site brings the electron to fully
reduce the ubisemiquinone to ubiquinol on Qi. From the
cytochrome c
1
, the electron is transferred to a soluble
cytochrome c
2
that is the physiological electron donor to the
oxidized P. From the Qo site, protons are released into the
periplasmic space of the cell. This cyclic mechanism of redox
reactions acts as a proton pumping system, moving protons
from the cytoplasmic to the periplasmic space. The formed
H
+
gradient is the driving force for synth esis of A TP that is
used to power the metabolic reactions in the cell [2].
In Rb. sphaeroides the ability of the RC to capture light
energy is largely increased by the presence of two LH
complexes: LH1 and LH2. The LH1 complex is intimately
associated with th e RC in a fixed stoichiometry to form the
core complexes (LH1–RC), while the LH2 is arranged
peripherally with respect to the core. Both LHs are
organized in circular supramolecular complexes, resulting
from the repetition of a minimal building block commonly

Correspondence to F. Francia, Department of Biology, Laboratory of
Biochemistry and Bi ophysics, Un iversity of Bologna, Via Irnerio n.42,
40126 Bologna, Italy. Fax: + 39 051 242576,
Tel.: + 39 051 2091300, E-mail:
Abbreviations: BChl, bacteriochlorophyll; cyt bc
1
, cytochrome bc
1
complex; ICM, intracytoplasmic membranes; LH, light-harvesting
complex; PMC, photosynthetic membrane complex; Q
A
,Q
B
, primary/
secondary electron acceptor; Qi, quinone reductase site of the cyt bc
1
complex; Qo, quinol oxidase site of the cyt bc
1
complex; RC, reaction
center.
*Present address: Department of Plant Biology, The Ohio State Uni-
versity, Columbus, OH, USA.
Present address: GSF Forschungszentrum, Institut fu
¨
r Humangene-
tik, Oberschleißheim, Germany.
(Received 6 November 2002, revised 12 February 2002, accepted 13
February 2002)
Eur. J. Biochem. 269, 1877–1885 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02834.x
referredtoasthea,b heterodimer. The a and b polypeptides

span the membrane with a single hydrophobic a helix. This
circular protein scaffold binds the pigments that are
maintained in a spatial orientation that maximizes the
efficiency of the energy transfer reactions.
Structures of the LH2 [3,4] and RC [5], as well as
of cyt bc
1
[6] are known at atomic r esolution but came
from different organisms; on the contrary, high resolu-
tion structural data of the core complex are not yet
available.
In Rb. sphaeroides and Rb. capsulatus photosynthetic
growth requires the presence of the PufX protein [7,8].
When an intact LH1–RC core complex is present, PufX is
essential to p romote an efficient ubiquinone/ubiquinol
exchange between the RC and cyt bc
1
[9], but is not
necessary when the LH1 system is absent or reduced in size
[10–12]. T his evidence points to a complex structu ral
relationship between the components of the photosynthetic
system, in which PufX plays a central role [13]. Recently,
several works have indicated that PufX is involved directly
in the supramolecular organization of the photosystem: (a)
the core complexes of Rb. sphaeroides are organized in a
dimeric form [14], in which the p resence of PufX induces a
specific orientation of the RC inside the LH1 complex as
well as the formation of a long range regular array of LH1–
RC in the photosynthetic membrane [15]; (b) biochemical
studies have shown that PufX is present in the LH1–RC

complex in a 1 : 1 stoichiometry with the RC, and that the
dimeric form of the core complex c ould only be isolated in
the presence of PufX [16]; (c) the PufX protein has a strong
tendency to i nteract with the LH1 a polypeptide, while no
interaction was detected with the LH1 b polypeptide [17];
(d) the deletion of PufX increases t he number o f LH1-
associated Bchls per RC, suggesting an increased number of
a,b heterodimers in the LH1 [18]. Moreover in the presence
of PufX, electron density maps of the dimeric LH1–RC
show unequivocally interruptions in the LH1 ring encircling
theRC.ThetopviewoftheLH1–RCcorecomplex
presents two rings of LH1 in close contact forming a pattern
which resembles the shape of the letter S ; each interrupted
ring contains an electron dense nucleus attributed to the RC
[14].
All of these experimental results are consistent with the
idea that PufX is responsible for these interruptions,
allowing a faster lateral diffusion of ubiquinone/ubiquinol
molecules toward/from the RC Q
B
site.
A previous work on Rb. sphaeroides demonstrated the
role of the C-terminal amino-acid residues of the LH1 a
polypeptide in the organization of the LH1–RC complex
[12]. In the present work, we have investigated the
possible involvement of the N-terminus and of the
C-terminus of PufX in protein–protein interactions stabil-
izing the LH1–RC complex. To this aim two sets of
mutant strains have been constructed. The N-terminal
domain has been progressively shortened by deletions

extending from the second residue of the primary
sequence, while the C-terminal portion has been progres-
sively shortened by introducing stop codons by site-
directed mutagenesis.
We have obtained information on the involvement of the
N- and C-terminal portions of PufX in its insertion in the
membrane and dimerization of the core complexes.
EXPERIMENTAL PROCEDURES
Bacterial strains, plasmid, gene transfer, growth
conditions, membrane preparations
Bacterial strains and the plasmid used in this w ork were as
described previously [16]. Growth conditions for Escherichia
coli and Rb. sphaeroides have also been described [16]. All o f
the Rb. sphaeroides strains were grown semi-aerobically.
Photoheterotrophic growth tests in liquid culture were
monitored with a Klett–Summerson colorimeter as
described by Farchaus et al. [7]; kanamycin and tetracycline
were added at 25 lgÆmL
)1
and 2 lgÆmL
)1
, respectively.
Cultures were illuminated by two 120 W incandescent light
bulbs; excessive warming was prevented by placing a 40-cm
water bath between the lamps an d the cultures. Intra-
cytoplasmic membranes (ICM) were prepared as described
previously [19].
PufX mutagenesis
The PufX N-terminal deletion and C-termin al stop codon
series were constructed using the pRKX plasmid [16] as

DNA template a nd introducing the desired mutation/
deletion with the method given by Ausbel et al. [20]. The
external primers used for the cited sequential PCR muta-
genesis anneal, respectively, 330 bp upstream and 441 bp
downstream the pufX gene on the plasmid. This fragment
contains the Hin dIII and ClaI (8 bp upstream and 171 bp
downstream the pufX gene, respectively) unique sites and
allows, after digestion with HindIII and ClaI, the ligation of
the final PCR product containing the mutated pufX gene
with the pRKX vector digested with the same restriction
enzymes. After transformations of E. coli S17-1 cells, single
colonies of the putative t ransformants were grown over-
night in 5 mL Luria–Bertani media with 10 l
M
tetracycline.
The p RKX derived harbouring mutated pufX gene co n-
structs f rom t he E. coli S17-1 cells were introduced into
Rb. sphaeroides DQ x/g cells by conjugation [16]. Muta-
tions were confirmed by sequencing the plasmids isolated
from transformed Rb. s phaeroides cells with the Qiagen
Minikit.
Isolation of core complexes and SDS/PAGE
Core complexes were extracted from (ICM) according to
the method described previously [16,21] except that the
NaBr washing step was performed a t 0.6 mgÆmL
)1
total
protein.
The concentration of LH1–RC complex in the isolated
bands was estimated on the basis of the total photooxid-

izable RC measured by flash kinetic spectrophotometry as
described before [16]. Aliquots containing the same number
of LH1–RC moles were treated with 10 vol. cold acetone/
methanol (7 : 2, v/v), vortexed for 2 min and centrifuged.
The organic phase was discarded, and the protein pellet was
dried at 40 °C for 30 min. The pellets were redissolved in
SDS/PAGE loading buffer to final concentration of 2 l
M
LH1–RC in all of the samples.
SDS/PAGE was carried out accordingly to Scha
¨
gger &
Von Jagow [22], with a separating gel o f 19.5% (w/v)
acrylamide, 0.5% (w/v) bis-acrylamide.
1878 F. Francia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
MS and sequencing of PufX54* protein
Proteins were separated by SDS/PAGE as described above,
and stained with Coomassie G250.
Gels were washed extensively with H
2
Otoremove
residual acid from the destaining process. The b and of
interest was cut out with a razor blade and transferred to
reaction tubes. Proteins were then subjected to a limited
protease treatment overnight (0.5–1 lgÆband
)1
endopro-
teinase LysC, Roche Molecular Biochemicals) [23].
Peptides were extracted from gel slices by altered
incubation with 10% formic acid and acetonitrile. Pooled

fractions were dried i n a speed vac concentrato r. D ried
peptides were re-dissolved in 10 lL 10% acetonitrile/0.1%
trifluoroacetic acid. Between 0.5 and 1 lLwereusedfor
MALDI-TOF a nalysis (adapted from [2 4]).
The r esidual sample was applied to a reversed-phase
HPLC system to separate peptides. Purified peptides were
subjected to automated Edman degradation (with kind
support of J. Kellermann, Max Planck Institute for
Biochemistry, Martinsried).
Time resolved spectroscopy on ICM
The kinetics o f cytochrome b
561
reduction induced by a
single actinic flash were measured under the following
conditions: ICM were resuspended in a buffer composed of
50 m
M
Mops, 100 m
M
KCl, pH 7.0; valinomycin and
nigericin were added at 10 l
M
to collapse the transmem-
brane proton gradient and to avoid spectral interference du e
to BChl and carotenoid electrochromic effects; 5 l
M
antimycin A was used to inhibit the Qi site of the cyt bc
1
.
Measurements were performed in a nitrogen atmosphere

under controlled redox conditions as described by Venturoli
et al. [25]. One micromolar each of phenazine methosulfate
and phenazine ethosulfate; 2 l
M
of 2,3,5,6-tetra-methyl-p-
phenylenediamine; 10 l
M
each of p-benzoquinone, duro-
quinone, 1,2-naphthoquinone, 1 ,4-naphthoquinone were
used as redox mediators. The experimental apparatus i s as
described in Francia et al. [16].
Traces of cytochrome b
561
reduction (Fig. 3) were ana-
lysed numerically in terms o f pseudo fir st-order kinetics
following an initial lag period, as described by Barz et al. [9].
In order to determine the best fitting parameters, the lag
period following the time of the flash was varied stepwise:
for each lag period the amplitude and r ate constant of the
exponential function were optimized using a nonlinear v
2
minimization routine [26] an d a plot of the minimized v
2
vs.
the lag period was constructed for each kinetic trace. For all
traces this procedure yielded a minimum reduced v
2
(v
2
min

)
between 0.8 and 1.2. The confidence interval in the
determined value of the lag was obtained by using an
F-statistic to determine the probability p of a particular
fractional increase in v
2
according to:
v
2
=v
2
min
¼ 1 þ½m=ðn À mÞ Fðm; n À m; 1 À pÞ
where m is the number of parameters, n is the number of
data points, and F is the upper (1–p) quantile for Fisher’s
F distributions with m and (n–m) d egrees of free dom [27].
Confidence intervals within 1 SD (P ¼ 0.68) calculated by
this procedure are given in Table 2. These intervals are
generally asymmetrical, due to the nonlinear nature of
deconvolution.
RESULTS
Construction of the PufX N-terminally deleted series
and C-terminally truncated series
To obtain the strains with the mutated PufX protein
reported in Table 1, two sets of plasmids were constructed.
The first series consists of a progressive deletion at the
N-terminus, extending from the second residue of the
primary sequence; the second series consists of a progressive
truncation of the C-terminal domain of PufX (Fig. 1)
obtained by the introduction of stop codons in the gene

sequence of pufX.Inallthecases,theRb. sphaeroides host
strain was DQ x/g [10].
The pseudo wild-type strain used in this work was
obtained reintroducing the complete puf operon via the
plasmid pRKX (in trans) into the host Rb. s phaeroides DQ
x/g, deprived of the chromosomal copy of the puf operon.
Table 1. Bacterial strains and plasmids. The plasmid host strain in all
the cases was Rb. sphaeroides DQx/g.
Strain Plasmid name
Wild-type pRKX
N-Terminus series
PufXD2–4 pRKXD2–4
PufXD2–7 pRKXD2–7
PufXD2–19 pRKXD2–19
PufXD2–26 pRKXD2–26
C-Terminus series
PufX54* pRKX54*
PufX68* pRKX68*
PufX72* pRKX72*
PufX76* pRKX76*
PufX81* pRKX81*
PufDX pRKDX
Fig. 1. Nature of the deletions and truncations on PufX. The helix
transmembrane r egion of the Pu fX p rotein , pre dicted w ith the program
PHDHTM
[32], is indicated at the top of the figure and represented as an
empty recta ngle in the primary sequ ences of PufX showed below. The
related Rb. sphaeroides strains are give n on th e lef t.
Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1879
Photosynthetic growth curves

Aliquots of bacteria corresponding t o 1 absorbance unit at
700 n m from precultures grown semi-aerobically in dark-
ness were transferred to 13 m L final volume of fresh media
in 15 mL glass tubes. Air was eliminated from the tubes by
using a vacuum water pump; tubes were then exposed to the
light in a 30 °C chamber. The results of this photosynthetic
assay a re shown in Fig. 2. Mutants of the N -terminally
deleted series (Fig. 2A) exhibit photosynthetic growth with
the exception of the P ufXD
2)26
strain. The curves in Fig. 2A
show a lag phase varying between 15 and 50 h. B y
comparing several independent growth curves for each
mutant (data not shown), it appeared that a similar, large
variability could be observed in any strain including wild-
type (compare Fig. 2A,B). Therefore the observed lag phase
did not show any correlation with the phenotype. Clearly
photosynthetic-negative phenotypes are e videnced by the
PufDX (as already reported previously [28]) and PufXD
2)26
curves. A lso t he C-terminus mutants P ufX76*, PufX72*
and PufX68* exhibit a nonphotosynthetic phenotype,
whereas the mutant with the shortest truncation (PufX81*)
is photosynthetically competent (Fig. 2B). Surprisingly, the
most extended truncation (mutant PufX54*) does not affect
the ability of photosynthetic growth.
Kinetics of cytochrome
b
561
reduction induced

by a single-turnover flash on ICM
The rate of electron transfer through the Qo site of cyt bc
1
can be measured in ICM by monitoring the reduction of the
cytochrome b
561
induced by a short actinic light fl ash in the
presence of the inhibitor a ntimycin A [ 29]. Reduction of
the cytochrome b
561
typically shows a lag period prior to the
onset of the reaction at its maximal rate. In wild-type ICM,
the initial rate of this reaction, as well as the lag phase,
depends on the redox state of the ubiquinone pool and on
the ubiquinone/RC stoichiometry [2,25]. For a normal size
of ubiquinone pool (% 25 ubiquinone molecules/RC
)1
),
upon decreasing the ambient redox potential (Eh) from 250
to 100 mV at pH 7.0, the initial rate of cytochrome b
561
reduction increases progressively, while the lag becomes
shorter. This behaviour has been attributed to the increased
availability of prereduc ed ubiquinone molecule s in t he pool
reactingattheQositeofcytbc
1
. K eeping t he Eh high
enough, the only ubiquinol molecule which can react at Qo
and reduce t he cytochrome b
561

is the one released by the
RC following photoexcitation, as the ubiquinone pool is
completely preoxidized [30]. Under this condition the lag
period is maximal, typically 1 m s in wild-type ICM. A
drastic increase of the lag phase, paralleled by a decrease in
the initial reduction rate is observed in pufX-deleted strains
as compared with wild-type [9]. Both of these effects are
maximal at Eh > 180 mV (i.e. when the ubiquinone pool is
fully oxidized) and reflect a dramatic impairment in t he
redox interaction betw een the Q
B
site of the RC and the Qo
site of the cyt bc
1
in the pufX-deleted strain.
We have measured the k inetics of cytochrome b
561
reduction of all the N- and C-terminal PufX mutants (at
Eh 180–220 mV) on ICM prepared from cultures grown
semi-aerobically the d ark. It must be pointed out that under
these growth conditions there is no photosynthetic selective
pressure that could induce t he suppression phenomenon
reported in [10]. Kinetic traces recorded from the mutants
PufXD
2)26
and PufX54* are shown in F ig. 3. The continuous
curves are best-fits to an exponential function; the lag dura-
tion was d etermined numerically as outlined in Experimental
procedures. T he properties of the complete N-terminally
deleted and C-terminally truncated series are liste d in

Table 2 . While the lag period of PufX54* is comparable to
that measured in a typical wild-type, the PufX68*, PufX72*,
PufX76* as well as the PufXD
2)26
exhibit an increased lag
period usually observed in the pufX-deleted strain.
The Pu fXD
2)4
,PufXD
2)7
and PufX81* show a lag period
like that of wild-type, indicating that a short deletion a t the
N- and C-terminus does not affect this parameter. Measure-
ments carried out on PufX D
2)19
revealed an intermediate lag
duration (see Table 2), making ambiguous the attribution of
the PufXD
2)19
strain to the wild-type or the PufDXcluster.
Isolation of the photosynthetic complexes
from the mutant strains
As described previously [16], the photosynthetic complexes
(PMCs) could be extracted by detergent solubilization from
Fig. 2. Growth curves of the control and mutated PufX strains under
photosynthetic conditions. The growth of the coltures was monitered by
a Klett–Summerson colorimeter. (A) Wild-type (j), PufXD2–4 ( s),
PufXD2–7 (n), PufXD2–19 (,), PufDX(h); the growth curve of
PufXD2–26 (not shown in the figure for visual clarity) coincides with
that of PufDX. (B) Wild-type (j), PufX54*(s), PufX68* (n),

PufX72* (,), PufX81* ( e); the growth curve of Pu fX 76* (not shown)
coincides with those of PufX68* and PufX72*, i.e. reveals inability of
photosynthetic growth.
1880 F. Francia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the membranes and purified by centrifugation in continuous
sucrose density gradients. Briefly, the final wild-type pattern
consists of four bands, named PMC1, PMC2, PMC3 a nd
PMC4 from top to bottom of the tubes (Fig. 4, tube 1).
PMC1, PMC2, PMC3 and PMC4 r epresent LH2, LH1
Ôempty ringsÕ, LH1–RC monomers and LH1–RC dimers,
respectively. A consequence o f the deletion of the pufX gene
is the lack of LH1–RC dimer bands in the gradients (see
Fig. 4A, tube 6).
Fig. 4 s hows the patter ns of the N -terminally deleted
(Fig. 4 A) and C-terminally truncated (Fig. 4B) series.
PufXD
2)4
and the PufX81* (the shortest deletion and
truncation, respectively) mutants exhibit the wild-type like
pattern, with all four bands present. In the mutant
PufXD
2)7
, a very faint band in the correct position of
PMC4 could be seen in the original photograph, whereas in
the other N-terminally deleted strains, PufX D
2)19
and
PufXD
2)26
PMC4 is undetectable. Also in the mutants

PufX68*, PufX72* and PufX76* PMC4 is not seen;
interestingly a fourth band not clearly separated from
PMC3, with a position intermediate between those of
PMC3 and PMC4 (between the LH1–RC monomer and the
dimer), is present in the PufX54* gradient profile (Fig. 4B,
tube 2).
SDS/PAGE of the isolated LH1–RC complex
The RC : PufX stoichiometry in i solated LH1–RC com-
plexes is 1 : 1 in t he wild-type strain. The same unitary
stoichiometry was determined in the monomeric (PMC3)
and dimeric (PMC4) core complex bands [16]. The presence
of the mutated PufX can therefore be assessed in PMC3
isolated from the mutants. Fig. 5A shows an S DS/PAGE of
isolated PMC3 from the N-terminally deleted s eries. Only
the small molecular weight r egion is s hown. In Fig. 5A,
lanes 2 and 3, corresponding to PMC3 from PufXD
2)4
and
PufXD
2)7
, respectively, above the dominant LH1 a and b
bands, a band attributable to the mutated PufX is clearly
visible. In lane 4 a faint band, attributable to PufXD
2)19
is
indicated by an arrow, while no band, except those of LH1
a and b, can be seen in the mutant P ufXD
2)26
and in the
PufDXstrain.

Table 2. Summary of experimental data.
Strain
Light
growth
Cytochrome b
561
reduction lag (ms)
a
Sucrose gradient-
isolated PMC
Detection of
PufX in PMC3
b
Wild-type Yes 0.7 (0.2–0.8) PMC
3
,PMC
4
Yes
PufXD2-4 Yes 1.1 (0.9–1.3) PMC
3
,PMC
4
Yes
PufXD2-7 Yes 0.5 (0.0–1.0) PMC
3
(PMC
4
)
c
Yes

PufXD2-19 Yes 3.8 (3.5–4.2) PMC
3
Yes
PufXD2-26 No 5.3 (4.3–5.8) PMC
3
No
PufX54* Yes 1.2 (0.5–1.4) PMC
3
,PMC
3/4
Yes
PufX68* No 10.7 (9.5–11.1) PMC
3
No
PufX72* No 6.3 (6.2–6.8) PMC
3
No
PufX76* No 4.7 (4.0–5.8) PMC
3
No
PufX81* Yes 0.7 (0.5–1.0) PMC
3
,PMC
4
Yes
PufDX No 8.8 (8.8–9.1) PMC
3
No
a
The confidence interval within 1 SD is given in parentheses.

b
Detected by SDS/PAGE on sucrose gradient-isolated PMC 3.
c
Detectable as
a weak band in the original photograph.
Fig. 3. Cytochrome b
561
reduction kinetics induced by single flash pho-
toexcitation in ICM. The continuous vertical line indicates the instant
when the actinic flash pulse was fired (time ¼ 0), the dotted vertical
line marks the beginning of t he cytochrome b
561
reduction at its
maximal rat e. Th e t ime i nter val b etween the continuous a nd the dotted
vertical lines corresponds to th e l ag p hase of the reduction kinetics. Lag
duration was evaluated by a numerical procedure as outlined under
experimental procedures. The experimental t race is represented b y a
continuous lin e connec ting the points sampled by the rec ording
apparatus; the best-fitting mono-exponen tial functio n is indicated by a
continuous cu rve. (A) ICM from strain PufXD2-26; (B) ICM from
strain PufX54*.
Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1881
In Fig. 5B, data for the C-terminally truncated series are
shown. The presence of PufX81* is evident in lane 2, while
no PufX band was observed in the mutants PufX76*,
PufX72* and PufX68* (lanes 3, 4, 5, respectively).
AthinbandveryclosetotheLH1a band, indicated by
the arrow, is apparent in the PufX54* mutant (lane 6 ).
This band was excised from the gel after SDS/PAGE and
the protein was identified by using the method described in

the Experimental procedures. Briefly, the band was
digested wi th 0.5 lg of the proteolytic enzyme e ndopro-
teinase Lys-C and parts of the resulting peptide mixture
were analysed by MS (MALDI-TOF). The peptide mass
fingerprint obtained corresponded to fragments 17–29
(TNLRLWVAFQMMK) and 5–16 (TIFNDHLNTNPK)
of the PufX p rotein. The identity of PufX in the band
isolated was examined further by subjecting part of t he
peptide mixture to separation by reversed-phase HPLC.
Purified p eptides w ere subjected to sequence analysis b y
Edman degradation. The sequence KTIFNDHLNTN,
corresponding to the 4–14 fragment of PufX, was identi-
fied.
DISCUSSION
Effects of N-terminal and C-terminal PufX deletion
on LH1–RC dimerization
In the LH1–PufX–RC core complex o f Rh. sphaeroides,the
RC : PufX stoichiometry is 1 : 1 [16]. The role o f PufX as a
structural organizer of the core complex has been discussed
recently in several works (rewiewed in [13]). Data on the
sequence of assembly of the LH1–PufX–RC complex in vivo
[31] and on protein–protein interactions between the single
polypeptides o f the complex [17] are consistent with the
hypothesis t hat PufX interrupts the continuity of the LH1
ring and switches the structure of the complex from a
Ôclosed Õ monomeric form to an ÔopenÕ dimeric form.
Moreover, linear dicroism studies have demonstrated the
role of PufX in the orientation of the RC inside the LH1
Fig. 4. Isolation of the PMCs on a sucrose gradient. The final detergent
extracts from the ICM were loaded on the top of a 10–40% sucrose

gradient a nd centrifuged for 1 9 h at 23 0 · 10
3
g. T he gradient was
buffered with 50 m
M
Na-glycylglycine to pH 7.8, the detergents octyl-
glucoside and Na-cholate were added to the gradien t at a final con-
centration of 0.6% and 0.2% (w/v), respectively. (A) T ube 1, wild-type;
tube 2, PufXD2–4;tube3,PufXD2–7; tube 4, PufXD2–19; tube 5,
PufXD2–26; tube 6, PufDX. (B) Tube 1, wild-type; tube 2, PufX54*;
tube 3, PufX68*; tube 4, PufX72*; tube 5, PufX76*; tube 6, PufX81*.
Fig. 5. SDS/PAGE on sucrose g radient-isolated core complexes. The
proteins o f the PMC3 bands isolated from the sucrose gradients (see
Fig. 4) were sub jected to SDS/PAGE accordin g to Scha
¨
gger & Von
Jagow [22]. The concentrations of acrylamide and bis-acrylamide were
19.5% and 0.5% (w/v), respectively, in the separating gel and 3.9%
and 0.1% in the stacking gel. For each lane, 24 pmol PMC3, corre-
sponding to the monomeric form of the core complex, was loaded.
Only the region of low molecular mass proteins is shown i n the figure.
(A)Lane1,wild-type;lane2,PufXD2–4; lane 3, PufXD2–7; lane 4,
PufXD2–19; lane 5, PufXD2–26; lane 6, PufDX. (B) Lane 1, wild-type;
lane 2, PufX81*; lane 3, PufX76*; lane 4, PufX72*; lane 5, PufX68*;
lane 6, PufX54*; la ne 7, Puf DX. The position of t he faint band
attributed to the PufXD2–19 protein is indicated by an arrow in lane
4 A, th e position of t he detec ted PufX54* is ind icated by a n arrow in
lane 6 B.
1882 F. Francia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
[15]. These results indicate that the PufX protein is in

contact with the LH1 and the RC subunits inside the core
complexes.
When secon dary structure prediction was p erformed on
PufX [32] the final output revealed a strong tendency to
build ahelices at both t he N- and C-termini [33] and a
transmembrane a helix in the central region (Fig. 1 ). On this
basis, and in view of the finding that the C-terminal part of
the LH1 a polypeptide plays an important role in the
structure of the core complex [12], we decided to investigate
the possible structural role of the N-terminus and the
C-terminus of PufX. To this aim, nine strains of
Rb. sphaeroides with mutated PufX were constructed.
The dimeric form of the core complex purified from ICM
[16] has been confirmed by electron microscopy [14]. We
consider, therefore, the pr esence of the d imeric form
(PMC4) upon isolation as an indication for dimerization
in v ivo. The shortest deletion in the PufXD
2)4
and the
shortest truncation in PufX81* do not impair the ability of
PufX to facilitate dimerization, as a clear PMC4 b and can be
detected in the gradient (Fig. 4). Interestingly in the gradient
of the N -terminus mutant PufX D
2)7
averyfaintPMC4
band is visible in the original gradient photograph (unde-
tectable in Fig. 4). Apparently this deletion strongly desta-
bilizes the dimer to th e extent that it cannot withstand fully
the m embrane d etergent extraction. The p resence o f t he
dimer in vivo in the mutant PufXD

2)7
and presumably in
PufXD
2)19
is therefore not excluded. We have shown
previously that in vitro an irreversible dissociation of the
dimeric to the monomeric form of the complex from the
wild-type exists: the dimer dissociates gradually into
the monomer when the octyl-glucoside concentration is
increased from 0.6 to 1.2% [16]. This result suggested that
hydrophobic interactions are involved in maintaining the
dimeric form. The data obtained on t he PufXD
2)7
and
PufXD
2)19
strains indicate that important protein–protein
hydrophobic interactions are made by the Pu fX N-terminus.
In the case of the longest N-terminal deletion (strain
PufXD
2)26
), the PufXD
2)26
protein i s not detectable in the
core complex (see below and Fig. 5A, lane 5). Correspond-
ingly only the monomeric form of the complex can be seen
in the gradient (Fig. 4A, tube 5).
Two main points of interest a rise from the results
obtained from the C-terminal truncation series. First, three
mutants (characterized by a nonphotosynthetic phenotype),

PufX76*, PufX72* and PufX68* show no dimers of the
isolated core complex, whereas from the P ufX54* s train a
fourth band, with different sedimentation characteristics on
sucrose gradients, has been isolated. In the following we
refer to this band, located in an intermediate position
between t he monomer (PMC3) and the dimer (PMC4), as
PMC3/4. We propose three alternative interpretations: (a)
PMC3/4 represents a dimeric form in which the LH1 rings
assume a d ifferent curvature, leading t o a different sedi-
mentation coefficient; (b) PMC3/4 is formed by two LH1
rings that lost one or two r eaction centers; (c) when t he
C-terminal part of PufX is deleted the equilibrium between
the monomer and the dimer is not attained during
sedimentation.
The second interesting point is that PufX76*, PufX72*
and PufX68* mutants are photosynthetically incompetent,
whereas the PufX54* mutant grows photosynthetically,
demonstrating that a complete removal of the C-terminus is
tolerated by the cell, while a partial truncation is photosyn-
thetically lethal. The absence of PufX in PufX76*, PufX72*
and PufX68* (Fig. 5B) could in principle either reflect an
impairment in the insertion into the membrane of the
shortened protein and/or in the assembly of PufX in the
LH1–RC, or resides at transcriptional/post-translational
level. The PufX54* protein possesses only the N-terminus
and the hydrophobic transmembrane h elix, whereas the
other mutants have in addition part of the C-terminus. We
suggest that the presence of a partial C-terminus leads to a
misfolding that impedes the insertion/assembly of PufX in
the membrane complex.

Parkes-Loach et al. [34] have recently reported that
mature forms of PufX extracted from cells of Rb. sphaer-
oides and Rb. capsulatus contains 12 and nine fewer amino
acids, respectively, at the C-terminal end of the protein than
are encoded by their pufX genes. These data are inconsistent
with our previous repo rt [16], where a PufX with a
C-terminal six-histidine tail has been used to determine
the RC : PufX stoichiometry by Western blot analysis with
anti-His6 antibodies. However the genetic background of
the strains used is different: in our studies (present paper and
[16]) both the LH2 and the LH1 antenna systems a re
present, while in the work of P arkes-Loach et al. an LH2
–
,
LH1
–
strain and an LH2
–
strain from Rb. sphaeroides and
from Rb. capsulatus, respectively, have been used to extract
PufX. We can suppose that the discrepancy is related to the
presence of the LH2 which could influence the shortening
processes of the assembled PufX protein.
The exchange of ubiquinone between the RC
and the cyt
bc
1
in the presence of mutated
PufX protein
The role of the PufX protein in facilitating the ubiquinone/

ubiquinol exchange between the Q
B
site of the RC and the
ubiquinone pool has been demonstrated in Rb. sphaeroides
wild-type strains [7,8]. It has been proposed that PufX
facilitates ubiquinone exchange by determining the struc-
tural supramolecular organization o f the LH1–PufX –RC
complex [12].
In this work, PufX has been detected by SDS/PAGE in
core complexes (Fig. 5) isolated from the N -terminus
mutants PufXD
2)4
,PufXD
2)7
,PufXD
2)19
and from the
C-terminus mutants PufX54*, PufX81*. The evidence that
these a re the only mutants which are ph otosynthetically
competent ( see Table 2) is in accordance with previous
results on the requirement of the PufX protein for
photosynthetic growth and suggests that the assembly of
the wild-type or mutated PufX protein in the core complex
is necessary for efficient light energy transduction. In the
other mutants examined, PufXD
2)26
, PufX68*, PufX72*
and PufX76* no PufX protein could b e d etected on SDS/
PAGE after isolation of the complex.
Assaying on ICM the reduction kinetics of the cyto-

chrome b
561
induced by a single actinic flash in the mutants
PufXD
2)4
,PufXD
2)7
, PufX54* and PufX81*, we found a
lag time betwee n the fl ash excitation a nd the onset of
cytochrome b
561
reduction close to that observed in wild-
type ICM. This is indicative of a fast ubiquinone exchange
between the reaction center Q
B
site and the cyt bc
1
Qo site.
In the case of the shortest N-terminal deletion and
C-terminal truncation (PufXD
2-4
and PufX81*, respectively)
Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1883
this result was expected; in these two mutants a d imeric
form of the core complex could be i solated. On the contrary,
we obtained evidence of a less stable dimer in mutan t
PufXD
2)7
and observed a band intermediate between that
of the monomeric and the dimeric form ( see above) in

mutant PufX54*. As in these last two mutants a short lag
was observed (see Table 2), apparently the presence of a
stable dimer is not a n ecessary requisite for a fast RC/bc
1
redox interaction, which is associated with a photosynthetic
phenotype.
As an alternative e xplanation the monomeric a nd the
dimeric form of t he LH1–PufX–RC could both be presen t
in vivo; i n the presenc e of an intact PufX the dimeric form
would prevail, while altered equilibria arising from muta-
tions on PufX could affect the stationary concentration o f
the dimer in the membranes.
In the PufXD
2)19
strain the dimeric form is even more
destabilized, as no PMC4 can be isolated. Measurements of
the lag in cytochrome b
561
reduction in ICM from this
mutant yielded values intermediate between those usually
obtained in t he wild-type and in the P ufDX s train, with
some variability between preparations from different cul-
tures. Considering t hat the same amount of LH1–RC has
been loaded in all lanes of the SDS/PAGE gel in Fig. 5A,
the weaker intensity of the PufXD
2)19
band (lane4) suggests
that the amount of PufX per LH1–RC complex is lower in
this mutant. T here fore it is likely that a mixture of
monomeric LH1–RC with and without PufXD

2)19
is
isolated on the sucrose gradient. The occurrence of a mixed
population of LH1–RC core complex in the ICM would
explain the variability i n the duration of the lag of
cytochrome b
561
reduction kinetics. The presence of the
PufXD
2)26
within the isolated core complex cannot be
excluded in the SDS/PAGE shown in Fig. 5A, due to a
possible overlapping with the a subunit of the LH1
complex. However a significant efficiency of PufXD
2)26
insertion in the core complex seems unlikely, due to the
nonphotosynthetic phenotype of this strain and to the
pronunced lag in the cytochrome b
561
reduction kinetics (see
Table 2 ), systematically found in chromatophores from the
pufX-deleted strain.
Organization of the Q-cycle complexes
The dimeric organization of the LH1–PufX–RC has been
demonstrated directly in the membranes of Rb. sphaeroides
by electron microscopy [14]. In this paper, the authors
tentatively attribute a positive electrondense region in the
two-dimensional projection o f the dimer to cyt bc
1
and

interpret the S-shaped structure of the projection map as a
supercomplex formed by the LH1–RC and cyt bc
1
in a 2 : 1
stoichiometr y.
Some considerations on this point can be made in the
light of our results. The presence in vitro of a less stable
dimer in the mutant PufXD
2)7
neither affects the photo-
synthetic capability of the bacteria nor the efficiency of
exchange of the ubiq uinol molecules between th e RC and
the bc
1
, a s judged from the reduction kinetics of cyto-
chrome b
561
measured in ICM. Also the mutant PufD
2)19
,
in which the dimeric form cannot be detected in the isolated
core complex, exh ibits a p hotosynthetic phenotype. In these
two mutants, t he photosynthetic phenotype suggests the
presence in viv o of an o pen monomeric complex (or a
prevalence of it with respect to the wild-type situation),
consisting of an incomplete single LH1 ring containing one
RC.
The photosynthetic ability in P ufX54* is consistent with
the fast RC/bc
1

ubiquinol exchange observed in ICM; on
the other hand, the structural organization of the core
complexes and/or the possible monomer–dimer equilibrium
seem to be appreciably perturbed a lso in this m utant as
judged from the d ifferent position of t he PMC3/4 band
(Fig. 4 B) after isolation of the photosynthetic complexes on
linear sucrose gradien t. It is possible that the dimer form is
not required as long as a r eorganized core complex can
efficiently shuttle quinones between the RC a nd the bc
1
complex. The PMC3/4 isolated complex stimulates our
interest, and further studies are in progress to understand
the nature of the PufX54* mutant.
In conclusion, our data indicate that both the N- and
C-terminal portions of the PufX protein play a complex role
in organizing the structure of the LH1–RC complex; the
N-terminal region would be responsible mainly for the
formation o f a stable dimer, whereas the C-terminal portion
would be involved mainly in PufX insertion/assembly. The
transmembrane helix region of PufX appears to be sufficient
to allow a fast quinone exchange between the core and the
cytochrome b
561
complex. Interestingly this c onclusion fits
well with the recent work of Parkes-Loach et al. [34],
showing that the interaction between the hydrophobic PufX
region and the LH1-a polypeptide has an inhibitory effect
on the formation of the LH1 complex. This result suggests
that the central core of the PufX protein is responsible of the
break in the continuity of the LH1 ring in vivo [14], allowing

a faster diffusion of the quinone molecules from/toward the
RC Q
B
site.
ACKNOWLEDGEMENTS
We thank B . A. Melandri and P. Turina for fruitful discussions,
C. Wey rauch, U. Schimanko and N. Mele for technical assistance.
F. F. and J . W. were recipients of M .P.I. postdoctoral fe llowships. This
work was supported b y grant PRIN/99, Bioenergetica e t rasporto di
membrana from the Italian MURST and from The Fonds der
Chemischen Industrie.
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