From the Laboratoire de Bioénergétique et
Ingénierie des Protéines (UPR 9036), CNRS, Institut de
Biologie Structurale et Microbiologie, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France and the ¶ Institut für Biochemie,
Medizinische Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
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ABSTRACT |
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The Rieske proteins of two phylogenetically
distant acidophilic organisms, i.e. the proteobacterium
Thiobacillus ferrooxidans and the crenarchaeon
Sulfolobus acidocaldarius, were studied by EPR. Redox
titrations at a range of pH values showed that the Rieske centers of
both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common
pK value of 6.2. This pK value is significantly
more acidic (by almost 2 pH units) than that of Rieske proteins in
neutrophilic species. The orientations of the Rieske center's g
tensors with respect to the plane of the membrane were studied between
pH 4 and 8 using partially ordered samples. At pH 4, the
Sulfolobus Rieske cluster was found in the "typical"
orientation of chemically reduced Rieske centers, whereas this
orientation changed significantly on going toward high pH values. The
Thiobacillus protein, by contrast, appeared to be in the
"standard" orientation at both low and high pH values. The results
are discussed with respect to the molecular parameters conveying acid
resistance and in light of the recently demonstrated long-range
conformational movement of the Rieske protein during enzyme turnover in
cytochrome bc1 complexes.
The cytochrome bc complex is the only energy-conserving
enzyme that is common to photosynthetic and respiratory electron
transport systems. The respective complex has been studied for several
decades in mitochondria and proteobacteria (bc1
complex) (1, 2) as well as in chloroplasts and cyanobacteria
(b6f complex) (3). The functional
core of the enzyme was found to be made up of three subunits containing
two b-type hemes (bH and bL;
cytochrome b or b6), a c-type heme
(cytochrome c1 or f), and a
[2Fe-2S] cluster called the Rieske center.
Recently, crystal structures of mitochondrial cytochrome
bc1 complexes from various sources were reported
(4-6). These structures found the soluble domain of the Rieske protein
in substantially differing positions, suggesting conformational
flexibility of this subunit within the complex. The conformational
flexibility seen in the structures together with independent evidence
(7, 8)1 indicated a
long-range conformational movement of the extramembrane domain of the
Rieske protein as an essential step in enzyme turnover. This movement
appears to swing the [2Fe-2S] cluster from a position close to heme
bL toward another one close to heme c1,
i.e. promoting electron transfer from the quinone in the
Qp site2 to
cytochrome c1. Such a domain movement represents
a hitherto unknown mechanism for bridging long electron transfer
distances between redox centers.
Cytochrome bc-type complexes appear to be spread over the
entire phylogenetic tree of bacteria since, in addition to proteo- and
cyanobacteria, the enzyme was found to be present in green sulfur
bacteria (10-12), in green filamentous bacteria (13), in Deinococci
(14) and in Firmicutes (15-19). The Rieske proteins in the cytochrome
bc complexes from all the above-mentioned species are rather
similar with respect to EPR spectroscopic and redox properties despite
the fact that significant variability is observed concerning the
remaining subunits of the complex. In all cases, the Rieske cluster is
characterized by a typical gav = 1.91 EPR spectrum;
sensitivity of the spectral features (especially in the region of
gx) to the presence of inhibitors and the redox state of the
quinone (3, 16-18, 20); and a dependence of redox midpoint potential
on pH, indicating pK values of ~8 and 9-10 on
redox-linked deprotonatable groups in the oxidized form of the cluster
(12, 17, 21-23).
In addition to the mentioned eubacterial species, Rieske proteins have
been found in the thermo- and acidophilic archaeon Sulfolobus
acidocaldarius. In Sulfolobus, cytochrome b
and a Rieske protein are present in a supercomplex with components of a
cytochrome oxidase-related enzyme (24). The Rieske protein contained in this fused cytochrome bc-cytochrome oxidase supercomplex has
subsequently been characterized in detail (purification, sequence, and
Em versus pH) (25). Intriguingly, the
Sulfolobus Rieske cluster falls out of the pattern of
characteristics observed on the cluster in bacteria. The EPR spectrum
was reported to be insensitive to the presence of inhibitors; its redox
potential does not correspond to those of the other mentioned systems;
and the pK values of the
Em-versus-pH curve are shifted by almost
2 pH units toward the acidic region. This raises the question of
whether the unusual properties of the Sulfolobus Rieske
protein are due to the large phylogenetic distance of its parent
organism (perhaps indicating a significantly different mode of
functioning of the whole enzyme) or whether all or part of these
particularities represent adaptation to the acidophilic growth
conditions. Furthermore, since a cytochrome c subunit
appears to be absent in Sulfolobus (25), the structural
organization of the Rieske protein in the complex may well differ from
that of typical cytochrome bc1 or b6f complexes.
To address the ensemble of these questions, we have carried out a
comparative study of the Rieske protein's electrochemical and
structural characteristics in two phylogenetically distant acidophilic
species, i.e. the crenarchaeon S.
acidocaldarius and the proteobacterium Thiobacillus
ferrooxidans. Both organisms strive at pH values of ~2 and use
molecular oxygen as a terminal electron acceptor in (otherwise rather
different) respiratory chains (for reviews, see Refs. 25 and 26). The
presence of a cytochrome bc complex in T. ferrooxidans has recently been demonstrated (27), and the enzyme
has been characterized in detail in the accompanying article (50).
T. ferrooxidans belongs to the Bacterial growth and isolation of membrane fragments from
T. ferrooxidans were carried out according to Elbehti and
Lemesle-Meunier (27). S. acidocaldarius (DSM 639) was grown
and harvested, and the membranes were prepared as described (29).
To obtain partially ordered membrane multilayers at various pH values,
membrane fragments from T. ferrooxidans and S. acidocaldarius were first washed in 50 mM sodium
acetate (pH 4), 2 mM EDTA, and 5 mM ascorbate
or in 50 mM Tricine (pH 8), 2 mM EDTA, and 5 mM ascorbate. The pellet was subsequently resuspended in
unbuffered water at pH 4, 7, or 8 and resedimented by
ultracentrifugation for 1 h at 300, 000 × g.
Oriented membrane multilayers were obtained as described by Rutherford
and Sétif (30). The membranes were resuspended in unbuffered
water at pH 4, 7, or 8; applied to sheets of Mylar; and dried in a
humidity-controlled atmosphere, under argon, for ~72 h at
4 °C.
Redox titrations of the Rieske cluster from T. ferrooxidans
were performed at 15 °C as described by Dutton (31) at several pH
values (between pH 4 and 8.1) using 50 mM sodium acetate,
30 mM MES, 30 mM MOPS, 10 mM
potassium phosphate, and 30 mM Tricine for titrating at pH
4, 6, 6.7, 7.4, and 8.1, respectively. pH values were controlled at the
beginning and end of each redox titration. The following redox
mediators were used: benzoquinone, potassium ferricyanide,
ferrocenemonocarboxylic acid, and 1,1'-ferrocenedicarboxylic acid (all
at 100 µM). Reductive titrations were carried out using sodium dithionite, and oxidative titrations were done using potassium hexachloroiridate. No hysteresis was observed.
EPR spectra were recorded at liquid helium temperatures with a Bruker
ESP 300e X-band spectrometer fitted with an Oxford Instruments cryostat
and temperature control system. All chemicals used were reagent-grade.
Fig. 1 shows a comparison of EPR
spectra obtained at neutral pH on membrane fragments from T. ferrooxidans and S. acidocaldarius in the
ascorbate-reduced state. Both spectra were characteristic for the
[2Fe-2S] Rieske cluster with its typical gy line at g ~ 1.9 and a broad gx trough in the region of g = 1.80 to 1.70. As shown in the accompanying article (50), the shape and position of
the gx trough of the Thiobacillus Rieske center were
sensitive to the redox state of the quinone and to the presence of
inhibitors, resembling the effects observed on many other Rieske
clusters from cytochrome bc complexes (3, 12). By contrast,
the Sulfolobus Rieske protein appeared to be unaffected by
the presence of the tested inhibitors (data not shown), as already
reported previously (32). As mentioned in the Introduction, a salient
particularity of the Sulfolobus Rieske cluster is the close
to 2 pH units-downshifted pK value of its Em-versus-pH dependence (32). Since
respective data were so far missing for the Thiobacillus
Rieske center, we have studied the pH dependence of the
Thiobacillus protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subgroup of
proteobacteria (28), and its cytochrome bc complex can
therefore be expected to be phylogenetically closely related to the
bc1 complex of purple bacteria, of
Paracoccus, or of mitochondria.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
EPR spectra recorded on ascorbate-reduced
membranes from T. ferrooxidans (spectrum
a) and S. acidocaldarius (spectrum
b) at pH 7. Instrument settings were as follows:
microwave frequency, 9.44 GHz; modulation frequency, 1.6 milliteslas
(mT); temperature, 15 K; and microwave power, 6.3 milliwatts.
Electrochemical Properties of the T. ferrooxidans Rieske
Center--
A series of redox titrations in the region between pH 4 and 8.1 monitoring the intensity of the gy EPR signal of the
Rieske center as a function of ambient redox potential was carried out
on isolated cytoplasmic membranes from T. ferrooxidans. All
titration data obtained could be fitted with simple n = 1 Nernst curves. Fig. 2 shows the redox
midpoint potential as a function of pH. The determined data points
yielded a pK value of 6.2 and an Em value
of +490 mV in the pH-independent region. Above the pK, the
redox potential was seen to decrease with a slope of about 60 mV/pH
unit. This behavior corresponded well (apart from a difference of 90 mV
in absolute values of the redox potential) with that of the
Sulfolobus Rieske cluster (32).
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Orientation Properties of the T. ferrooxidans and S. acidocaldarius Rieske g Tensors-- To obtain structural information, the Rieske clusters from both species were studied in partially oriented membrane multilayers. In general, studies on oriented membrane multilayers are performed at roughly neutral pH values. Due to the acidophilic character of both species, however, orientations were performed at several pH values in the range from pH 4 to 8. Good two-dimensional orientations (as judged by the anisotropy of EPR signals from all observable paramagnetic species in the membrane) were obtained at all pH value with membranes from Sulfolobus and at high pH values with membranes from Thiobacillus. Unexpectedly, at pH ~4, only very mediocre anisotropies were observed upon orienting Thiobacillus membranes. Since this effect was observed systematically in all of several attempts to produce oriented samples from Thiobacillus at low pH, we concluded that this failure arises from intrinsic properties of the Thiobacillus membranes at low pH values (see also "Discussion").
Fig. 3 shows EPR spectra of partially
ordered membrane multilayers from T. ferrooxidans and
S. acidocaldarius at pH 8 (panel a) and pH 4 (panel b) recorded parallel (solid lines) and
perpendicular (dashed lines) to the membrane plane with the
magnetic field. Anisotropic gy and gx signals of both
species could easily be discerned. The gz signal was obscured by a strong signal at g = 2 arising from radical-type paramagnetic species in the membranes.
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Substantial differences were observed both (i) between the low and high
pH experiments in Sulfolobus (cf. Fig. 3,
a versus b) and (ii) between
Sulfolobus and Thiobacillus at high pH values (Fig. 3a). (i) At pH 4, the Sulfolobus gy
signal was slightly more intense when the magnetic field was parallel
to the membrane, whereas at pH 8, a significantly stronger signal was
observed with the field perpendicular to the membrane. (ii) Thiobacillus at pH 8, by contrast, showed a stronger
gy signal parallel to the membrane. These differences indicate
differing conformations of the Rieske proteins as a function of both
species and pH value. The full dependence of the Sulfolobus
Rieske center's gy signal amplitudes versus angle
is shown in the polar plots of Fig.
4a. At pH 4, the polar plot of
the gy direction was characterized by a pronounced maximum
parallel to the membrane and a broad, badly defined maximum close to
90°. The gx trough (Fig. 4b) yielded only a
single, well defined maximum perpendicular to the membrane. gy
and gx directions parallel and perpendicular, respectively, to
the membrane correspond to what has been seen in cytochrome
bc1 complexes from mitochondria (33) and purple
bacteria (34) and in cytochrome bc-type complexes from
Chlorobium limicola (12) and Chloroflexus
aurantiacus.3
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At pH 8, drastically different polar plots for the gy direction of the Sulfolobus Rieske center were obtained (Fig. 4a). The perpendicular component had become largely dominant at pH 8, whereas the parallel component (although probably still present) could not be discerned anymore. The gx direction still showed a single maximum perpendicular to the membrane. It is of note, however, that the relative intensity of the gx trough as compared with the gy line significantly decreased on going from pH 4 to 8 (cf. Fig. 3, b versus a). Since a corresponding decrease was observed for the parallel component of the gy line, the perpendicular gx trough and the parallel gy line must be attributed to the same paramagnetic species. The intensity of the gx trough corresponding to centers pointing their gy direction at steep angles with respect to the membrane was obviously below detection (see "Discussion").
A different behavior was observed for the Thiobacillus Rieske center at pH 8. The polar plots of gy and gx showed clear maxima parallel and perpendicular to the plane of the membrane, respectively, i.e. corresponding to the typical orientation of neutrophilic bc1 complexes measured at neutral pH values.
As stated above, oriented samples from Thiobacillus at pH 4 yielded only slightly anisotropic signals for all paramagnetic centers
present in the membrane. In the best orientations, a weak anisotropy of
the Rieske cluster's gy and gx signals could be
observed. The resulting directions were similar to those observed at pH
8, i.e. gy parallel and gx perpendicular to
the membrane. This indicates that the orientation pattern of the
dominant species in Thiobacillus does not change between pH
4 and 8. The low quality of orientation at pH 4, however, does not
allow us to decide whether conformational heterogeneity is present at
this pH (as observed for the Sulfolobus Rieske protein).
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DISCUSSION |
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Electrochemical Properties
As shown in Fig. 2, the redox midpoint potential of the T. ferrooxidans Rieske center remains independent of pH up to ~6 at Em = +490 mV and decreases with an apparent slope of 60 mV/pH unit above this pK value. Apart from a shift of
90 mV (Em,low pH = +400 mV for
Sulfolobus) (32), the pH dependence of the
Thiobacillus Rieske center thus strongly resembles that of
the protein from Sulfolobus (Fig.
5) (32). In the titration of the isolated
Rieske protein from Sulfolobus, a second pK value
at pH 8.5 (32) has been determined. The examination of the
Thiobacillus protein for the presence of a second
pK value at higher pH was not feasible by experiments
performed on membrane fragments.
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All Rieske centers studied previously in bacteria, mitochondria, and
chloroplasts4 fall into one
of two distinct classes (Fig. 5) with respect to electrochemical
behavior: (a) the ubiquinone/plastoquinone group with an
Em value of about +300 mV and (b) the
menaquinone group with a redox potential of about +120 mV in the
pH-independent regions. In contrast to this difference in absolute
potential, the global shape of the
Em-versus-pH curve and especially the
pK value of close to 8 appear to be common to all
ubiquinone-, plastoquinone-, and menaquinone-oxidizing Rieske centers
studied so far. In a few selected systems, the presence of a second
pK value at significantly higher pH has been demonstrated
(22, 17, 35) or suggested (12) on the basis of the detailed shape of
the Em-versus-pH curve in the
pH-dependent region, analogous to the results obtained with
the Sulfolobus Rieske protein. These two pK
values have tentatively been attributed (17, 35) to the
N protons on the two histidine ligands to the cluster.
Although this interpretation appears sensible, no experimental
confirmation has been presented so far. Since the two pK
values are roughly similar in ubiquinone/plastoquinone and menaquinone
systems, they apparently are not involved in controlling the absolute
redox midpoint potentials in the different species (in contrast to what had been proposed previously). The respective redox potentials in the
ubiquinone/plastoquinone and menaquinone groups were recently shown to
be predominantly adjusted by the presence or absence of
hydrogen-bonding interactions between a specific amino acid side chain
and an acid-labile sulfur as well as one of the cysteine sulfur ligands
of the [2Fe-2S] cluster (36, 37). Hydrogen-bonding interactions have
previously been proposed as factors strongly influencing
Em values of Rieske-type proteins on the basis of
electron nuclear double resonance and electron spin echo envelope
modulation data (38).
The Rieske centers of T. ferrooxidans and of the archaeon S. acidocaldarius do not belong to either of the two described groups (Fig. 5). Their Em-versus-pH curves showed pK values close to 6.2, i.e. almost 2 pH units lower than that of the above-mentioned species. The strongly similar pH dependences of Rieske proteins in the phylogenetically extremely distant species S. acidocaldarius and T. ferrooxidans dismiss the hypothesis that the low pK in the archaeon could reflect a phenomenon related to the evolution of the Rieske proteins. They rather argue strongly in favor of the downshifted pK being an adaptive response to the acid (pH ~2) solvent environment of the periplasmic Rieske proteins in the two acidophiles. This raises the question of whether (a) the observed pK shift is required to assure the functional mechanism of the enzyme or whether (b) it simply confers stability to the Rieske protein at pH 2.
Is the First pK Value Involved in the Functional
Mechanism?--
At first sight, a functional implication of the
pK value of 8 in the neutrophilic species appears unlikely
since this value is at least 1 pH unit above the highest pH reached
during turnover of the cytochrome bc complexes. This gap
goes up to 4 pH units for the studied acidophilic species
(pK of ~6 versus medium pH of ~2). It is of
note, however, that the reported pK values were determined
under equilibrium conditions. We have observed that the presence of the
inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone in
the Qp site of the plastidic
b6f complex downshifts the
pK value of the Rieske protein's
Em-versus-pK curve by 2 pH
units (39). Such a pK shift makes perfect sense in light of
the x-ray structures of the mitochondrial bc1
complexes, which show that the N proton on
His181 points into the Qp pocket and is
therefore likely to be affected by occupancy of this site. Respective
alterations of pK values, e.g. by the presence of
reduced quinone in the Qp site, can therefore not be
excluded. The dependence of conformational parameters of the Rieske
cluster on pH described in this work further indicates that
protonation/deprotonation reactions probably play a role in the
conformational rearrangement of the Rieske protein during turnover.
However, as discussed below, the deprotonatable groups involved in
domain movement of the Rieske protein are not necessarily identical to
the groups giving rise to the pK values of the
Em-versus-pH curve.
Structural Integrity of the Rieske Proteins at Low pH
Values--
The [2Fe-2S] cluster of the Rieske proteins from
neutrophilic purple bacteria and from chloroplasts (see above) is
irreversibly lost at pH values below
4.5 This instability at low
pH values is a common property of iron-sulfur clusters due to the
presence of the "acid-labile" bridging sulfur atoms. In addition to
coping with the lability of these sulfurs, Rieske proteins from
acidophilic species have to overcome the problem that the histidines
are labile ligands at low pH due to protonation of the -nitrogen of
the imidazole moiety. The breakage of the metal histidine bond at
acidic pH values has been extensively studied in blue copper proteins
(40). To assure the integrity of the [2Fe-2S] cluster in the acidic
region, the respective Rieske proteins need to shield their histidine
ligands from solvent access. An excellent example how this may be
achieved is provided by the blue copper protein rusticyanin from
Thiobacillus (41), which caps the solvent-exposed histidine
ligand to the copper ion by two Trp residues, thereby lowering the
pK values of both N
and N
by
several pH units.6 Placing
the two histidine ligands to the [2Fe-2S] Rieske cluster in a more
hydrophobic environment would correspondingly render the
N
-Fe bond more stable at low pH values, but also
downshift the pK value of the proton on N
.
The observed pK shifts can therefore be considered as being entailed by the stabilization of the imidazole-iron bond.
A major difference between the Sulfolobus Rieske protein and those from neutrophilic eubacterial sources is the insertion of 10 amino acids (compared with the bovine protein) in the case of the SoxF protein and 29 amino acids for the SoxL protein between the two conserved cluster-binding motifs (43). It is tempting to speculate that this stretch folds such that the environment of the histidine ligands is rendered more hydrophobic. The lack of sequence data for the Thiobacillus Rieske protein unfortunately precludes analogous speculations regarding this organism at the present time. Due to the large phylogenetic distance between Sulfolobus and Thiobacillus, we would tend to assume that the strategy employed to achieve acid stability differs between the two species. Detailed structural information on the Thiobacillus and Sulfolobus Rieske proteins can be expected to significantly improve our understanding of the adaptation to acidic conditions.
Orientation
S. acidocaldarius-- As shown in Fig. 4, the orientation of the Sulfolobus Rieske cluster's g tensor with respect to the membrane varies with pH. At low pH values, the majority of Rieske centers point their gy axis roughly parallel to the membrane plane, i.e. similar to the orientation typically observed for neutro- and alkalophilic organisms. A minority of centers, however, were observed with the gy direction at high angles with respect to the membrane. A respective minority fraction was not seen in the cytochrome bc1 complexes from mitochondria (33), purple bacteria (34), and Gram-positive bacteria (17), but was seen in the bc-type complexes from the green sulfur bacterium C. limicola (12) and C. aurantiacus.3 It is of note that in all studies on the orientation of the Rieske cluster published so far, the oriented samples have been prepared at roughly neutral pH, i.e. well below the pK values of the Em-versus-pH curve. At high pH values (pH 8), the orientation dependence of the Sulfolobus Rieske gy line changed substantially, and a sizable fraction of the centers appeared to point their gy direction at rather steep angles with respect to the membrane. The most straightforward interpretation of the observed orientation dependences at low and high pH values (Fig. 4) is to assume that at least two differently oriented populations of Rieske centers coexist in these samples. At pH 4, a large majority of centers pointed gy parallel to the membrane, whereas at pH 8, the population directing gy at high angles to the membrane had substantially increased at the expense of the "standard" orientation. The fact that an intermediate orientation dependence was observed at pH 7 (data not shown) corroborates this hypothesis. Concomitantly with the decrease in the parallel gy component, a decrease in the intensity of the (perpendicular) gx signal was observed. A corresponding new gx trough at low angles with respect to the membrane, however, could not be identified. This is probably due to the fact that (a) parallel components are characterized by a much lower intrinsic signal amplitude than perpendicular components (44) and that (b) the gx trough of this conformation of Rieske centers can be expected to be very broad, as was also observed for the purple bacterial complex (9).
In Sulfolobus, two Rieske proteins with somewhat differing primary structures are present (25). Part of the observed heterogeneity may therefore arise from these two distinct proteins. At least one of those, however, must show pH-dependent changes in the orientation of its g tensor. This raises the question of whether the respective pH effects are a unique feature of Sulfolobus Rieske proteins. We have therefore studied the pH dependence of the orientation properties of the Rieske center in a purple bacterium and have observed the same phenomenon, although upshifted by 2 pH values.7 Thus, the Sulfolobus Rieske protein(s) behave like their purple bacterial counterparts in altering the equilibrium between differing orientations as a function of pH.
The mitochondrial and purple bacterial Rieske proteins were recently discovered to perform a long-range movement in order to transfer an electron from quinol to cytochrome c1. The Sulfolobus bc-type complex lacks a c-type heme subunit and rather seems to be fused to its physiological electron acceptor, i.e. cytochrome oxidase. The lack of the c-type heme subunit thus, in principle, raises the question of whether the Sulfolobus Rieske protein displays differences as compared with its eubacterial counterparts. The data shown above indicate similar properties of the Rieske protein from both the archaeon Sulfolobus and mitochondria. This suggests a comparable functional mechanism involving movement of the Rieske center in Sulfolobus, most probably shuttling the electron directly to the binuclear copper center of the oxidase subunits.
It is of note that the apparent pH correlation of the orientation and redox effects may well be coincidental. Orientation by partial dehydration necessarily occurs in a buffer-free medium (since the presence of buffers at typical concentrations hampers orientation), and slight deviations of pH values from the starting conditions during drying cannot be excluded. It is therefore not possible to determine an exact pK value for the orientation effects.
From an inspection of the recently published structures (5, 6), the
histidine ligands to the [2Fe-2S] cluster are certainly tempting
candidates for controlling the association of the Rieske protein with
the Qp site. Ding et al. (45) have proposed
several possible arrangements and different interactions of the
ubiquinone and ubihydroquinone in the Qp site and the
resulting interactions with the adjacent [2Fe-2S] Rieske cluster. In
particular, the possibility of hydrogen bonding from the -nitrogen
of one or both histidines of the [2Fe-2S] cluster to the carbonyl
oxygen of ubiquinone and the phenoxyl of the ubihydroquinone or other occupants of the Qp site has been pointed out. As mentioned
above (see "Electrochemical Properties"), occupation of the
Qp site by the inhibitor
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone shifts the
pK of the Rieske cluster from 8 to ~6 in spinach
chloroplasts (39), indicating strong interactions of the quinone analog
with the residues responsible for the pH dependence of the [2Fe-2S] cluster.
The pK values of these residues, however, concern the oxidized state of the cluster. From the shape of the Em-versus-pH curve, one must conclude that this pK value is much higher in the reduced state of the [2Fe-2S] cluster and that therefore the respective residues are always protonated when the Rieske center is reduced. The EPR experiments discussed above were performed with the Rieske center in the paramagnetic, i.e. reduced, state. We therefore favor a model assuming that the residues responsible for the pH-dependent orientation properties differ from those affecting the pH dependence of the redox midpoint potential.
In an oriented study performed on the cytochrome b6f complex from spinach chloroplasts, heterogeneous populations of g tensor orientations of the Rieske cluster have already been observed (20). In this work, several possible rationalizations for this phenomenon have been proposed, including structural heterogeneity. Guided by the observation of differing relaxation properties of the clusters giving rise to the differing orientation dependences, however, a purely physical interpretation was favored. In light of the recently observed conformational mobility of the Rieske protein (4-8)1 and the data reported above, we now tend to reinterpret the respective data as in fact arising from conformational heterogeneity, possibly affected by the pH value of the sample. A reexamination of the orientation characteristics of the Rieske protein in the cytochrome b6f complex has therefore been initiated.
T. ferrooxidans-- Despite several efforts, we did not succeed in producing well oriented multilayers from Thiobacillus cytoplasmic membranes at low pH values (pH ~4). At neutral pH values, by contrast, Thiobacillus membranes oriented readily, yielding strongly anisotropic angular dependences of signal amplitudes. We therefore concluded that (a) the failure to obtain oriented samples at low pH arose from sample-inherent properties and that (b) these properties change in a pH-dependent manner, permitting satisfactory orientation at neutral pH values. The existence of permanently charged membrane surfaces at low pH values invoked to explain maintenance of neutral interior pH values even in the resting state of cells (46) is possibly involved in the experimental problems in dehydrating Thiobacillus membranes at acidic pH.
Unexpectedly, at neutral pH (i.e. well above the
physiological ambient pH values of Thiobacillus), the
orientation of the Rieske cluster's gy value was found to be
parallel to the membrane, i.e. the reverse of what was
observed for the Sulfolobus cluster and for the Rieske
centers in the neutrophilic purple bacterium at comparatively high pH
values. Whereas the Sulfolobus Rieske protein with respect
to orientation characteristics therefore behaves like a standard Rieske
protein in other examined organisms (apart from the
acidophilicity-induced pH shifts), the Thiobacillus protein
stands out as an exception. It is tempting to attribute this unique
feature of the Thiobacillus Rieske cluster to the singular
growth conditions of this organism. Ferrous iron as sole source of
electrons does not provide sufficiently reducing equivalents to enter
the chain upstream of the cytochrome bc1
complex. As discussed in the accompanying article (50), this complex is therefore liable to work in reverse gear, possibly entailing
substantial deviations in functional parameters from typical cytochrome
bc1 complexes.
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ACKNOWLEDGEMENT |
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We are grateful to R. Toci (Fermentation Unit, Laboratoire de Chimie Bactérienne, Marseille, France) for growing T. ferrooxidans.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 33-4-91-16-44-86; Fax: 33-4-91-16-45-63; E-mail: lemesle{at}ibsm.cnrs-mrs.fr.
1 M. Brugna, S. Rodgers, A. Schricker, G. Montoya, M. Kazmeier, W. Nitschke, and I. Sinning, submitted for publication.
3 M. Brugna and W. Nitschke, unpublished results.
4 In a recent study of the soluble Rieske fragment from spinach chloroplasts, Zhang et al. (47) observed differing pK values and differing absolute Em values when using optical (pK ~6.5) or EPR (no pH dependence in the neutral region) spectroscopy. The EPR data are consistent with the earlier study on thylakoids (23). Zhang et al. rationalized this discrepancy by invoking temperature-dependent pK and Em values. This explanation appears unlikely to us for the following reasons. (a) None of the other Rieske proteins examined by optical methods at room temperature showed this kind of temperature effect (22, 35, 48, 49). (b) An Em value obtained by CD at room temperature for the b6f complex Rieske protein (42) corresponds well with the EPR titrations and is in conflict with the data reported by Zhang et al. (c) In contrast to what is stated by Zhang et al., a pK of 6.5 is not closer to the value of free histidine than that observed in the other systems. Since the histidine residue serves as ligand to the [2Fe-2S] cluster, the pK value in question corresponds to the second deprotonation reaction. The pK value of this deprotonation in free histidine is far beyond pH 10, and a downshift of this pK value in the Rieske proteins by ~1.5 pH units, to our mind, would require substantial structural differences in the vicinity of the histidine ligands. This is, however, not observed (9). We could not help noticing that the optical titration reported by Zhang et al. was carried out in the absence of redox mediators, and we are therefore presently initiating a reexamination of this pK value in the spinach complex using CD spectroscopy at room temperature.
5 A. Riedel and W. Nitschke, unpublished results.
6 M.-T. Giudici-Orticoni, F. Guerlesquin, M. Bruschi, and W. Nitschke, submitted for publication.
7 M. Brugna, S. Rodgers, I. Sinning, and W. Nitschke, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Qp site, quinone-binding site in cytochrome bc-type complexes located close to the positive side of the membrane; Em, redox midpoint potential; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid.
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