From the Laboratoire de Bioénergétique et Ingéniérie des Proteines, CNRS, Institut de Biologie Structurale et Microbiologie, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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ABSTRACT |
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The redox components of the cytochrome
bc1 complex from the acidophilic
chemolithotrophic organism Thiobacillus
ferrooxidans were investigated by potentiometric and
spectroscopic techniques. Optical redox titrations demonstrated the
presence of two b-type hemes with differing redox midpoint potentials
at pH 7.4 ( Thiobacillus ferrooxidans is the main
bacterium used in the industrial extraction of copper and uranium from
ores, using the microbial leaching technique (1). The sole
energy-producing process used by this obligate chemolithotrophic
bacterium for growth and cell maintenance involves the oxidation of
reduced sulfur compounds and/or ferrous ion under acidic conditions,
using O2 as the oxidant (2-4). When grown on
Fe2+, a comparatively small amount of energy is released by
the oxidative reaction. The bacterium nevertheless fixes its own
CO2, and Fe2+ oxidation must therefore be
coupled to reduction of the NAD(P) required for this fixation and other
anabolic processes. It has been suggested that an uphill electron
transfer, established at the expense of the energy derived from the
oxidation of Fe2+ by oxygen, may be involved in the
reduction of NAD(P)+ from Fe2+ (for a review,
see Ref. 5).
Various respiratory chain components have been detected in T. ferrooxidans. The optical spectra measured at 77 K on whole cells
indicated the presence of multiple cytochromes c, one
cytochrome b, and cytochrome oxidase (6). Using optical and
EPR techniques combined with potentiometric and kinetic studies,
Ingledew and Cobley (7) have detected an a1-type
cytochrome, multiple cytochromes c, cytochrome b,
two copper-containing centers, high and low spin ferric hemes, and a
ferredoxin center. In these experiments, only the The electron transfer chain from Fe2+ to O2 is
presently considered to involve a Fe2+-cytochrome
c oxidoreductase, rusticyanin, at least one cytochrome c (the 14-kDa soluble cytochrome c and/or the
c4-type cytochrome), and an
a1-type cytochrome. Ingledew (5) has put forward
the idea that the uphill electron transfer from Fe2+ to
NAD+ may involve a putative bc1
complex, according to a chemiosmotic mechanism, possibly via a Q-cycle
mechanism operating in reverse. This mechanism requires the presence of
two separate and independent quinone reaction sites: the ubiquinol
oxidation site (Qp
site)1 located on the
positive side of the membrane and the ubiquinone reduction site
(Qn site) located on the negative side of the membrane (20-22). The Qp site is formed by cytochrome b
(in the vicinity of heme bL) and the iron-sulfur protein
and is the target of specific inhibitors like myxothiazol,
stigmatellin, DBMIB, and UHDBT (for a review, see Ref. 23). The binding
of myxothiazol and stigmatellin results in an alteration of the
absorbance spectrum of heme bL of cytochrome b,
which is shifted toward the red. The binding of stigmatellin, DBMIB,
and UHDBT furthermore induces spectral changes in the EPR spectrum of
the Rieske cluster. The Qn site is associated with
cytochrome b (in the vicinity of heme bH) and is
the target of other specific inhibitors like antimycin A, funiculosin, HQNO, and diuron. The binding of these Qn site inhibitors
to the bc complexes induces a red or a blue (in the case of
funiculosin) shift in the absorbance spectrum of heme bH.
These shift properties have been used to measure the binding of the
inhibitors to their specific sites in the bc complexes
(24).
We have recently provided evidence for the presence of a
bc1 complex in T. ferrooxidans based
on the observation of the typical EPR spectrum of the Rieske center,
which is one of the crucial redox centers of
bc1-type complexes (25). This was the first and
so far only evidence for the existence of a bc1
complex in an acidophilic proteobacterium. In the present study, we
characterized the redox centers of the enzyme and the effect of the
binding of the specific bc1 complex inhibitors
on cytochrome b and the Rieske iron-sulfur protein in more detail.
Organism and Culture Growth Procedure--
T.
ferrooxidans was kindly supplied by Dr. D. Morin (Bureau des
Recherches Géologiques et Minières (BRGM), Orléans,
France). The strain was isolated from drainage water at the Salsigne
sulfur mine (France). Large-scale growth of the organism was performed at pH 1.6 in the 9 K medium described by Silverman and Lundgren (26),
supplemented with 1.6 mM
CuSO4·5H2O, using a homemade polypropylene
fermentor with a capacity of 400 liters. Cells were harvested according
to Bodo and Lundgren (27) and stored as pellets at Preparation of Membrane Fragments and Spheroplasts--
Membrane
fragments were prepared as described by Elbehti and Lemesle-Meunier
(19). For the preparation of spheroplasts, cell paste (5 g, wet weight)
was washed three times in 0.5 M NaCl at 0 °C and
resuspended in 20% (w/v) glucose. The cell suspension was incubated at
37 °C in a shaking water bath for 2 h. The cells were
centrifuged at 6000 × g for 10 min and washed three
times in 30 mM Tris-HCl (pH 8). After each washing step,
one freezing (in liquid nitrogen) and thawing (room temperature) step
was performed. The cells were then resuspended in 30 mM
Tris-HCl (pH 8), 10 mM EDTA, and 20% glucose and incubated
for 20 h at 37 °C in the presence of lysozyme (1 mg/ml). After
being centrifuged at 16,000 × g for 15 min, the
spheroplasts obtained were washed twice in 20 mM
Optical and EPR Spectroscopy--
The visible and UV absorption
spectra were determined with an Aminco DW2 recording spectrophotometer
using 1-cm light path cuvettes. The heme content was determined from
the reduced-minus-oxidized difference spectrum using the following
millimolar extinction coefficients for the
The spectral shift of the b hemes was measured as the spectral
difference between the dithionite-reduced enzyme in the presence and
absence of saturating amounts of inhibitor. Spheroplasts were first
reduced by adding a few grains of sodium dithionite, and the base line
was recorded. Inhibitor was then added to the sample cuvette, and the
same volume of solvent was added to the reference cuvette. The spectra
were recorded at 25 °C in either 50 mM potassium phosphate buffer (pH 7.4) or 20 mM
For redox potentiometry, optical titrations were carried out at
25 °C as described by Dutton (30), using a Kontron Uvikon 922 double-beam spectrophotometer equipped with a Spectralon integrating sphere. Membrane fragments were suspended in the same buffers (pH 7.4 and 3.5) as mentioned above; pH values were controlled at the beginning
and end of each redox titration. The following redox mediators were
used: 1,1'-ferrocenedicarboxylic acid, ferrocenemonocarboxylic acid,
1,4-benzoquinone,
N,N,N',N'-tetramethyl-p-phenylenediamine, 2,5-dimethyl-p-benzoquinone, 1,2-naphthoquinone,
1,4-naphthoquinone, phenazine ethosulfate, indigo carmine,
anthraquinone-2,6-disulfonate, and anthraquinone-2-sulfonate. Reductive
titrations were carried out using sodium dithionite, and oxidative
titrations were done using sodium hexachloroiridate
(Na2Cl6Ir).
Electrochemical Properties of the b- and c-type Hemes of T. ferrooxidans--
Membrane fractions prepared from the BRGM strain of
T. ferrooxidans were examined for their content of the
various cytochromes at pH 7.4. The optical difference spectra shown in
Fig. 1 demonstrate the presence of
cytochrome a (597 nm), cytochrome(s) c (552 nm), and cytochrome b (560 nm). It can be seen that b-type hemes
are substoichiometric to c-type hemes and can only be discerned as a
shoulder on the red flank of the c-type hemes'
Fig. 2 shows the results of a redox
titration of b-type (at 562 nm; circles) and c-type (at 550 nm; diamonds) hemes carried out on membrane preparations at
pH 7.4 (closed symbols) and 3.5 (open symbols).
In the case of cytochrome b and at pH 7.4, the data could be
fitted with two components with midpoint potentials of
Fig. 2 (inset) shows differences in spectra recorded during
the titration (at pH 7.4) representing the low ( Inhibitor Binding to the Thiobacillus Cytochrome bc1
Complex--
The typical inhibitors of the bc1
complexes (antimycin A, HQNO, myxothiazol, and stigmatellin) have been
found to inhibit a proton-motive force-dependent uphill
electron transfer from reduced cytochrome c to
NAD+ in spheroplasts from T. ferrooxidans.2 The study
of inhibitor-induced changes in spectral properties of the redox site
allows the detection of binding of the respective inhibitors and the
identification of the affected heme center by its characteristic
absorption maximum (24, 31). Typically, difference spectra between
dithionite-reduced bc1-containing samples in the
presence and absence of inhibitor yield symmetrical S-shaped curves
crossing the base line at a wavelength corresponding to the maximum of
the absorption band of the heme affected by the inhibitor binding
(31-35).
Binding experiments were performed at acidic and neutral pH values (3.5 and 7.4, respectively) on Thiobacillus spheroplast preparations reduced by an excess of dithionite. Spheroplasts are
closed vesicles, and the internal pH is always constant (almost neutral
around pH 6.5) whatever the external pH (36). Fig.
3 (A and B) shows
the effect of antimycin A, HQNO, and stigmatellin on the optical
spectra of cytochrome b recorded at pH 7.4 and 3.5, respectively.
At pH 7.4, the S-shaped symmetrical curves obtained upon adding
antimycin A and HQNO crossed the base line at ~562 nm, showing a
maximum at 566 nm and a minimum at 559 nm (Fig. 3A). This
shape reflects a shift of a heme
At pH 3.5, the difference spectra obtained after adding stigmatellin to
reduced spheroplasts exhibited a symmetrical S-shape crossing the base
line at 564-565 nm, with a maximum at 569 nm and a minimum at 557 nm
(Fig. 3B). Similar to what has been observed concerning the
action of this inhibitor on submitochondrial particles or
bc1 complex isolated from beef heart
mitochondria (34, 35), the red shift induced by stigmatellin therefore
has a maximum and a minimum at higher wavelengths than those induced by
antimycin and HQNO. This indicates that the heme affected by the
binding of stigmatellin has an Characteristics of the T. ferrooxidans Rieske Iron-Sulfur
Protein--
The EPR spectra of membrane preparations from T. ferrooxidans, reduced by ascorbate at pH 7.4 or pH 3.5, are shown
in Fig. 4. The g value of the
derivative-shaped gy signal (gy = 1.90) and the fact
that this center was reduced by a weakly reducing agent such as
ascorbate are characteristic for the so-called Rieske center,
representing one of the four redox centers of the bc
complexes (21). A gx signal is observed at g = 1.74 in the
spectra at pH 7.4 (Fig. 4, spectrum A); lowering the pH to
3.5 resulted in broadening of this signal and a slight shift toward
lower magnetic fields (spectrum A'). It has been shown previously that the shape of the EPR signal is affected by the redox
state of the quinone pool in beef heart submitochondrial particles (39)
and in chromatophores from Rhodobacter sphaeroides (40). In
both cases, a significant sharpening of the gx trough was seen
to occur upon oxidation of the quinone pool. Fig. 4 suggests that, at
acidic pH values, the quinone pool in T. ferrooxidans membranes was at least partially reduced by the added ascorbate, whereas it remained oxidized at pH 7.4. This is in line with the fact
that the Em value of ubiquinone increased by 230 mV
on going from pH 7.4 to pH 3.5, yielding a theoretical
Em,3.5 value of ~300 mV.
When the inhibitors stigmatellin, UHDBT, and DBMIB (known to bind at
the Qp site in close vicinity of the Rieske center) were added, the shape and intensity of the Rieske EPR signals were altered,
in particular the gx signal (Fig. 4). At pH 7.4, DBMIB
(spectrum B) and UHDBT (spectrum D) shifted the
gx signal to slightly lower fields. Addition of stigmatellin
resulted in a distinct sharpening of the gx trough and of the gy line (spectrum C). At pH 3.5, the
gx trough, which was rather broad in the absence of inhibitor
(spectrum A'), was sharpened upon addition of all examined
inhibitors (spectra B'-D'). The
inhibitor-induced spectra at pH 3.5 appeared to be rather similar to
those observed at pH 7.4.
The uphill electron transfer between Fe2+ and
NAD+, required to allow CO2 fixation via the
Calvin cycle, has only been scanty studied so far. The only information
on the compounds that may function in this respiratory chain was
obtained by Ingledew and Cobley (7). In particular, these authors have
performed redox titrations of the cytochromes in membrane preparations;
clear results, however, were obtained only for cytochrome
a1. With respect to c-type hemes, their results
indicated the presence of more than one species, but no clear
inflection points were obtained. While titrating an a-type
cytochrome at 440-479 nm, they found evidence for the presence of two
additional components at pH 3.2 with Em values of
280 and 150 mV, which they attributed to b-type hemes. No clear results
were obtained from measurements carried out at pH 7.4 for the
b- and c-type cytochromes. Electron paramagnetic
resonance spectra of the electron transport particles revealed the
presence of two copper-containing centers, high and low spin ferric
hemes, and a ferredoxin center. The presence of a Rieske protein has
not been detected in these studies.
We have recently shown that ferrous ion-grown cells from T. ferrooxidans contain a bc1-type complex
(25) functioning in reverse mode,2 as postulated by
Ingledew (5). The present study reports on the characteristics of this
bc1 complex, allowing us to speculate about a
possible reverse electron transfer mechanism.
Membrane-bound c-type Cytochromes--
The global absorption band
at 551-552 nm obtained from membrane preparations arises from three
different c-type cytochromes with apparent molecular masses
of 46, 30, and 21 kDa (19). The EPR spectrum of the 21-kDa cytochrome
c was characteristic of a dihemic
c4-type cytochrome, and its terminal sequence
was found to be identical to that of the soluble cytochrome
c4 present in the same strain of T. ferrooxidans (10).3 It
is well known from studies on other species that cytochrome c4 shows a strong affinity toward the membrane,
and similar to what we found in Thiobacillus, this
hemeprotein is generally detected both in the soluble and membrane
fractions of cells (41). The EPR spectrum of the purified 46-kDa
cytochrome did not resemble those typically found for cytochrome
c1.4
Moreover, Bonnefoy and co-workers (42) have cloned and sequenced the
gene coding for a c-type cytochrome, named cyc2,
in the T. ferrooxidans ATCC 33020 strain, which is 67%
identical and 86% similar to the N-terminal amino acid sequence of the
46-kDa cytochrome of our strain. This suggests that the cyc2
gene encodes a protein equivalent to the 46-kDa cytochrome of our
strain (19, 42). No significant homologies to other cytochromes were
detected in data banks, indicating that cyc2 does not
correspond to cytochrome c1. Moreover, the
cyc2 gene is cotranscribed with cytochrome
c4 (cyc1), subunits 1-4 of
cytochrome oxidase, and the rusticyanin genes, suggesting that all
these components belong to the downhill electron transfer chain toward
reduction of oxygen (43).5
Only the 30-kDa cytochrome therefore remains as a possible candidate for cytochrome c1. Since cytochrome
c4 was present in the membrane fragments (as
discussed above), it must contribute to the optical Cytochrome b and Sensitivity to the Specific Inhibitors of the
bc1 Complex--
The redox titration of cytochrome
b at pH 7.4 shows the existence of a low midpoint potential
heme bL (Em =
In this study, we present data on the spectral shifts induced by these
inhibitors in spheroplasts from T. ferrooxidans. The results
show that the two distinct classes of inhibitors, antimycin and HQNO on
one hand and stigmatellin on the other hand, induce distinguishable red
shifts in the absorption band of cytochrome b in this
species. This demonstrates that the binding of antimycin and HQNO
occurs on cytochrome b near a heme center with an
The experimental results furthermore show that antimycin and HQNO
induced a red shift in the absorption band of cytochrome b
irrespective of the pH of the medium, although yielding more symmetrical S-shaped curves at pH 7.4 than at pH 3.5, whereas stigmatellin induced a significant red shift only when the suspension medium was at pH 3.5; the red shift was comparatively weak at pH 7.4. These findings indicate that, at neutral pH, the binding domain for
stigmatellin is modified and is no longer able to accommodate the
inhibitor. This in turn suggests that the antimycin- and HQNO-binding domains are located on the cytoplasmic side of the membrane, which is
in contact with a near-neutral pH (around pH 6.5) (36), whereas the
stigmatellin-binding domain is located on the periplasmic side of the
membrane, which is in contact with an acidic pH.
The Rieske Iron-Sulfur Protein--
The EPR studies performed on a
highly concentrated, ascorbate-reduced membrane preparation of T. ferrooxidans clearly showed the presence of a [2Fe-2S] Rieske
center in this bacterium: the spectral parameters (gy = 1.90 and gx = 1.74) and the inhibitor sensitivity are characteristic
of this compound. The gx trough at g = 1.74 is at
unusually high magnetic fields as compared with other cytochrome
bc1 complexes and rather resembles the spectral
parameters reported for the chloroplast b6f complex. The gx signal
observed at pH 7.4 was seen to broaden and shift toward lower magnetic
fields when the pH became more acidic (pH 3.5), most probably
reflecting the fact that the quinone pool is oxidized at neutral pH,
but reduced at acidic pH. It is noteworthy that in all
bc1 complexes studied so far, the gx
signal of the Rieske protein is sharp in the presence of oxidized
quinone, but shifts toward higher fields and broadens considerably when
ubiquinone becomes reduced. In the
b6f complex, the gx trough
was seen to shift toward lower fields in the presence of reduced
plastoquinone. The T. ferrooxidans Rieske center exhibited
unusual characteristics with regard to inhibitor sensitivity at both
studied pH values. In fact, the three inhibitors induced a shift of the
gx trough to lower field positions, contrary to what is usually
observed in both the bc1 (34, 44) and
b6f (45) complexes. So far, similar
downfield shifts of the gx signal of the Rieske protein have
only been observed in Heliobacterium chlorum (46). In
addition, the [2Fe-2S] center of the T. ferrooxidans
Rieske protein shows further particular features. 1) Its midpoint
potential is ~150 mV higher than that typically measured in
cytochrome bc1 complexes (47). This is
consistent with the fact that higher redox potentials have been
obtained with almost all of the electron transfer components of
T. ferrooxidans as compared with other species. 2) The
pK value of its pH-dependant redox midpoint potential is
more acidic than that found in neutrophilic systems, but turned out to
be close to that observed on the Rieske protein from another acidophilic bacterium, i.e. Sulfolobus acidocaldarius
(47).
Membrane preparations from T. ferrooxidans were found to
transfer electrons from quinol to cytochrome c via the
bc1 complex at neutral pH; in spheroplasts and
at acidic external pH values, the bc1 complex
functions in reverse via a proton-motive force-dependent mechanism.2 Fig. 5 shows the
thermodynamic profile of the T. ferrooxidans bc1
complex when the value of the external pH is 7.4 (Fig. 5B) or 3.5 (Fig. 5C) compared with the profiles of the
bc1 and b6f complexes (Fig. 5A, continuous and dashed
boxes, respectively). Fig. 5 visualizes the positions of hemes
bL and bH close to the bulk phase and the
cytosol, respectively, as is the case for the typical
bc1 complexes and as is suggested by our results
to also apply to the T. ferrooxidans bc1
complex. As can be seen from Fig. 5, the difference in
Em values at pH 7.4 (external pH) between the
hydroquinone/quinone couple and the Rieske center is much higher than
in usual
bc1/b6f
complexes. On the other hand, the Em values of the
cytochrome b redox centers are closer to those observed in
the b6f complex than in the
bc1 complex. Under physiological conditions,
i.e. at acidic external pH, the bc1
complex functions in the reverse direction, and its thermodynamic profile is reminiscent of that of the
b6f complex, except for the
169 and + 20 mV for bL and bH,
respectively). At pH 3.5, by contrast, both hemes appeared to titrate
at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of
the b hemes'
-bands, providing evidence for the binding of
antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near
heme bH (located on the cytosolic side of the membrane) and
of stigmatellin near heme bL (located on the periplasmic
side of the membrane). The inhibitors stigmatellin,
5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected
the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1
or b6f complexes. The results
obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for
bc1 complexes, contains unique properties that
are most probably related to the chemolithotrophicity and/or
acidophilicity of its parent organism. A speculative model for reverse
electron transfer through the T. ferrooxidans complex is proposed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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-band of
cytochrome b was detected at pH 3.2. The presence of
ubiquinone-8 has also been reported (8). Some of these compounds have
been isolated and purified: a soluble 14-kDa cytochrome
c552 (9), a soluble
c4-type cytochrome (10), the blue copper protein rusticyanin (11-13), an iron-sulfur protein
Fe2+-cytochrome c552 oxidoreductase
(14, 15), an a1-type cytochrome oxidase (16),
and three membrane-bound cytochromes c: (a high molecular
mass cytochrome c, the size of which depends on the strain
(17-19), and a 30-kDa (19) and a 22.3-kDa (18) or 21-kDa (17, 19)
cytochrome c.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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70 °C. About
12 g of cells (wet weight) were obtained from 300 liters of cell culture.
-alanine/H2SO4 buffer (pH 3.5) and 20%
glucose and finally resuspended in the same buffer. The Gram coloration
method was used to verify formation of spheroplasts (28). Protein
concentrations were measured using the method of Lowry et
al. (29).
-peak:
550-540 nm = 16.5 mM
1
cm
1 (cytochrome c),
552-575 nm = 24 mM
1
cm
1 (cytochrome b), and
603-630 nm = 21 mM
1
cm
1 (cytochrome oxidase).
-alanine/H2SO4 buffer (pH 3.5), with each
buffer containing 0.1 mM phenylmethylsulfonyl fluoride and
0.1 mM
-aminocaproic acid. EPR spectra were recorded at
liquid helium temperature on a Bruker ESP 300e X-band spectrometer equipped with an Oxford Instruments liquid helium cryostat and temperature control system.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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-peak in the full
redox difference spectrum.
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Fig. 1.
Difference spectra between reduced (by
dithionite) and oxidized (by Na2Cl6Ir) membrane
fragments from T. ferrooxidans resuspended in 50 mM potassium phosphate buffer (pH 7.4) at 6.4 mg/ml.
169 and 22 mV,
each contributing approximately equally to the total absorbance. These
would correspond to cytochrome bL (
170 mV) and
bH (20 mV) and imply a ratio of ~1:1 assuming similar extinction coefficients. At pH 3.5, by contrast, a good fit could already be obtained using only a single n = 1 component
with a midpoint potential of 20 mV. These results suggest that the two cytochrome b hemes exhibit rather close redox midpoint
potentials at pH 3.5. The data points for the c-type hemes were fitted
with four n = 1 components with midpoint potentials of
150, 360, 410, and 485 mV at pH 7.4 and 240, 350, 430, and 560 mV at pH
3.5.
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Fig. 2.
Redox titration of b and c hemes carried out
on membrane fragments (at a protein concentration of 3.5 mg/ml) from
T. ferrooxidans at pH 7.4 and 3.5. Data points
represent signal amplitude at 562 nm (b-type hemes
(circles)) and 550 nm (c-type hemes (diamonds))
as a function of ambient redox potential. The inset shows
representative difference spectra recorded during the titration at pH
7.4 (spectrum A, 238 mV minus
127 mV; spectrum
B,
127 mV minus +103 mV; spectrum C,
238 mV minus
+585 mV). Open symbols, pH 3.5; closed symbols,
pH 7.4.
238 mV minus
127
mV; spectrum A) and high (
127 mV minus +103 mV;
spectrum B) potential components of cytochrome b
as well as the full redox difference attained during the titration
(spectrum C). A slight shift toward shorter wavelengths was
observed on going from the low to the high potential spectra of
cytochrome b (see below)
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Fig. 3.
Red shifts in reduced cytochrome b
induced by the binding of the inhibitors antimycin, HQNO,
myxothiazol, and stigmatellin in T. ferrooxidans
spheroplasts. The protein (7 mg/ml) was resuspended in 50 mM potassium phosphate buffer (pH 7.4) (A) or 20 mM -alanine/H2SO4 buffer (pH
3.5) (B). Sample and reference cuvettes were reduced with
dithionite prior to addition of inhibitor. Antimycin A (10 µM), HQNO (20 µM), myxothiazol (10 µM), or stigmatellin (10 µM) was added to
the sample cuvette, and the same volume of solvent was added to the
reference cuvette.
-band with a maximum at ~562 nm
toward longer wavelengths (bathochromic effect). These features are
consistent with the results of experiments carried out on the purified
mitochondrial bc1 complex and on
submitochondrial particles of various origins regarding the red shift
of the heme 562 nm absorption band induced by these inhibitors (31-33,
37, 38). At pH 3.5, the S-shaped curves induced upon adding antimycin A
and HQNO also crossed the base line at ~562 nm, but the curves were
less symmetrical than those obtained at pH 7.4, with minima at 553 and
552 nm for antimycin and HQNO, respectively, and maxima at 566 nm for
both these inhibitors (Fig. 3B).
-band with a maximum at a higher
wavelength than the heme reacting to antimycin and HQNO,
i.e. at ~564-565 nm. At pH 7.4, only a very minor shift
was observed in the presence of stigmatellin. The demonstration of the
presence of two spectrally different hemes corroborates the slight
shift observed during titration. The hemes sensitive to HQNO and
antimycin A on one side and stigmatellin on the other side correspond
to the high and low potential hemes, respectively.
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Fig. 4.
EPR spectra recorded on membrane fragments
(at a protein concentration of 100 mg/ml) from T. ferrooxidans reduced by ascorbate at pH 7.4 (spectra
A-D) and at pH 3.5 (spectra
A'-D') in the absence of inhibitor
(spectra A and A') and in the
presence of DBMIB (spectra B and B'),
stigmatellin (spectra C and C'), and
UHDBT (spectra D and D').
Instrument settings were as follows: temperature, 15 K; microwave
frequency, 9.42 GHz; microwave power, 6.3 milliwatts; and modulation
amplitude, 1.6 milliteslas (mT). Rieske centers typically
start to saturate below 25 K (e.g. see Ref. 45). The lower
temperature (15 K) was chosen in order to increase signal amplitude.
Within experimental precision, no spectral distortions induced by the
saturating conditions were observed.
DISCUSSION
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ABSTRACT
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-peak at 552 nm.
By comparison with the reported midpoint potential of soluble
cytochrome c4 from T. ferrooxidans
(10), the components titrating at 360 and 410 mV at pH 7.4 and at 350 and 430 mV at pH 3.5 are attributed to the two hemes of cytochrome
c4. It is, however, difficult at present to
decide which of the two redox species at 240 and 560 mV (pH 3.5) and at
150 and 485 mV (pH 7.4) belongs to cytochrome c1
and which one to the 46-kDa cytochrome.
169 mV) and a high
midpoint potential heme bH (Em = +20
mV); when the pH is lowered to 3.5, the heme bL midpoint
potential increases in order to reach a value close to that of heme
bH, which in turn is pH-independent. The two distinct types
of bc1-specific inhibitors represented by
antimycin A and HQNO on one hand and stigmatellin on the other hand
induce a shift in the absorption spectrum of cytochrome b.
The occurrence of a bathochromic or hypsochromic shift in the
absorption spectrum of a hemic component induced by a ligand means that
the binding of the ligand affects the electronic surroundings of the
heme. The red shifts induced by center N inhibitors show a maximum at
564 nm and a minimum at 556 nm in beef heart bc1
complex, whereas those induced by center P inhibitors have a maximum at
568 nm and a minimum at 560 nm (32). The values of these maxima and
minima associated with a specific inhibitor may be slightly different,
depending on the organism or on the material used (i.e.
purified bc1 complex or submitochondrial
particles). In all cases studied, the maxima and minima of the red
shifts induced by the Qn center inhibitors were
consistently observed at a lower wavelength than those induced by the
Qp center inhibitors; this indicates that Qn
site inhibitors predominantly influence the surroundings of the
bH heme, whereas Qp site inhibitors influence
that of the bL center (31-35). The red shift induced by
stigmatellin had a maximum at 568 nm and a minimum at 555-557 nm with
a shoulder at 562 nm, which indicates that this inhibitor mainly
influences the heme bL center (34).
-band maximum at ~562 nm, whereas stigmatellin binds to another heme b
center peaking at ~564 nm. The striking similarities to typical cytochrome bc1 complexes therefore provide
evidence for the existence of a dihemic cytochrome b in
T. ferrooxidans.
Em between the two b hemes. Based on these data, hypotheses describing the reverse electron transfer mechanism in the
T. ferrooxidans bc1 complex can be formulated
(Fig. 5): a proton-motive force would be required to enable electrons
from the iron-sulfur protein (Em = 480 mV at pH 3.5)
and protons from the periplasmic side of the membrane to move toward
center P, where reduction of ubiquinone (Em ~ 340 mV at pH 3.5) would take place. The hydroquinone thus formed at the
acidic external pH (Em ~ 340 mV) would move to the
Qn center in contact with the cytosolic neutral pH
(Em ~ 100 mV), reducing heme bH, which
then would in turn reduce heme bL. The electron transfer
between the two b hemes would be facilitated by their close midpoint
potential values at acidic pH. In subsequent cycles, hydroquinone would
be reduced by 1 electron from the Rieske center and 1 electron from
heme bL (the electron transfer between reduced heme
bL and quinone being thermodynamically favorable).
Experiments are in progress to obtain a better understanding of the
reverse electron transfer mechanism in the T. ferrooxidans
bc1 complex.
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Fig. 5.
Thermodynamic profile of the redox centers of
the T. ferrooxidans bc1 complex when the
value of the external pH is 7.4 (B) or 3.5 (C) compared with profiles of typical
bc1 and
b6f complexes
(A). Continuous and dashed
boxes, profiles of the bc1 and
b6f complexes, respectively;
Q, quinone; QH2, hydroquinone.
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ACKNOWLEDGEMENTS |
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We are grateful to P. Brivet and G. Brasseur for fruitful discussions, C. Appia and V. Bonnefoy for communicating results prior to publication, and R. Toci (Fermentation Plant Unit, Laboratoire de Chimie Bactérienne, Marseille, France) for growing Thiobacillus. We also thank S. Touloudjian for technical assistance.
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FOOTNOTES |
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* This work was supported in part by a grant from l'Agence de l'Environnement et de la Maitrise de l'Energie, le Bureau des Recherches Géologiques et Minières, and COGEMA (France).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.:
33-04-91-16-44-86; Fax: 33-04-91-16-45-63; E-mail:
lemesle{at}ibsm.cnrs-mrs.fr.
2 A. Elbehti and D. Lemesle-Meunier, manuscript in preparation.
3 D. Lemesle-Meunier and P. Tron, unpublished results.
4 D. Lemesle-Meunier and W. Nitschke, unpublished results.
5 C. Appia and V. Bonnefoy, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Qp and Qn sites, quinone-binding sites in cytochrome bc-type complexes located close to the positive and negative sides of the membrane, respectively; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; UHDBT, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole; HQNO, 2-heptyl-4-hydroxyquinoline N-oxide; Em, redox midpoint potential.
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REFERENCES |
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