From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078
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ABSTRACT |
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The three-dimensional structure of
the mitochondrial cytochrome bc1 complex
suggests that movement of the extramembrane domain (head) of the Rieske
iron-sulfur protein (ISP) may play an important role in electron
transfer. Such movement requires flexibility in the neck region of ISP,
since the head and transmembrane domains of the protein are rather
rigid. To test this hypothesis, Rhodobacter sphaeroides
mutants expressing His-tagged cytochrome bc1
complexes with cysteine substitution at various positions in the ISP
neck (residues 39-48) were generated and characterized. The mutants with a single cysteine substitution at Ala42 or
Val44 and a double cysteine substitution at
Val44 and Ala46 (VQA-CQC) or at
Ala42 and Ala46 (ADVQA-CDVQC) have
photosynthetic growth rates comparable with that of complement cells.
Chromatophore membrane and intracytoplasmic membrane (ICM)
prepared from these mutants have cytochrome bc1 complex activity similar to that in the complement membranes, indicating that flexibility of the neck region of ISP was not affected
by these cysteine substitutions. Mutants with a double cysteine
substitution at Ala42 and Val44 (ADV-CDC) or at
Pro40 and Ala42 (PSA-CSC) have a retarded
(50%) or no photosynthetic growth rate, respectively. The ADV-CDC or
PSA-CSC mutant ICM contains 20 or 0% of the cytochrome
bc1 complex activity found in the complement ICM. However, activity can be restored by the treatment with
The cytochrome bc1 complex
(ubiquinol-cytochrome c reductase) is an essential segment
of the energy-conserving electron transfer chains of mitochondria and
many respiratory and photosynthetic bacteria (1). This complex
catalyzes electron transfer from ubiquinol to cytochrome c
and concomitantly translocates protons across the membrane to generate
a membrane potential and pH gradient for ATP synthesis. Although the
cytochrome bc1 complexes from different sources
vary in their polypeptide compositions, they all contain four redox
prosthetic groups: two b-type cytochromes (b566 or bL and
b562 or bH), one
c-type cytochrome (cytochrome c1),
and one high potential Rieske iron-sulfur cluster
[2Fe-2S].1 The
proton-motive Q cycle model (2) has been favored for electron transfer
and proton translocation in the complex. The key feature of this model
is the presence of two separate ubiquinone- or ubiquinol-binding sites:
a ubiquinol oxidation site near the P side of the inner mitochondrial
membrane and a ubiquinone reduction site near the N side of the membrane.
Recently, the cytochrome bc1 complex from beef
heart mitochondria was crystallized and its three-dimensional structure
solved at 2.9-Å resolution (3, 4). The structural information obtained not only answered a number of questions concerning the arrangement of
the redox centers, transmembrane helices, and inhibitor binding sites
but also suggested movement of an extramembrane domain within the
iron-sulfur protein (ISP) during electron transfer (4). This suggestion
arose from observation of an uneven electron density in the
I4122 crystal data of native bovine cytochrome
bc1 complex. A particularly low electron density
area is observed in the intermembrane space portion of the complex,
where the extramembrane domains of ISP and cytochrome
c1 reside (4). This movement hypothesis was
further supported by the finding that the position of the iron-sulfur
cluster in the complex is affected by ubiquinol oxidation site
inhibitor binding (5, 6, 8) and by the crystal form (7, 9).
The anomalous light scattering signal of the [2Fe-2S] cluster is
enhanced in co-crystals with stigmatellin or UHDBT but is diminished in
the co-crystal with
(E)-methyl-3-methoxy-2-(4'-trans-stilbenyl) acrylate (5). Thus, binding of stigmatellin or UHDBT arrests the
movement of the extramembrane domain of ISP, fixing the iron-sulfur cluster 27 Å from heme bL and 31 Å from heme
c1 (referred to as the "fixed state" of ISP), the same
position it occupies in the I4122 crystal of
native bovine cytochrome bc1 complex. The
position of the iron-sulfur cluster changes from the fixed state to
somewhere closer to heme c1 (referred to as the "released
state" of ISP) upon
(E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)
acrylate binding. The recent report of Iwata et al. (9),
showing the iron-sulfur cluster at two different positions in two
crystal forms, further supports the presence of a variable position of
in the "released state" of the iron-sulfur cluster.
Movement of the head domain of ISP during electron transfer in
cytochrome bc1 complex can be explained as
follows. The [2Fe-2S] cluster is reduced by the first electron of
ubiquinol at a position 27 Å from heme bL and 31 Å from
heme c1 (ISP in "fixed state"). Since a reduced
[2Fe-2S] cluster cannot donate an electron to cytochrome
c1 before the second electron of ubiquinol is
transferred to heme bL, it was postulated that either the
change of the ubiquinone binding position during reduction of
bL or the electron transfer from
bL to bH causes a
conformational change in cytochrome b, which forces or
allows reduced [2Fe-2S] to move close enough to heme c1
(ISP in "released state") for electron transfer (5, 7). This model
would also explain why ubisemiquinone, a more powerful reductant than
ubiquinol, reduces bL, but not the [2Fe-2S] cluster, during ubiquinol oxidation.
ISP has three domains: the membrane-spanning N-terminal domain
consisting of residues 1-62 (tail), the soluble C-terminal extramembrane domain consisting of residues 73-196 (head), and the
flexible linking domain comprising residues 63-72 (neck). ISP is
associated with the complex primarily via the membrane-spanning N-terminal domain (4, 7, 9). The [2Fe-2S] cluster is located at the
tip of the head domain (12, 13). Since the three-dimensional structures
of the head and tail domains are the same in the fixed and released
states, movement of the head domain of ISP in the
bc1 complex requires flexibility in the neck region.
If movement of the head domain of ISP is required for
bc1 catalysis and the neck region of ISP confers
the required mobility, decreasing the flexibility of the neck region of
ISP should affect bc1 complex activity. This
hypothesis can be tested by site-directed mutagenesis followed by
biochemical and biophysical characterization of mutant expressing
cytochrome bc1 complexes with altered ISP necks.
However, site-directed mutagenesis in bovine heart mitochondria is not
practical. R. sphaeroides is an ideal system to study the neck region of ISP by molecular genetics approach. The four-subunit complex is functionally analogous to the mitochondrial enzyme; the
largest three subunits are homologous to their mitochondrial counterparts; and this system is readily manipulated genetically. In
addition, the recent generation of R. sphaeroides expressing His6-tagged cytochrome bc1 complex
greatly speeds up the preparation of the bc1
complex from wild-type or mutant cells (14).
The R. sphaeroides ISP neck is composed of residues 39-48
(corresponding to residues 63-72 of bovine ISP) with a sequence of
NPSADVQALA. Ala42, Asp43, Val44,
Ala46, and Ala48 are the conserved amino acid
residues. We have previously generated mutants with increased ISP neck
rigidity by double or triple proline substitution of the conserved
residues. The results demonstrated that flexibility in the ISP neck is
important in bc1 catalysis (14). The ALA-PLP and
ADV-PPP mutant membranes have, respectively, 30 and 0% of the
cytochrome bc1 complex activity found in the complement membrane. The ALA-PLP mutant complex has a larger activation energy than the wild-type complex, suggesting that movement of the head
domain decreases the activation energy barrier of the bc1 complex.
To better define the structure and function relationship of the neck
region of ISP in the bc1 complex, we recently
generated mutants expressing His6-tagged
bc1 complex with single or double cysteine
substitution at various positions in the ISP neck. We predict that
formation of a disulfide bond between a pair of genetically engineered
cysteines in the neck will decrease its flexibility and thus decrease
electron transfer activity. Measuring the bc1 activity in these cysteine-substituted mutants should give insight into
the dynamic state of the ISP neck. Herein we report procedures for
generating R. sphaeroides mutants expressing
His6-tagged cytochrome bc1 complexes
with altered ISP necks by introducing single cysteines or a pair of
cysteines at different positions. The photosynthetic growth behavior,
the cytochrome bc1 complex activity, and the EPR
characteristics of the Rieske [2Fe-2S] cluster in membranes and the
purified state from complement and mutant strains are examined and
compared. The effect of sulfhydryl reagents on cytochrome bc1 complexes from complement and mutant
membranes is also examined.
Materials--
2-Mercaptoethanol ( Generation of R. sphaeroides Strains Expressing the
bc1 Complexes with Altered ISP--
Mutations were
constructed by site-directed mutagenesis using the Altered Sites system
from Promega. The oligonucleotide primers used for mutagenesis were as
follows: ADV(42-44)-CDC,
GCTGATCAACCAAATGAATCCGTCGTGCGACTGCCAGGCCCTCGCCTCCATCTTCGTCG; A42C, CAAATGAATCCGTCGTGCGACGTGCAGGCCCTCGCCTCCATCT; V44C,
ATGAATCCGTCGGCCTACTGCCAGGCCCTCGCCTCCATCT; VQA(44-46)-CQC,
AACCAAATGAATCCGTCGGCCGACTGCCAGTGCCTCGCCTCCATCTTCGTCGATGTGA; PSA(40-42)-CSC,
TGGCCGCTGATCAACCAAATGAATTGCTCGTGCGACGTGCAGGCCCTCGCCTCCATCTT; ADVQA(42-46)-CDVQC,
TCGTGCGACGTCCAGTGCCTCGCCTCCATCTT.
The method for construction of ISP mutants is essentially the same as
previously reported by Tian et al. (14). The
ADVQA(42-46)-CDVQC mutant was constructed by annealing the
oligonucleotide primer with single-stranded pSELNB3503 carrying a A42C
mutation in ISP. The presence of engineered mutations were confirmed by
DNA sequencing before and after photosynthetic or semiaerobic growth of
the cells. Expression plasmid
pRKDfbcFmBCHQ was purified from an
aliquot of a photosynthetic or semiaerobic culture using the Qiagen
plasmid Mini Prep kit. Since R. sphaeroides cells contain
four types of endogenous plasmids, the isolated plasmids are not pure
and concentrated enough for direct sequencing. Thus, a 1.2-kilobase
pair DNA segment containing the mutation sequence was amplified from
the isolated plasmids by polymerase chain reaction and purified by 1%
agarose gel electrophoresis. The 1.2-kilobase pair polymerase chain
reaction product was recovered from the gel by a gel extraction kit
from Qiagen.
Growth of Bacteria--
Escherichia coli was grown at
37 °C in an enriched medium (TYP) in order to shorten growth time
and increase plasmid yield (15). For photosynthetic growth of the
plasmid-bearing R. sphaeroides BC17 cells, an enriched
Sistrom's medium containing 5 mM glutamate and 0.2%
casamino acids was used. The pH of the medium was adjusted to 7.1 with
a mixture of 6 N NaOH and 2 N KOH to increase
the sodium and potassium ion content of the medium to a more optimal level (16). Photosynthetic growth condition of R. sphaeroides was essentially as described previously (14); cells
harboring mutated fbc genes on the pRKDfbcFBCQ
plasmid were grown photosynthetically for one or two serial passages to
minimize any pressure for reversion. For semiaerobic growth of R. sphaeroides, an enriched Sistrom's medium supplemented with 20 amino acids and extra rich vitamins was used. These semiaerobic
cultures were grown in 0.5 liters of enriched medium in 2-liter Bellco
flasks with vigorous shaking (220 rpm) for 26 h. The inoculation
volumes used for both photosynthetic and semiaerobic cultures were
always at least 5% of the total volume. Antibiotics were added to the
following concentrations: ampicillin (125 mg/liter), tetracycline (10 mg/liter for E. coli and 1 mg/liter for R. sphaeroides), kanamycin sulfate (30 mg/liter for E. coli and 20 mg/liter for R. sphaeroides), trimethoprim (87.5 mg/liter for E. coli).
Enzyme Preparations and Activity Assays--
Cells were
harvested, washed, and passed twice through a French pressure cell at
846 p.s.i. as described previously (17). The chromatophore
fraction was pelleted by ultracentrifugation of broken cells in a
Beckman Ti 50.2 rotor at 48,000 rpm for 2 h at 4 °C. Membrane
was washed with 50 mM Tris-Cl, pH 8.0, containing 1 mM MgSO4 and stored at
Ubiquinol-cytochrome c reductase activity was measured at
23 °C in a 1-ml assay mixture containing 100 mM
sodium/potassium phosphate buffer, pH 7.4, 0.3 mM EDTA, 100 µM cytochrome c, 25 µM
2,3-dimethoxy-5-methyl-6(10-bromodecyl)-1,4-benzoquinol, and an
appropriate amount of membrane or purified cytochrome
bc1 complex. Chromatophores or ICM were diluted
with 50 mM Tris-Cl, pH 8.0, containing 20% glycerol and 1 mM MgSO4 to a final concentration of cytochrome
b of 5 µM. No detergent was added to the
diluted mixture in order to preserve the bc1
activity. 5 µl of diluted membrane was added to the assay mixture.
Activity was determined by measuring the reduction of cytochrome
c (the absorbance increase at 550 nm), using a millimolar
extinction coefficient of 18.5 cm Treatment of Membranes with Reaction of Genetically Engineered Cysteine Residues in Membranes
with NEM or PCMB--
Freshly prepared NEM (100 mM in
H2O) was added to the Molecular Modeling--
Molecular modeling was carried out in a
Indigo II Silicon Graphics Station. A peptide with NCSCQVQALA sequence,
corresponding to the ISP neck sequence of the R. sphaeroides
PSA-CSC mutant, was built using the Builder Module from Insight II
software from Molecular Simulation, Inc. Whether this peptide has
acceptable geometry for disulfide bond formation was examined by
minimizing the structure as follows: (a) continued
iterations using steepest descents minimization until the maximum
derivation was less than 10 kcal/Å; (b) continued
iterations using conjugate minimization until the maximum derivation
was less than 1.0 kcal/Å; (c) continued iterations using
va09a minimization until the maximum derivation was less than 0.01 kcal/Å; (d) creation of a disulfide bond; (e) minimization of the peptide using the Optimize command in Builder; and
(f) visual examination of geometry and comparison with a
control (no disulfide bond) using the print energy per residue command.
Other Biochemical and Biophysical Techniques--
Protein
concentration was determined by the method of Lowry et al.
(18). Cytochrome b (19) and cytochrome
c1 (20) were determined according to published
methods. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to Laemmli (21) using a Bio-Rad Mini-protean dual
slab vertical cell. Western blotting was performed using rabbit
polyclonal antibodies against cytochrome b, cytochrome
c1, ISP, and subunit IV of the R. sphaeroides bc1 complex. The polypeptides separated in
the SDS-PAGE gel were transferred to polyvinylidene difluoride membrane
for immunoblotting. Goat anti-rabbit IgG conjugated to alkaline
phosphatase or protein A conjugated to horseradish peroxidase was used
as the second antibody.
EPR spectra were recorded with a Bruker ER 200D apparatus equipped with
a liquid N2 Dewar at 77 K. Instrument settings are detailed
in the figure legends.
Characterization of the Mutants Carrying Cysteine Substitutions in
the Neck Region of the Iron-Sulfur Protein--
Six R. sphaeroides mutants expressing His6-tagged cytochrome
bc1 complexes with single or double cysteine
substitutions at various positions in the ISP neck region were
generated to test the hypothesis that neck flexibility allows ISP head
domain movement required for bc1 catalysis. The
flexibility of the neck should decrease when a disulfide bond is formed
between a pair of substituted cysteines.
Of the six mutants, two, A42C and V44C, are single substitutions, in
which Ala-42 or Val-44 is replaced with cysteine. The other four are
double cysteine substitutions, PSA-CSC, ADV-CDC, VQA-CQC, and
ADVQA-CDVQC, in which Pro40 and Ala42,
Ala42 and Val44, Val44 and
Ala46, or Ala42 and Ala46 are
replaced with cysteines. A plate mating technique was used (22) to
transfer the pRKDfbcFmBC6HQ plasmid
from E. coli S17 to R. sphaeroides BC17. The
mating took place in less than 16 h on the LB/SIS plates. R. sphaeroides BC17 cells harboring
pRKDfbcFmBC6HQ plasmid were selected
by spreading the conjugated cell mixture on enriched Sistrom's plate
containing tetracycline and kanamycin sulfate. It took 4 days for the
A42C, V44C, ADVQA-CDVQC, and VQA-CQC mutant colonies to show up on the
plate, the same time period as that required for complement colonies.
However, it took about 7 days for the ADV-CDC and PSA-CSC mutant
colonies to appear. This slower growth rate on the plates is an
indication of zero or reduced bc1 activity in
the virtual absence of environmental selection pressure. A similar
phenomenon was observed with several cytochrome b mutants
that had no bc1
activity.2
When mid-log phase, aerobically grown complement and mutant cells were
inoculated into enriched Sistrom medium and subjected to anaerobic
photosynthetic growth conditions, the A42C, V44C, VQA-CQC, and
ADVQA-CDVQC mutants grow at a rate comparable with that of complement
cells, the ADV-CDC mutant has a retarded (50%) growth rate, and
PSA-CSC does not grow photosynthetically (Table I). Chromatophores from the A42C, V44C,
and ADVQA-CDVQC mutant cells have cytochrome bc1
complex activity comparable with that of the complement chromatophores.
The VQA-CQC and ADV-CDC mutant chromatophores have, respectively, 77 and 68% of the bc1 complex activity found in
complement chromatophores. ICM from the PSA-CSC mutant have no
ubiquinol-cytochrome c reductase activity. This was
expected, since bc1 complex is required for
photosynthetic growth and this mutant does not grow
photosynthetically.
To determine whether the loss (or decrease) of the cytochrome
bc1 complex activity in the mutant membranes
results from a lack of or improper assembly of ISP protein in the
membrane, the amount of ISP and its EPR characteristics in mutant and
complement membranes were compared. Western blot analysis with
antibodies against R. sphaeroides cytochrome b,
cytochrome c1, ISP, and subunit IV revealed that
the amount of these four subunits in the six mutant membranes is the
same as that in the complement membrane (Fig.
1, lanes 2-8). Absorption
spectral analysis shows that the content of cytochrome b and
c1/c2 in all of these
mutant membranes is the same as that in complement membrane. These
results indicate that the mutations did not affect the assembly of ISP
protein into the membrane. The [2Fe-2S] cluster in all of these
mutant membranes has an EPR spectrum identical to that observed in
complement chromatophores, with resonance at gx = 1.80 and
gy = 1.9 (Fig. 2). Thus the
mutations did not change the microenvironment of the iron-sulfur
cluster.
Effect of Mutation on the Disulfide Bond Formation in the Neck
Region of the Iron-Sulfur Protein--
The bc1
complex activity decreased by 23, 32, 14, and 100% in the VQA-CQC,
ADV-CDC, ADVQA-CDVQC, and PSA-CSC mutant membranes, respectively, with
no change in the amount of ISP incorporated into the membrane and the
EPR characteristics of the [2Fe-2S] cluster. Did this activity loss
result from increased neck region rigidity due to the formation of a
disulfide bond? We addressed this question by comparing cytochrome
bc1 complex activity in mutant and complement
membranes with and without
When ICM from the PSA-CSC mutant, which has no
bc1 activity, was treated with
Formation of a disulfide bond between Cys40 and
Cys42 in the ISP neck of the PSA-CSC mutant is also
supported by molecular modeling using the peptide sequence of
NCSCQVQALA, corresponding to the neck sequence of the PSA-CSC mutant.
In the bovine bc1 structure (4), the distance
between C-
The possibility that the lack of bc1 activity in
the PSA-CSC mutant ICM is due to the formation of a intermonomer
disulfide bond between one of the two cysteines at positions 40 or 42 and a cysteine in the other monomer is ruled out because
bc1 complex activity in the A42C mutant
chromatophore or ICM is the same as that in complement membranes and is
not activated by the addition of
When the VQA-CQC and ADVQA-CDVQA mutant chromatophores, which have,
respectively, 77 and 86% of the cytochrome bc1
complex activity of the complement chromatophore, were treated with
The finding that the ADV-CDC mutant ICM has only 20% as much
bc1 complex activity as does its chromatophore
membrane is rather surprising (Table I), because
bc1 complex activities in chromatophore and ICM
are comparable in all of the other cysteine-substituted mutants and in
complement cells (Table I). The addition of
It should be noted that the loss of bc1 complex
activity in the ADV-CDC mutant ICM is not due to the formation of an
intermonomer disulfide bond, because the distance between potential
cysteine pairs in the two monomers is over 30 Å. Recall that the
bc1 activity in the single cysteine substitution
mutants A42C and V44C was not affected by the addition of
Since disulfide bonds occur in ICM, but not in the chromatophore, of
the ADV-CDC mutant, probably disulfide bond formation in this mutant
ISP neck region depends on the oxygen supply in the cell culture and is
related to the functionality of the bc1 complex
in the membranes. When these mutant cells are grown in the dark with an
ample oxygen supply, no bc1 activity is detected in the cytoplasmic membrane due to the formation of a disulfide bond in
the ISP neck region. The addition of Effect of Mutation on Binding Affinity of the Iron-Sulfur Protein
in the bc1 Complex--
Although chromatophores from
mutants with a double cysteine mutation in the ISP neck region
(VQA-CQC, ADV-CDC, and ADVQA-CDVQC) have ISP amounts, EPR properties,
and ISP neck flexibility similar to those in complement chromatophores,
the bc1 activities are 23, 32, and 14% less,
respectively. Is this activity loss due to a decreased binding affinity
of ISP to the whole complex? Assuming that ISP binding is sensitive to
detergent treatment, any difference in subunit composition of purified
His-tagged bc1 complexes from mutant and
complement strains will indicate detergent lability and binding
affinity differences between normal and mutant ISPs.
When chromatophores from these three mutant cells, VQA-CQC, ADV-CDC,
and ADVQA-CDVQC, at a cytochrome b concentration of 25 µM were mixed with various amounts of dodecylmaltoside
(up to 0.44 mg/nmol of cytochrome b), the cytochrome
bc1 complex activities decreased as the
concentration of dodecylmaltoside (DM) increased (Fig.
3A). More than 95% of the
bc1 complex activity in all of these mutant
chromatophores was inactivated by 0.44 mg of DM/nmol of cytochrome
b. Under identical conditions, complement chromatophores did
not lose activity (Fig. 3A), indicating that the mutant
cytochrome bc1 complexes (two-cysteine
substitution) are more labile to detergent.
Since DM solubilizes the bc1 complex from
R. sphaeroides chromatophores, it is important to establish
that DM denaturation of the bc1 complex in
mutant chromatophores is due to ISP dissociation from the complex and
not due to a decreased affinity for membrane. This is done by
centrifuging DM-treated chromatophores from mutants and complement
cells at 100,000 × g for 30 min to separate the solubilized from the unsolubilized fractions and measuring cytochrome content, ISP amount, and bc1 activity in both
fractions. Since the amount of cytochrome bc1
complex protein (cytochromes b and c1, ISP, and subunit IV) in the supernatant
fractions of the three mutant chromatophores is comparable with that in
the supernatant fraction from complement chromatophores at a given
concentration of detergent, it is apparent that DM solubilization of
the cytochrome bc1 complex is not affected by
mutation (data not shown). However, the specific activity of cytochrome
bc1 complex, based on b content in
the supernatant fractions from mutant chromatophores, decreased as the
detergent concentration increased (Fig. 3B). Under identical conditions, the bc1 activity in supernatant
fractions from complement chromatophores does not drastically decrease.
The extraction efficiency, based on cytochrome b content, is
the same for all mutants and complement membranes (Fig.
3C).
To confirm that the loss of bc1 activity in the
DM-solubilized fractions resulted from dissociation of ISP from the
complex, the supernatant fractions, extracted by 0.5 or 1% of DM, were passed through a Ni2+-NTA column. The column effluents and
eluates were examined for bc1 complex subunit
composition. Two subunits, corresponding to cytochromes b
and c1, were found in the Ni2+-NTA
column eluates (Fig. 4, lanes
5-7). ISP and subunit IV were detected in the column effluents by
Western blot analysis (Fig. 1, lanes 9-11). On the other
hand, four subunits (cytochrome b, cytochrome
c1, ISP, and subunit IV) in unit stoichiometry
are in the Ni2+-NTA column eluates from
detergent-solubilized complement chromatophores (Fig. 4, lane
2). These results indicate that a double cysteine substitution in
the ISP neck region decreases the binding affinity of ISP for the
bc1 complex. Therefore, detergents dissociate
ISP from the complex. Since no ISP is found in the Ni2+-NTA
column eluates from the DM (0.5%)-solubilized mutant chromatophore fractions having 40% of the untreated cytochrome
bc1 complex activity, the activity detected in
this fraction reflects the ability of dissociated ISP to reconstitute
into a functionally active bc1 complex after
dilution of detergent in the assay mixture. Perhaps the activity
decrease in untreated mutant chromatophores indicates the extent of ISP
dissociation from the complex. However, the 14% decrease in
bc1 complex activity in the A42C or V44C mutant chromatophores is probably due to a deviation in the activity assay and
not to a mutational effect, since their activities are not labile to
detergents. Indeed, all four subunits of the bc1 complex are absorbed on the Ni2+-NTA column after the
detergent solubilization (Fig. 4, lanes 2 and 3).
The purified complexes from these two mutants have specific activities
comparable with that of the complement (Table I).
Although the restored bc1 complex activity in
the PSA-CSC mutant ICM treated with
The ability of detergent-dissociated ISP to reconstitute into
functionally active bc1 complex depends on the
detergent concentration in the reconstituting mixture (or in the assay
mixture) and the status of the dissociated ISP. It has been reported
(23) that including reducing reagents, such as dithiothreitol or
-mercaptoethanol (
-ME). The restored activity is diminished upon
removal of
-ME but is retained if the
-ME-treated membrane is
treated with the sulfhydryl reagent N-ethylmaleimide or
p-chloromercuribenzoic acid. These results indicate that
the loss of bc1 complex activity in the ADV-CDC
or PSA-CSC mutant membranes is due to disulfide bond formation, which
increases the rigidity of ISP neck and, in turn, decreases the mobility
of the head domain. Using the conditions developed for the isolation of
His-tagged complement cytochrome bc1 complex, a
two-subunit complex (cytochromes b and c1) is obtained from all of the double
cysteine-substituted mutants. This suggests that introduction of two
cysteines in the neck region of ISP weakens the interactions between
cytochromes b, ISP, and subunit IV.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-ME),
N-ethylmaleimide (NEM), and
p-chloromercuribenzoic acid (PCMB) are from Sigma. All other
chemicals are of the highest purity commercially available.
80 °C in the
presence of 20% glycerol. The His6-tagged cytochrome
bc1 complexes were purified from chromatophores by the method of Tian et al. (14). His6 tag is
located at the C terminus of the cytochrome
c1 subunit.
1
mM
1. Nonenzymatic oxidation of
2,3-dimethoxy-5-methyl-6(10-bromodecyl)-1,4-benzoquinol, determined
under the same conditions in the absence of enzyme, was subtracted. The
purified bc1 complex activity assay is
essentially as described previously (14).
-ME--
-ME was added to
membrane preparations (40 µM cytochrome b) to
a final concentration of 100 µM. After incubation on ice
for 10 min, aliquots were removed from the mixture for assay.
-ME was removed from the treated membrane by washing with 50 mM
Tris-Cl, pH 8.0, and 1 mM MgSO4 and
centrifuging at 80,000 rpm for 30 min with a Beckman TL
ultracentrifuge. This process was repeated three times.
-ME-pretreated chromatophore or
ICM (concentration is at 40 µM cytochrome b)
to a final concentration of 500 µM. The mixture was
flushed briefly with nitrogen, sealed, and incubated at room
temperature for 15 min before measuring cytochrome
bc1 complex activity. The stock solution of PCMB
was prepared by first dissolving PCMB powder in 0.5 N NaOH
followed by neutralization. Modification of cysteine with PCMB was
carried out as described for NEM. Excess NEM and PCMB was removed by
repeated washing and centrifuging, and enzymatic activity was redetermined.
RESULTS AND DISCUSSION
Characterization of ISP neck cysteine mutants
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Fig. 1.
Western blot analysis of the cytochrome
bc1 complexes in mutant and chromatophore
membranes. Membrane samples containing 75 pmol of cytochrome
b were loaded into each well and subjected to SDS-PAGE. The
proteins in the gel were transferred electrophoretically to a
polyvinylidene difluoride membrane and reacted with antibodies against
R. sphaeroides cytochrome b (Cyt b),
cytochrome c1 (Cyt c1),
ISP, and subunit IV (Sub IV). After transfer, membrane was
cut into two pieces so that the upper part contained proteins with a
molecular mass greater than 23 kDa, including cytochromes b
and c1, and the bottom part contained the low
molecular mass proteins, including ISP and subunit IV. Since antibodies
against R. sphaeroides cytochrome bc1
complex available in our laboratory have antibody titers for
cytochromes b and c1 much higher than
those for ISP and subunit IV, the top section of the membrane was
developed with a horseradish peroxidase system, whereas the bottom
section was developed with an alkaline phosphatase system. Lane
1, prestained molecular mass standards; lane 2, the
complement chromatophores; lanes 3-7, the A42C, V44C,
VQA-CQC, ADV-CDC, and ADVQA-CDVQC chromatophores; lane 8,
the PSA-CSC ICM; lanes 9-12, effluent from the
Ni2+-NTA column, which is the unbound portion from
detergent (1% DM)-solubilized membrane fraction of the VQA-CQC,
ADV-CDC, PSA-CSC, and ADVQA-CDVQC mutants.
View larger version (20K):
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Fig. 2.
EPR spectra of the [2Fe-2S] cluster of the
Rieske iron sulfur protein in mutant and complement membranes.
Chromatophore membranes from the A42C, V44C, VQA-CQC, ADV-CDC, and
ADVQA-CDVQC mutants and complement cells and ICM from the PSA-CSC
mutant were incubated with a small amount of ascorbate on ice for about
10 min and frozen in liquid nitrogen. EPR spectra were recorded at 77 K
with the following instrument settings: microwave frequency, 9.37 Hz;
microwave power, 20 milliwatts; modulation amplitudes, 20 G; modulation
frequency, 100 kHz; time constant, 0.1 s; scan rate, 20 G/s.
-ME treatment. Also, we used molecular
modeling to examine the feasibility of disulfide bond formation in the
PSA-CSC mutant.
-ME, about
60% of the activity found in complement ICM was restored (Table
II). It should be emphasized that the observed activity restoration is not due to nonenzymatic reduction of
cytochrome c by
-ME, because less than 0.05 µM
-ME is present in the assay mixture, and the
restored activity is sensitive to antimycin. This restored cytochrome
bc1 complex activity is diminished when
-ME
is removed by repeated centrifugation and suspension. However, the
restored bc1 activity is retained if the
-ME-treated membrane is reacted with sulfhydryl modifying reagents,
such as NEM or PCMB, at 5-fold molar excess to
-ME, followed by
repeated centrifugation and suspension (Table II). Activity restoration is not observed when the PSA-CSC mutant membrane is treated with NEM or
PCMB without prior reduction with
-ME. These results indicate that
the complete lack of the bc1 activity in the
PSA-CSC mutant membrane results from decreased mobility of the ISP head
domain, due to a neck region made rigid by the formation of a disulfide bond between Cys40 and Cys42. The addition of
-ME reduces this disulfide bond, restoring flexibility to the neck
region. Modification of
-ME released SH- groups with NEM or PCMB
has no effect on the flexibility of the neck region but blocks
reformation of the disulfide bond, thus guaranteeing retention of the
restored activity.
The effect of -ME, NEM, and PCMB on the cytochrome bc1
complex activities in chromatophore and ICM membranes from mutant and
complement cells
atoms of Ala64 and Ala66
(corresponding to residues 40 and 42 of R. spheroides ISP)
is 5.2 Å, and the side chains of the two residues point in the same direction relative to the backbone of this stretch. Molecular modeling,
based upon this structural information, indicates that a disulfide bond
can be formed. This would result in an 11-membered ring structure. A
distance of 5.2 Å for disulfide bond formation is slightly longer than
the 4.1 Å observed for the two C-
atoms of the disulfide bond,
between Cys160 and Cys144 of ISP, in bovine
bc1 crystals (4).
-ME. Furthermore, the distance
between the two cysteines at position 40 and 40' in ISP from different
monomers appears to be over 33 Å, as estimated from the distance
between Ala64 of one ISP monomer and Ala64' in
the symmetrically related monomer in the bovine
bc1 structure. Intermonomer disulfide bonding
over such a long distance is impossible.
-ME, the bc1 activities were unchanged (Table
II). Also, when
-ME-pretreated mutant chromatophores were reacted
with NEM or PCMB, no change in activity was observed (Table II). These
results proved that no disulfide bonds are formed between the cysteines
at positions 44 and 46 or positions 42 and 46. The lack of disulfide
bond formation in the ISP neck region of these two mutant
chromatophores is also supported by the bovine
bc1 crystal structure, which shows that the
distances between C-
atoms of Val68 and
Ala70 of ISP (Val44 and Ala46 in
R. sphaeroides) and between C-
atoms of Ala66
and Ala70 (Ala42 and Ala46 in
R. sphaeroides) are 7.35 and 12 Å, respectively. These
exceed the distance that permits disulfide bond formation. Since no
disulfide bond is formed, the flexibility of the ISP neck should not
change in these mutant chromatophores. Thus, the decreased
bc1 activity in these two mutant chromatophores
cannot be attributed to decreased mobility of the head domain of
ISP.
-ME to ADV-CDC mutant
membrane has no effect on bc1 complex activity in chromatophores, while increasing the activity in ICM to the level
observed in untreated chromatophores (Table II). The restored activity
in the mutant ICM is diminished by removal of
-ME. The addition of
5-fold molar excess NEM or PCMB to the
-ME-treated mutant ICM
preserves the restored activity in the membrane. These results indicate
that the loss of bc1 activity in the ADV-CDC mutant ICM, compared with that in its chromatophore, results from the
decreased mobility of the head domain of ISP, due to formation of a
disulfide bond between Cys42 and Cys44 in the
neck region of ISP. However, a similar explanation does not account for
the lower (32% less than complement) bc1
complex activity in ADV-CDC mutant chromatophore or in
-ME-treated
ICM, because neither contain disulfide bonds. Apparently, some other effect of cysteine substitution decreased the
bc1 activity.
-ME. In
the bovine bc1 complex structure, the distance
between C-
atoms of Ala66 and Val68
(corresponding to residues Ala42 and Val44 in
R. sphaeroides) is 7 Å, and the orientations of the side
chains are opposite each other. However, since the neck region is
flexible, a different crystal form might result in a different
conformation. This comparison is at the "noise" level of homologous
sequence similarities, e.g. bovine versus
R. sphaeroides. Therefore, formation of a disulfide bond
between Cys42 and Cys44 in ADV-CDC mutant ICM
is not impossible.
-ME to the cytoplasmic membrane
can restore bc1 activity to the level observed
in the chromatophore membrane. This is consistent with the fact that bc1 complex is not required for dark aerobic
growth of this organism because the cells can utilize quinol oxidases.
When the O2 supply is limited, the cells adjust their
protein expression to prepare for photosynthetic growth, as indicated
by the appearance of chlorophyll. The bc1
activity in ICM increases slightly (0.3 µmol of cytochrome c reduced per min per nmol of b; Table I),
because either some part of the bc1 complexes
become active by disulfide bond cleavage or the newly synthesized
complexes stay in a conformation that prevents disulfide bond formation
between Cys42 and Cys44. When oxygen is
completely depleted from the culture, the cells undergo photosynthetic
growth and require active bc1 complex. Apparently, no disulfide bonds are formed in the mutant
chromatophores, and bc1 activity increases
greatly (1.5 µmol of cytochrome c reduced per min per nmol
of b; Table I).
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Fig. 3.
The effect of dodecylmaltoside concentration
on the activity and the solubilization efficiency of
bc1 complex from membranes of the double
cysteine-substituted mutants and the complement cells. The
bc1 complex activities in the extraction mixture
(A) as well as in the supernatant fractions (B)
obtained from membranes treated with various concentrations of
dodecylmaltoside are expressed as the percentage of that of the
untreated sample. Extraction efficiency (C) was calculated
as the ratio of the cytochrome b present in the
detergent-solubilized supernatant fraction to the total amount of
cytochrome b in the extraction mixture. The -ME-restored
bc1 activity in the PSA-CSC mutant ICM was used
as 100% activity for this mutant membrane. Membranes from the
complement (
), VQA-CQC (+), ADV-CDC (
), ADVQA-CDVQC (
), and
PSA-CSC (
) cells were adjusted to 25 µM cytochrome
b with 50 mM Tris-Cl, pH 8.0, containing 20%
glycerol. Dodecylmaltoside was added to give the final concentrations
as indicated.
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Fig. 4.
SDS-PAGE of purified His6-tagged
bc1 complexes. Aliquots of cytochrome
bc1 complexes(75 pmol of cytochrome
b), recovered from the Ni2+-NTA column of
DM-solubilized mutant and complement membrane fractions were loaded
into each well of a SDS-PAGE gel. Lane 1, prestained
molecular weight standards; lane 2, cytochrome
bc1 complex from the wild-type complement
strain; lanes 3-8, the bc1 complexes
from mutants A42C, V44C, VQA-CQC, ADV-CDC, ADVQA-CDVQC, and PSA-CSC,
respectively.
-ME is more labile to DM
treatment than are the bc1 activities in
VQA-CQC, ADV-CDC, and ADVQA-CDVQC mutant chromatophores (Fig. 3), the
effectiveness of dodecylmaltoside in solubilizing
bc1 complex from PSA-CSC mutant ICM and the
bc1 complex subunit composition of the
Ni2+-NTA column effluents and eluates (Fig. 1, lane
11, and Fig. 4, lane 8) are the same as that for the
three double cysteine-substituted mutant chromatophores. Probably the
binding affinity of ISP for the bc1 complex in
PSA-CSC is weaker than in the other double cysteine mutant
chromatophores. Thus, ISP is more easily dissociated from the complex
by detergent. Since only 60% of the bc1
activity found in complement chromatophore is restored to the PSA-CSC
mutant ICM upon the addition of
-ME, it is likely that 40% of the
ISP in this mutant ICM is dissociated from the
bc1 complex.
-ME, during isolation of ISP from the complex protects free ISP from
oxidation by oxygen and that the addition of phospholipid to the
detergent-containing solution reduces effective detergent
concentration. Therefore, we examined the effect of
-ME and
phospholipid on the reassociation of ISP into functionally active
bc1 complex in the DM-solubilized chromatophore
fraction from the ADV-CDC mutant cells. As described above,
-ME has
no effect on the bc1 activity in this mutant
chromatophore or on the solubilization efficiency of DM.
-ME
preserves the activity of mutant ISP after its dissociation from
bc1 complex. In the presence of
-ME, when the
mutant chromatophore is treated with 1.2% dodecylmaltoside, the
cytochrome bc1 complex activity in the
supernatant fraction is 10-fold higher than that in the supernatant
fraction obtained from mutant chromatophore without pretreatment with
-ME. The activity of the supernatant with
-ME is about 50% of
that in the chromatophore before detergent solubilization. When the
detergent-solubilized fraction obtained from the
-ME-treated mutant
chromatophore is incubated with phospholipid (10 mg/ml), the cytochrome
bc1 complex activity increases to 95% of that
in the untreated chromatophore. Without prior addition of
-ME, the supernatant has minimal activity (2%), which only increases slightly after the addition of phospholipid. These results indicate that the
addition of
-ME to the ADV-CDC chromatophore prior to detergent solubilization prevents denaturation of dissociated ISP and subunit IV
and thus enables them to reconstitute with cytochromes b and c1 to form functionally active
bc1 complex after excess detergent is removed by
phospholipid and dilution with the assay mixture.
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ACKNOWLEDGEMENT |
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We thank Dr. Roger Koeppe for critical review of this manuscript.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCS9605455, National Institutes of Health Grant GM 30721, and Oklahoma Agricultural Experiment Station Grant OKL 01819.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.: 405-744-6612;
Fax: 405-744-7799; E-mail: cayuq{at}okway.okstate.edu.
2 H. Tian, S. White, L. Yu, and C-A. Yu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
[2Fe-2S] cluster, iron sulfur cluster of Rieske iron-sulfur protein;
bL, low
potential heme b;
bH, high potential heme b;
DM, dodecylmaltoside;
NTA, nitrilotriacetic acid;
ICM, intracytoplasmic membrane(s);
PAGE, polyacrylamide gel electrophoresis;
ISP, iron-sulfur
protein;
-ME,
-mercaptoethanol;
NEM, N-ethylmaleimide;
PCMB, p-chloromercuribenzoic acid.
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REFERENCES |
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