(Received for publication, July 20, 1995; and in revised form, September 20, 1995)
From the
The cytochrome b subunit (subunit I) of the
ubiquinol-cytochrome c reductase (bc complex) is thought to participate in the formation of two
quinone/quinol reaction centers, an oxidizing center (Q
)
and a reducing center, in accordance with the quinone cycle mechanism.
Threonine 160 is a highly conserved residue in a segment of subunit I
that was shown to bind quinone and is placed near the putative Q
site in current models of the bc
complex. Rhodobacter sphaeroides cells expressing bc
complexes with Ser or Tyr substituted for Thr
grow
photosynthetically at a reduced rate, and cells expressing the mutated
complexes produce an ``elevated'' level of the bc
complex. The Ser substitution also affects the
interaction of subunit IV with subunit I. Replacement of Thr
by Ser results in about a 70% loss of the activity in the
purified complex, whereas substitution by Tyr lowers the activity by
more than 80%. Both replacements lower the apparent K
for ubiquinol. Electron paramagnetic
resonance (EPR) spectroscopy shows that in the Ser substituted complex,
the environments of the Rieske iron-sulfur cluster in subunit III and
the high potential cytochrome b (b
) in
subunit I have been modified. The spectra of the Ser
and
Tyr
iron-sulfur clusters have become redox-insensitive,
with a line shape resembling that of the native complex in the fully
reduced state. The EPR signal of b
in the
Ser
complex is shifted from g = 3.50 to g = 3.52, but otherwise the line shape is very similar
to the spectrum of the native complex. Most of these results are
consistent with current ideas regarding the structure and function of
Q
in the bc
complex, except for the
alteration of the b
EPR feature, because this
heme is not thought to be located in proximity to Q
.
Immunoblotting analysis showed that the Ser or Tyr substituted complex
contained significantly less than a stoichiometric amount of subunit
IV. The enzymatic activity of mutated bc
complex
was found to be activable by the addition of purified subunit IV. These
results indicate that Thr
plays an important role in the
structure and/or function of the bc
complex.
The ubiquinol-cytochrome c reductase (or the bc complex) is a key component of the
energy-conserving electron transfer chains of mitochondria and many
respiratory and photosynthetic bacteria. It catalyzes the oxidation of
ubiquinol by cytochrome c and the coupled vectorial
translocation of protons to generate a transmembrane pH gradient.
Although many aspects of the structure and function of this enzyme
complex have been
elucidated(1, 2, 3, 4, 5, 6, 7) ,
the nature of the protein structures involved in the binding and
reactions of ubiquinone and ubiquinol remain an active area of inquiry,
and the identification of the residues involved in subunit interaction
is in the initial stages.
The cytochrome bc complex from the purple photosynthetic bacterium Rhodobacter
sphaeroides has been purified and characterized in several
laboratories(8, 9, 10, 11, 12, 13) .
The purified complex contains four protein subunits with five redox
cofactors: two b-type cytochromes (b
and b
), one c-type cytochrome
(cytochrome c
), one high potential iron-sulfur
cluster (the Rieske [2Fe-2S]), and at least one ubiquinone
(Q). (
)This bc
complex is functionally
analogous to mitochondrial ubiquinol-cytochrome c reductase,
and the three largest subunits are homologous to their mammalian
counterparts (14) . The degree of sequence similarity is
particularly striking among the cytochrome b polypeptides from
a wide range of species (2) .
The cytochrome b polypeptide contains the two b hemes and participates in
the ubiquinol oxidation and ubiquinone reduction reactions of the Q
cycle(6) . He et al.(15) have identified two
regions of the bovine cytochrome b subunit that may
participate in quinone binding using specific labeling with
photoreactive quinone analogs. We have created a number of relatively
conservative amino acid replacements in the region of the R.
sphaeroides protein that corresponds to the first of these
putative quinone-binding regions in the bovine cytochrome b.
While screening the chromatophore membranes isolated from
photosynthetically grown R. sphaeroides cells expressing the
various mutated forms of cytochrome b, we noted that the
substitution of a serine for threonine at position 160 resulted in the
loss of about two-thirds of the ubiquinol-cytochrome c reductase activity in the membranes relative to membranes from
control cells. Combined with the fact that this threonine residue is
one of the most conserved amino acids in the primary structure of
cytochrome b, this result may indicate that Thr is an important residue involved in the structure and function of
the bc
complex. Here we report the purification
and further characterization of the complexes containing conservative
substitutions at position 160 of cytochrome b, which affect
the activity of the enzyme and the properties of redox centers residing
in separate subunits. In general, the results are consistent with
current models, placing this region of cytochrome b at or near
the quinol-oxidizing center and the intersubunit interface with the
iron-sulfur subunit. However, the substitution of serine for threonine
160 also has an effect on cytochrome b
, which is
not generally thought to interact directly with the quinol-oxidizing
center. Thr
may also play a role in the proper
interaction of subunit IV and cytochrome b.
Figure 1:
Plasmids
constructed for mutagenesis, transfer, and expression of the R.
sphaeroides fbc operons containing mutated fbcB genes
encoding altered cytochromes b. A, pSELNB3503 was
derived from pALTER-1 (pSELECT-1) by insertion of a 3.5-kilobase pair R. sphaeroides DNA fragment containing the fbc operon
(see ``Experimental Procedures''). fbcF, fbcB, and fbcC denote the genes encoding the
iron-sulfur subunit, the cytochrome b subunit, and the
cytochrome c subunits, respectively. The
restriction sites marked by asterisks were engineered in
vitro to facilitate verification and transfer of the segment of
the fbcB gene containing the mutations (see text). Only
selected restriction sites are shown. B and C,
broad-host-range plasmids pRKDNB3503, containing the engineered
wild-type fbcFBC operon, and pRKDNB35KmBP were constructed to
facilitate subcloning, conjugal transfer to R. sphaeroides,
and expression of cytochrome bc
complexes
containing altered cytochromes b.
The cytochrome bc complexes were purified from chromatophores by
a modification of the procedure of McCurley et
al.(11) . Dodecyl maltoside was used to solubilize the bc
complexes and 20% glycerol was included during
the solubilization and loading of the first column (after Andrews et al.(12) ) to help stabilize mutated bc
complexes in case they prove to be less stable
to the extraction conditions. Chromatophore suspensions were adjusted
to about 18 µM cytochrome b by addition of 50
mM Tris-HCl (pH 8.0 at 0 °C) containing 20% glycerol, 1
mM MgSO
, and 1 mM phenylmethylsulfonyl
fluoride. Dodecyl maltoside (10% solution in 50 mM Tris-HCl
(pH 8.0 at 0 °C) containing 20% glycerol and 1 mM MgSO
) was added to the chromatophore suspension to
0.525 mg/nmol cytochrome b, and the mixture was stirred for 30
min at 0 °C and then centrifuged at 27,000
g for
30 min. The hard precipitates at the bottom of the centrifuge tubes
were discarded, and the loose pellets and supernatants were collected.
4 M NaCl was added to a final concentration of 0.1 M,
and the suspension was stirred for 1 h at 0 °C. The mixture was
centrifuged at 200,000
g for 90 min. The supernatants
were collected and applied to a DEAE-Biogel A column equilibrated with
50 mM Tris-HCl (pH 8.0 at 0 °C) containing 20% glycerol,
100 mM NaCl, 1 mM MgSO
, 5 mM NaN
, and 0.01% dodecyl maltoside (TMGD buffer
containing 100 mM NaCl). The column was washed with, in
sequence, 3.33 volumes of TMGD buffer containing 100 mM NaCl,
2 volumes of TMD buffer (TMGD buffer without glycerol) containing 150
mM NaCl, and 2 volumes of TMD buffer containing 200 mM NaCl. The crude cytochrome bc
complex was
eluted from the column with TMD buffer containing 300 mM NaCl.
The collected bc
complex was diluted with one-half
volume of TMD containing 40% glycerol and applied to a DEAE-Sepharose
CL-6B column equilibrated with TMD buffer containing 100 mM NaCl. The column was washed with 2 column volumes each of TMD
buffer containing 150 mM NaCl, 200 mM NaCl, and 250
mM NaCl. The nearly pure cytochrome bc
complex was eluted with TMD buffer containing 375 mM NaCl and concentrated using a Centriprep-10 concentrator to a
final concentration of 100 µM cytochrome b or
greater. Glycerol was added to about 20%, and the purified complex was
stored at -80 °C. The purity of the bc
preparations estimated by SDS-PAGE was 90-95%.
SDS-PAGE was performed according to Laemmli (42) and to
Schägger and von Jagow (43) using a Bio-Rad
Mini-Protean dual slab vertical cell. Disaggregation of the sample and
resolution was best obtained using a freshly prepared solubilization
buffer containing 5% SDS, 10 mM EDTA (pH 8.0), 5%
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 10%
glycerol, followed by separation on a 16.5% polyacrylamide gel as
described(43) . Samples were incubated for 1-2 h at
30-37 °C immediately prior to loading onto the gel.
Chromatophores were extracted with acetone/methanol (36) before
solubilization. Western blotting was carried out as described (44) using rabbit antibody raised against purified subunit IV (33) or against purified subunit II (cytochrome c)(45) . The polypeptides separated in the
SDS-PAGE gel were transferred to a 0.2-µm 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.
Low temperature EPR spectra were
obtained with a Bruker ER 200D spectrometer equipped with an Air
Products flow cryostat. Some spectra were recorded at 77 °K using a
liquid N Dewar. Instrument setting details are provided in
the legends of the relevant figures.
The
mutation Thr
Tyr (T160Y) was also subsequently
prepared. R. sphaeroides cells expressing this variant of
cytochrome b are also able to grow photosynthetically at a
reduced rate. The cytochrome b content of membranes from these
cells is also elevated relative to membranes from complement cells (Table 1) but to a slightly lesser extent than membranes
containing the T160S complex. The specific ubiquinol-cytochrome
reductase activity of these membranes was even lower than that of
membranes containing the T160S complex, only 10-20% of that found
in complement membranes.
The bc complexes were
extracted with dodecyl maltoside and purified by a modification of
described methods (see ``Experimental Procedures'') from
chromatophore membranes of Rs BC17 cells expressing the
Thr
Ser or Tyr mutations and from membranes of
BC17 expressing the cloned wild-type complex (complement cells). The
T160S complex is spectrally identical to the complement complex (Fig. 2) but retains the lower activity seen in the
chromatophores. The absorbance spectrum of T160Y bc
is also similar to that of the complement bc
, but the ratio of b to c
is lower for T160Y. The biochemical properties
of the purified enzymes are summarized in Table 2. SDS-PAGE (Fig. 3) shows that the T160S and T160Y complexes contain the
same four subunits as the wild-type and complement enzymes, but the
T160S complex has a reduced level of subunit IV (estimated by
densitometry to be about one half the level present in the complement bc
complex). Indeed, addition of purified,
recombinant subunit IV to the enzyme caused a 70% increase in the
activity of the T160S bc
complex (Fig. 4).
Addition of subunit IV to the complement bc
complex also produced some stimulation of the activity. By
comparison, addition of exogenous subunit IV to purified three-subunit
complex (completely lacking subunit IV(46) ) resulted in a
5-fold stimulation of the basal specific activity under the conditions
used (Fig. 4). Apparently not all of the recombinant subunit IV
preparation used was in an active conformation for interaction with
depleted bc
complexes, because full stimulation of
the three-subunit bc
required more than three
molecules of subunit IV per bc
complex.
Examination of additional preparations of wild-type, complement, and
mutant bc
complexes by SDS-PAGE (not shown)
indicated that the relative amount of subunit IV present varies
somewhat for each preparation, but purified T160S bc
had a consistently lower ratio of IV relative to subunits I-III
than complement or wild-type complexes.
Figure 2:
Reduced minus oxidized optical absorption
spectra of purified bc complexes.
Ferricyanide-oxidized and dithionite-reduced spectra of bc
complexes diluted to 2.5 µM cytochrome b were recorded at room temperature in 50
mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM
MgCl
, 0.01% dodecyl maltoside, and 20% glycerol, and the
difference spectra were calculated.
Figure 3:
SDS-PAGE of purified bc complexes and Western analysis for subunit IV. Aliquots of
mutated, complement, and wild-type bc
complexes
containing approximately 75 pmol cytochrome b were analyzed on
duplicate gels by SDS-PAGE using the tricine buffer system (A). One gel was transferred to a polyvinylidene difluoride
membrane and probed with antibody raised against R. sphaeroides subunit IV (B). Lanes 1, bc
from wild-type R. sphaeroides; lanes 2, bc
from R. sphaeroides BC17 complement
strain; lanes 3, prestained molecular mass standards; lanes 4, bc
from R. sphaeroides BC17 expressing the T160Y mutation; lanes 5, bc
from R. sphaeroides BC17 expressing
T160S mutation. The concentration of the sample in lane 1 was
overestimated prior to loading on the gel.
Figure 4:
Activation of T160S and three-subunit bc complexes by addition of exogenous, recombinant
subunit IV. Cytochrome bc
complexes of T160S
(
) and the three-subunit core complex (
) diluted to
2.5
µM cytochrome b were mixed with varying amounts
subunit IV in a total volume of 30-50 µl of 50 mM Tris-HCl (pH 8.0 at 0 °C), 250 mM NaCl, 1 mM
MgCl
, 0.01% dodecyl maltoside, and 10% glycerol and
incubated on ice for 1-2 h. 3-µl aliquots were withdrawn for
assay of ubiquinol-cytochrome c reductase activity. The amount
of subunit IV added was based on determination of the protein content
by the method of Lowry(35) . The concentration of bc
complex present was redetermined after dilution
by optical difference spectroscopy and was based on the cytochrome c
content.
In order to examine the
relative amount of subunit IV initially present in the membranes of the
mutant and complement cells, mutant and complement cells were grown
side by side, and chromatophore membranes were prepared at the same
time. Examination of these chromatophores by SDS-PAGE and subsequent
Western blots developed with antibodies raised against subunit IV
indicates that the membranes from the cells expressing T160S cytochrome b contain a significantly lower ratio of subunit IV to
cytochrome b when compared with the complement membranes. It
was estimated to be about 40% of the ratio found in the complement
cells by densitometry corrected for the apparent relative transfer
efficiency (see Fig. 5). T160Y membranes contain a modestly
lower ratio of subunit IV to cytochrome b (estimated to be
80% of the complement level). Complement membranes, in turn,
appear to contain a lower ratio of subunit IV than membranes from
wild-type R. sphaeroides (not shown). It thus appears that R. sphaeroides cells overproducing the fbc genes due
to the presence of multiple copies of the operon on a plasmid and/or
other factors either do not induce a concomitant increase in the levels
of subunit IV or have a more rapid turnover of that subunit. This
effect is most pronounced in the case of the T160S substitution.
Figure 5:
Western analysis of chromatophore
membranes from mutated and complement bc complexes. Membrane samples containing 75 pmol (lanes
1-4) or 150 pmol (lanes 6-9) cytochrome b were extracted with acetone/methanol to remove pigments,
redissolved in loading buffer containing SDS and 2-mercaptoethanol, and
subjected to tricine SDS-PAGE on duplicate gels. One gel was
transferred electrophoretically to a polyvinylidene difluoride
membrane. The membrane was cut into two pieces so that the upper part
contained proteins with a molecular mass greater than about 23 kDa, and
the bottom section contained the low molecular mass proteins. The top
section of the membrane was probed with antibodies to R.
sphaeroides subunit II (cytochrome c
) and
developed with a horseradish peroxidase system, whereas the bottom
section was probed with antibodies to subunit IV and developed with an
alkaline phosphatase system. Only the Western analysis of the membrane
is shown. Lane 1, chromatophores (75 pmol b) from R. sphaeroides BC17 expressing the T160S mutation; lane
2, chromatophores (75 pmol b) from BC17 expressing the
T160Y mutation; lane 3, chromatophores (75 pmol b)
from BC17 complement; lane 4, chromatophores (75 pmol b) from
Q strain (46) lacking subunit IV; lane 5, prestained molecular mass standards; lane 6,
chromatophores (150 pmol b) from BC17 expressing the T160S
mutation; lane 7, chromatophores (150 pmol b) from
BC17 expressing the T160Y mutation; lane 8, chromatophores
(150 pmol b) from BC17 complement; lane 9, chromatophores (150 pmol b) from
Q strain (46) lacking subunit IV.
Figure 6:
EPR spectra of the antimycin-sensitive
ubisemiquinone radical in the T160S cytochrome bc complex. Oxidized complement and T160S cytochrome bc
complexes containing about 300 µM cytochrome b in 50 mM Tris-HCl (pH 8.0), 300
mM NaCl, 1 mM MgCl
, 0.01% dodecyl
maltoside, and 20% glycerol were mixed with a fumarate/succinate
mixture (40:1) such that the final concentration of succinate was
3 mM and a catalytic amount of bovine
succinate-ubiquinone reductase. After recording the spectra over a
range of microwave powers, the samples were thawed and a 2-fold excess
of antimycin A was added. The EPR spectra of the T160S complex at 0.2
milliwatts in the absence (1) and presence (2) of
antimycin are shown together with their difference (1-2;
the antimycin-sensitive radical) spectrum. The inset shows the
uncorrected power saturation curves of the samples. EPR instrument
parameters were a microwave frequency of 9.32 GHz, a modulation
amplitude of 4 G, a time constant of 0.05 s, a scan rate of 2.5 G/s,
and a temperature of 140 K.
Figure 7:
EPR spectra of the iron-sulfur cluster in
T160S and complement bc complexes. A,
cytochrome bc
complexes (300 µM cytochrome b) were partially reduced by the addition of
sodium ascorbate to 1 mM. The samples were incubated on ice
for about 20 min and frozen in liquid nitrogen. EPR spectra were
recorded at 8 K. The instrument settings were: microwave frequency,
9.27 G; microwave power, 2 milliwatts; modulation amplitude, 6.3 G;
time constant, 0.1 s; scan rate, 20 G/s. B, the samples used
in A were thawed on ice and reduced by the addition of a small
excess of sodium dithionite from a buffered solution. The samples were
refrozen, and the EPR spectra were recorded at 10 K. Instrument
settings were the same as in A.
Figure 8:
EPR spectra of b cytochromes in
T160S and complement bc complexes. The
ascorbate-reduced bc
complexes were prepared as
described in the legend to Fig. 7. The EPR spectra were recorded
at 15 K with the following instrument settings: microwave frequency,
9.20 GHz; microwave power, 20 milliwatts; modulation amplitude, 20 G;
time constant, 0.1 s; scan rate, 5 G/s.
Threonine 160 is a highly conserved residue in the primary
sequence of cytochrome b, present in all known mitochondrial
and eubacterial cytochromes b, except those from nematodes (2) . Its position in the primary sequence is near residues
that are altered in mutations conferring resistance to Q
center inhibitors (Gly
, Ile
, and
Thr
), and it is contained within a segment of the
cytochrome b polypeptide that corresponds to a peptide
specifically labeled by azido-quinone in the bc
complex from beef heart(15) . Current interpretations of
experimental data and modeling efforts place this region in an
amphipathic helix, designated as helix-cd, located on the positive side
of the membrane and forming part of the Q
center of the
complex. This region should also be close to cytochrome b
, which is reduced by semiquinone in the Q
center. Indeed, a mutation at the position corresponding to
Ile
in Rhodobacter exhibits an altered circular
dichroism spectrum for cytochrome b
of
yeast(56) . Furthermore, the Rieske [2Fe-2S] cluster
should also be located nearby, although it is bound to a different
subunit of the complex, because it is reduced by quinol in the Q
center and binds certain Q
center inhibitors. As a
conserved residue in a perhaps pivotal region of the complex,
Thr
proves to be a good candidate for investigation using
the method of site-directed mutagenesis combined with biochemical and
biophysical characterization.
Expression of the T160S mutation in
cytochrome b results in a bc complex
having a significant loss of activity partially compensated by a small
decrease in the apparent K
. Interestingly, cells
expressing this mutated cytochrome b during photosynthetic
growth appear to induce an increased level of the three largest
subunits of the bc
complex. This induction could
be a regulatory response of cells expressing the mutated complexes to
lowered electron transfer activity or an elevated ``redox
poise.'' Alternatively, the effect could be caused by a
spontaneous second-site mutation that leads to enhanced expression and
is positively selected by photosynthetic growth. The latter possibility
is unlikely, however, because three T160S clones independently isolated
following mutagenesis all displayed elevated levels of the bc
complex during the first round of
photosynthetic growth following initial amplification by aerobic growth
in the dark. The purified T160S complex contains a significantly
reduced amount of subunit IV, relative to the bc
complex purified from complemented cells. Addition of recombinant
subunit IV to the partially depleted preparations results in an
increase in activity; however, the final activity of reconstituted
T160S bc
complex still remains lower than that of
the complement bc
complex. Given the apparent low
content of subunit IV in the T160S bc
preparations, the less than 2-fold activity increase seen upon
incubation with added exogenous subunit IV is surprising. One possible
explanation is that a large fraction of the purified T160S bc
complex is in a conformation that no longer
interacts with subunit IV, perhaps due to aggregation or partial
denaturation. Another possibility is that the low level of subunit IV
found in the T160S membranes in the first place, as well as any
conformational changes, in some measure results from a decreased
affinity of subunit IV for the modified cytochrome b. The
possibility that Thr
in cytochrome b is directly
or indirectly involved in the binding of subunit IV to the bc
complex is currently under investigation. In
contrast to the expression levels observed with T160S, membranes
isolated from T160Y contain levels of subunit IV more comparable with
the complement chromatophores. Possibly this more hydrophobic
substitution has an effect on the interaction of subunit IV with
cytochrome b different from that of the T160S change.
The
EPR spectra of the T160S complex display altered signatures for both
the Rieske iron-sulfur center and cytochrome b.
Interestingly, the broad g = 3.50 signal attributed to b
is shifted to lower field, whereas the g = 3.75 signal due to b
appears to be
essentially unchanged in the spectra of the mutated complex. This is
somewhat surprising, because it is b
that is
thought to be located near the quinol-oxidizing center and the positive
side of the membrane, whereas b
resides in the
central portion of the membrane or closer to the negative side and the
quinone-reducing center of the
complex(3, 5, 6) . Thus, the effect of the
T160S substitution on the b
spectrum would
appear to be indicative of a long range interaction between the
oxidizing center and b
or of a geometry placing
the two in closer proximity than current models. It is noted that
long-range effects of mutations at other positions in cytochrome b have been previously reported, e.g. the redox midpoint
potentials of cytochrome c
were affected by
substitutions in cytochrome b(27) .
The
substitutions at T160 in cytochrome b also affected the EPR
signature of the Rieske [2Fe-2S] center, which is located in
a separate subunit of the complex. The iron-sulfur subunit is thought
to bind in the general vicinity of b on the
positive side of the membrane to form part of the quinol-oxidizing
center, because the cluster is a primary electron acceptor from the
quinol. The particular line shape observed for the [2Fe-2S]
cluster is thought to be mediated by the oxidation state of the
ubiquinone present in the Q
center(11, 12, 50, 51, 52, 53, 57) .
When oxidized quinone is present, the EPR signal is sharper than when
quinol is present. The change is most apparent in the case of the g
resonance, which in the bc
from R. sphaeroides is found at g = 1.81
when oxidized ubiquinone is present but shifts to 1.76 and becomes much
broader when ubiquinol is present. The substitutions at Thr
resulted in a broadened [2Fe-2S] EPR signature with g
=1.76, which is unaltered by accessible
changes in the redox state of the samples. There was no detectable
difference between the EPR spectra of the mutated complexes and the
``reduced state'' spectrum of the complement or wild-type bc
complex. The effect of the mutations on the
iron-sulfur cluster spectrum suggests that the Thr
residue of cytochrome b interacts with the
quinol-oxidizing center and/or the [2Fe-2S] cluster. This
idea is consistent with current models of the structure and function of
this part of the complex.
The effect of the T160 substitutions on
the iron-sulfur cluster is also reminiscent of the change observed for
the substitution of Leu for Phe (F144L) in the cytochrome b from R. capsulatus(58) . The F144L bc
complex in R. capsulatus chromatophores was reported to have a very low turnover rate and a
dysfunctional Q
center (having less than 10% of the
wild-type activity for the ubiquinol to cytochrome b
reduction step). The EPR spectrum of F144L was broadened and
insensitive to redox state with g
assigned a value
of 1.765. It was suggested that these properties of the F144L complex
resulted from a reduced affinity for quinone and quinol exhibited by
the Q
center of the mutated complex. In a subsequent study
of the effect of the extraction of ubiquinone from chromatophore
membranes on the iron-sulfur cluster, Ding et al.(52) found that the g
signal became
very broad and was located at approximately 1.765 upon depletion of
ubiquinone from R. capsulatus chromatophore membranes. These
workers were able to distinguish the ``depleted state''
spectrum having g
1.765 from the
``reduced state'' spectrum with g
= 1.777 and found that the line shape of the
quinone-depleted state was broadened considerably beyond that seen in
the presence of either ubiquinone or ubiquinol. With our EPR
instrumentation we cannot assign g values with such a high
degree of precision given the broadness of the g
feature in the reduced and the putative quinone-depleted states.
However, there was no significant difference in the width or depth of
the g
= 1.76 signals of the R.
sphaeroides T160S and T160Y bc
complexes
relative to the spectrum of the complement bc
in
the reduced state. Thus, the changes in the iron-sulfur cluster EPR
spectra resulting from the T160 substitutions in the R. sphaeroides system do not exhibit the extremely broad line shape reported for
the quinone-depleted state and are probably not due to a complete
absence of quinone and quinol binding to the Q
center. The
nature of these interactions will be the subject of future
investigations. One possibility, which would account for the reduced
turnover of the mutant complexes and their ``reduced state''
EPR spectra, is that ubiquinol binds more tightly to the
quinol-oxidizing center of the mutated complexes than ubiquinone,
raising its effective redox potential beyond the optimal range for
transfer to the [2Fe-2S] cluster. Destabilization of the
transient ubisemiquinone state required at the Q
center
would also inhibit turnover.