(Received for publication, January 17, 1995; and in revised form, September 21, 1995)
From the
Seven new structures of cytochrome b have been recently
identified by isolating and sequencing revertants from cytochrome b respiratory deficient mutants (Coppée, J.
Y., Brasseur, G., Brivet-Chevillotte, P., and Colson, A. M.(1994) J. Biol. Chem. 269, 4221-4226). These mutations are
located in the center N domain (Q).
All the revertants
exhibited a modified heme b maximum, confirming
that part of the NH
-terminal region is in the vicinity of
the extramembranous loop between helices IV-V and heme b
. Based on measurements performed on the
maximal activities occurring in each segment of the respiratory chain,
the decrease observed in the NADH oxidase activities of several
revertants was correlated with some bc
complex
activity impairments; this may also explain why a moderate decrease in bc
complex activity does not limit the succinate
oxidase activity. The decrease in the rate of reduction of cytochrome b via the center N pathway is responsible for the impairment
of the bc
complex activity of these revertants.
The three double-mutated revertants (S206L/N208K or -Y; S206L/W30C) are temperature-sensitive in vivo, and their mitochondria like that of the original mutant S206L are thermosensitive in vitro. Isolating the W30C mutation does not yield a thermosensitive phenotype: the replacement of serine 206 by leucine is therefore responsible for the thermoinstability of these strains; this temperature sensitivity is reinforced by additional mutations N208K or N208Y, and not by W30C. These data suggest that serine 206 and asparagine 208 are involved in the thermostability of the protein.
When bc complex activity is lost after
incubating mitochondria at a nonpermissive temperature (37 °C),
heme b is still present, but can no longer be reduced by
physiological substrate. The progressive loss of bc
complex activity seems to be initially linked to a change in the
tertiary structure of cytochrome b, which occurs drastically
at center N and much more slowly at center P, as shown by kinetic study
on the two cytochrome b redox pathways.
The cytochrome bc complex is an integral
multisubunit membrane enzyme that spans either the inner mitochondrial
membrane or the plasma membrane of bacteria. It transfers electrons
from ubiquinol to cytochrome c, and this electron transfer is
linked to proton translocation across the membrane. This mechanism is
best described by the Q-cycle model introduced by Mitchell (1) and recently reviewed by Brandt and Trumpower(2) .
This mechanism requires two distinct quinone reaction sites, the
hydroquinone oxidation (Q
) and the quinone reduction
(Q
) centers, which are located on opposite sides of the
membrane, and linked to cytochrome b
(b
) and b
(b
) heme, respectively. The bis-heme
cytochrome b therefore plays a central role, since it is
responsible for the electrogenic electron transfer through the
membrane. An eight-transmembrane
helix folding model accounts for
the topological organization and heme arrangement (3, 4, 5) .
Upon comparing about 800
cytochrome b mitochondrial sequences, Degli Esposti et
al.,(6) found only 9 invariant amino acids, and this
number was not affected by adding bacterial sequences. Upon examining
the most highly conserved amino-acids among 900 sequences, including
the four histidine ligands to the hemes, Brandt and Trumpower (2) pointed out that 10 are located at center N and 14 at
center P. These well conserved amino acids are natural targets for
site-directed mutagenesis, which has been successfully carried out on
bacteria (7-14; for review, see (15) ). In some cases
however, inducing mutation of invariant amino acids did not impair the
function (11, 12) and only a few of the evolutionary
conserved residues chosen as targets seem to be essential for the
functioning of the bc complex.
In eucaryotes,
cytochrome b is the only subunit of the bc complex which is encoded by the mitochondrial genome, while all
other subunits (nine in yeast) are of nuclear origin: this means that
an eucaryotic organism is not the most suitable for performing
site-directed mutagenesis on cytochrome b. This obstacle can
be circumvented by selecting respiratory growth-deficient mutants of
cytochrome b(16) and numerous intragenic revertants
of these
mutants(17, 18, 19, 20, 21) ,
which has yielded many interesting phenotypes and led to identifying
structurally and/or functionally implicated residues without any
preconceived notions as to which amino acids will be involved.
Our previous paper (20) has described the isolation of non-native intragenic revertants selected from two cytochrome b respiratory deficient mutants located at the center N domain, namely mutant S206L and frameshift 204. Serine 206 is not an invariant residue, but replacing it by leucine would be lethal in the context of a strictly aerobic organism.
These revertants have pseudo-wild type
phenotypes, showing discrete but significant differences from the wild
type, especially in the case of those which are thermosensitive (they
do not grow at 37 °C with respiratory substrate as carbon source).
The aim of this study was to specify the role of the amino acids
involved in these modifications and to determine the structure-function
relationships of the cytochrome b center N domain, in
particular as regards the bc complex activity and
its thermal stability.
Figure 1:
Immunoblot
analysis of some of the largest complex III subunits in mitochondria
isolated from the wild type strain and from the cytochrome b-deficient mutant frameshift 204. Cells were grown at 28
°C; total mitochondrial protein (100 µg) and purified wild type bc complex (20 µg) were separated on 14%
SDS-PAGE and transferred to nitrocellulose. Mitochondrial proteins were
probed with polyclonal antibodies against isolated wild type bc
complex; as seen with the purified bc
complex (lane 1), the antibody
strongly reacts with the Core protein I (CPI), Core protein II (CPII)
and Rieske iron sulfur protein (FeS). Lane 1, wild type
purified bc
complex (20 µg); lane 2,
mitochondria isolated from the frameshift mutant 204 (100 µg); lane 3, mitochondria isolated from the wild type strain (100
µg).
When grown at 28 °C, the
revertants isolated from the two cytochrome b respiratory
deficient mutants S206L and frameshift 204 exhibited phenotypes which
were almost similar to that of the parental strain (box). In particular, these revertants had
identical growth yields on galactose and ethanol as substrates, while
the two original mutants did not grow at all on respiratory
substrate(20) . From these data, the phosphorylation
efficiencies in vivo were deduced with the revertants grown on
ethanol, which were found to be identical to that of the wild type
(±5%), as summarized in Table 1. This identity was
confirmed by directly measuring the coupling characteristics using the
polarographic method (with ethanol as substrate) on the mitochondria
isolated from these strains. The phosphate to oxygen ratios (moles of
ATP synthesized from added ADP/atom of consumed oxygen) were between 1
and 2 (Table 1), as expected with the yeast S.
cerevisiae, which has only two phosphorylation sites(30) .
At this growth temperature of 28 °C, only differences in the doubling time of some revertants were observed when cells were grown on respiratory substrates such as ethanol (Table 1). Moreover, three revertants exhibiting these significantly modified doubling times also turned out to be thermosensitive strains, because they did not grow on ethanol at 37 °C (Table 1). The increase in the doubling time of the three double-mutated revertants might reflect some changes in the kinetic parameters of these mutants, which were tentatively investigated in this study.
With a view
to explaining the discrepancy between the decrease in the NADH oxidase
and the stability of succinate oxidase activities with some revertants,
the maximal activities of each of the four segments of the respiratory
chain were investigated in the wild type strain, the revertants, and
the two original mutants which exhibited nil specific growth rate on
ethanol(20) . In each trial, the cytochrome b and aa contents of the strain were determined as
previously (20) and used to express the change in the specific
activity at the complex III and IV level (turnover). As shown in Table 2, point modifications in cytochrome b gene in the
respiratory deficient mutants lead to a drastic decrease not only in
complex III activity but also in complex II and IV activities in the
case of S206L and frameshift 204 mutants. The situation is quite
different with the revertants isolated from these two box mutants (Table 2), since complex II shows no change as
compared with the wild type value. In all the cases studied, NADH-DB
activity (segment I) remained stable (±10%), as did the complex
IV activity. With the three thermosensitive double mutants isolated
from S206L, the only significant variation was that observed at the
complex III level, which decreased by 30-40% in comparison with
the wild type value (box
), while S206T and
S206V revertants recovered activities similar to that of the wild type
strain (Table 2). These variations observed at the complex III
level were correlated with, and were probably mainly responsible for,
those recorded in the NADH oxidase activity.
The maximum activity
values measured in each segment of the respiratory chain (Table 2) provide a means of accounting for the differences
observed between the unmodified succinate and the decrease in the NADH
oxidase activities of the revertants exhibiting a decrease in complex bc activity. Assuming that the excess ubiquinone
is a mobile carrier linking dehydrogenases with cytochrome chains,
Kröger and Klingenberg (31) described the
overall oxidase activity (V) as
follows.
On-line formulae not verified for accuracy
V (or Vred) was defined as the maximum velocity of
the ubiquinone-reducing system and V
(or V
) as the maximum velocity of the
ubiquinol-oxidizing system. A similar formulation has been described
based on the enzymatic kinetic formalism, taking into account the
measured concentration of endogenous quinones in the chain (in excess
as in the above Q-pool hypothesis) and the presence of excess
oxidant(32) .
In , V is
proportional to the maximum velocity of dehydrogenases, and V
might be expressed as the maximum rate of the
cytochrome chain (bc
complex if taking V to stand for succinate or NADH-cytochrome c reductase
activity, the whole cytochrome chain (CIII + CIV) in the presence
of oxygen, taking V to stand for succinate or NADH oxidase
activity). If V
is the modified bc
complex activity with the revertant and V
the resulting succinate or NADH-cytochrome c reductase activity, then the relative rate of the revertant
(referred to that of the wild type V
) can be
expressed from , as
follows.
On-line formulae not verified for accuracy
If only complex III activity is modified in revertants, this
relative rate will vary hyperbolically with the relative activity V/V
of complex
III, and the concavity of the curve will depend on the value of V
/V
where V
is the succinate or NADH-ubiquinone reductase maximum activity
and V
, the complex III activity in the wild type
strain. As it can be deduced from the legend of Table 2, this
ratio V
/V
is lower with
succinate than with NADH as substrate. Hence, clearly
illustrates the fact that partial loss of bc1 complex activity
affects NADH-cytochrome c reductase activity to a greater
extend than succinate cytochrome c reductase activity: when
the bc1 complex activity is reduced to 50% of the wild type
value, the NADH-cytochrome c reductase activity declines to
about 60%, while the succinate-cytochrome c reductase activity
only declines to 85% of the respective wild type values.
The fact that a decrease occurred in the NADH oxidase activity and not in the succinate oxidase activity can therefore be explained in terms of a decrease at the complex III level and the restricted flux occurring in complex II in comparison with those recorded in complexes I and III; complex III is rate-limiting as regards the NADH oxidase but not the succinate-oxidase activity.
In order to test the phenotype arising from the reverse mutation alone in the revertant S206L/W30C, a recombinant strain carrying the wild type serine in position 206 and the mutated codon in position 30 was obtained(20) . This strain carrying only the mutation W30C grows at 37 °C on nonfermentable substrate such as the parental strain. Similarly, the other two revertants, S206V and S206T, as well as the revertants from the original frameshift cytochrome b-deficient mutant in position 204, namely H204Y and H204S/G205C, are also nonthermosensitive strains. It therefore seems likely that the replacement of serine 206 by leucine might confer thermosensitivity on the cells. However, since mutant S206L is respiratory growth-deficient and is unable to grow on non fermentable carbon sources at any temperature, it is impossible to determine whether or not it may be thermosensitive in vivo.
To determine whether the
activity of the bc complex is affected by the
temperature when the cells are grown at the permissive temperature (28
°C), under the conditions where the complex is well folded and
assembled, the DBH
-cytochrome c reductase activity
was measured as a function of the incubation time at nonpermissive
temperature (37 °C). As shown in Fig. 2, the in vitro
bc
complex activity of the wild type box
and of the nonthermosensitive revertants
(exemplified by S206V) were quite stable. The three in vivo thermosensitive revertants, as well as the original mutant S206L,
are liable to be inactivated at high temperature (Fig. 2): this
inactivation is particularly fast with the proximal revertant
S206L/N208K, which loses half of its bc
complex
activity after exposure of its mitochondria at 37 °C for only 1 h (t
) and which is completely devoid of bc
complex activity after 4 h. The distal
revertant S206L/W30C, which exhibits a t
of about
4 h, is the least severely disturbed thermosensitive strain. The
behavior of the isolated mutation W30C was also tested after incubating
mitochondria at 37 °C, and the activity was found to be as stable
as that of the parental strain, in line with the results obtained in
the in vivo growth study.
Figure 2:
Temperature sensitivity of the
ubiquinol-cytochrome c reductase activity in the wild type
strain and various mutants. Mitochondria were incubated at 37 °C
for the indicated period of time in phosphate buffer (50 mM potassium phosphate, 50 µM EDTA, pH 7.4), in the
presence of 2 mM KCN. Cells were grown at permissive
temperature (28 °C) with galactose medium and harvested in the
stationary phase. Mitochondria were isolated from the wild type strain
(), one nonthermosensitive revertant S206V (
), the
nonthermosensitive recombinant strain S206/W30C (
), the original
mutant S206L (
), and the three thermosensitive revertants (two
proximal revertants, S206L/N208K (
) and S206L/N208Y (
),
and the distal revertant, S206L/W30C (+). DBH
cytochrome c reductase activity was measured at 25 °C as
described under ``Materials and Methods''; the specific
activities of mitochondria before incubation are given in Table 2. With the four thermosensitive mutants, t
values can be deduced from the curves, which give the incubation
time necessary to decrease the bc
complex activity
by 50%; these t
were as follows: 1 h 10 min for
proximal revertant S206L/N208K (
), 2 h 35 min for the proximal
revertant S206L/N208Y (
), 4 h 10 min for the distal revertant
S206L/W30C (+), and 2 h 40 min for the original mutant S206L
(
).
Control experiments were carried
out in which mitochondria isolated from thermosensitive and
nonthermosensitive strains were incubated at 0 °C and at the
permissive temperature 25 °C for several hours: no significant
decrease in the bc complex activity was observed.
With all the revertants, the temperature sensitivity in vitro at the bc complex level was found to be
correlated with that observed in vivo. This thermoinactivation
of bc
complex activity is an irreversible
phenomenon: the decrease in activity cannot be restored by incubating
the mitochondria at permissive (25 °C) nor at freezing temperature
whatever the incubation time may be (data not shown). This cytochrome b conformational change is not an elastic process and reflects
some irreversible structural modification.
Experiments such as that
described in Fig. 2involving the thermoinactivation of bc complex activity of thermosensitive strains
were also carried on in the presence of protease inhibitor
phenylmethylsulfonyl fluoride, and similar results were obtained which
suggests that this thermoinactivation of bc
complex activity may not be due to proteolysis of some subunits
of the complex (results not shown). This was confirmed by SDS-PAGE and
Western blot analysis using polyclonal antibodies against whole bc
complex, which did not show any decrease in the
subunits after mitochondria had been incubated for several hours at 37
°C with or without protease inhibitors such as phenylmethylsulfonyl
fluoride and diisopropyl fluorophosphate (data not shown).
In order
to determine what happens with the cytochrome b heme when the
cytochrome bc complex activity decreases or is
abolished, the reducibility of cytochrome b was checked using
a physiological substrate, succinate, and a nonphysiological one,
dithionite, after exposure of mitochondria for 7 h at 37 °C. The
results obtained with the wild type and two revertants are given in Fig. 3(A-C): with all the strains, the heme was
still present after incubating the mitochondria at the nonpermissive
temperature (upper trace, dithionite-reduced minus oxidized
mitochondria), even with the revertant S206L/N208K, which had
completely lost its activity; however cytochrome b is not
reducible in that strain (Fig. 3B) neither with
succinate alone (middle trace) nor with succinate in the
presence of antimycin (bottom trace, which corresponds to the
reduction of cytochrome b via the center P pathway). The
distal revertant S206L/W30C is an intermediate case between the
parental strain (box
) and revertant
S206L/N208K: cytochrome b is partially reduced by succinate
alone, and through the center P pathway, which is in agreement with the
fact that this mutant is still partially active after being exposed for
7 h at 37 °C (Fig. 2). The control data obtained at 25
°C (Fig. 3D) show that revertant S206L/N208K
exhibits functional cytochrome b after mitochondria have been
incubated for 7 h at that permissive temperature. The loss of activity
in the bc
complex is therefore correlated with the
inability of cytochrome b to accept electrons from ubiquinol
(via physiological substrate), since incubation at the nonpermissive
temperature did not alter the attachment of cytochrome b hemes.
Figure 3:
Physiological and nonphysiological
reducibility of cytochrome b in the wild type strain and
thermosensitive revertants isolated from the respiratory deficient
cytochrome b mutant S206L, after incubating mitochondria at
the permissive (25 °C) or nonpermissive (37 °C) temperature.
Cells were grown at 28 °C and spectra recorded at 25 °C. A, mitochondria of the wild type strain (box) incubated for 7 h at 37 °C at the
concentration of 0.32 nmol of cytochrome b/ml; B,
mitochondria of the thermosensitive revertant S206L/N208K incubated for
7 h at 37 °C at 0.29 nmol of cytochrome b/ml; C,
mitochondria of the thermosensitive revertant S206L/W30C incubated for
7 h at 37 °C at 0.41 nmol of cytochrome b/ml; D,
mitochondria of the thermosensitive revertant S206L/N208K incubated for
7 h at permissive temperature (25 °C) at 0.33 nmol of cytochrome b/ml. Upper trace, difference spectra of
dithionite-reduced mitochondria in the sample cuvette versus potassium ferricyanide-oxidized mitochondria in the reference
cuvette: this is the nonphysiological reduction. Middle trace,
difference spectra of succinate-reduced versus potassium
ferricyanide oxidized mitochondria: this is the physiological
reduction. Bottom trace, difference spectra of
succinate-reduced mitochondria in the presence of antimycin (20
µM) versus potassium ferricyanide-oxidized
mitochondria.
The reduction kinetics of cytochrome b with NADH as electron donor were studied via these two pathways
and the results are given in Fig. 4. The data obtained before
any incubation at the nonpermissive temperature (at t =
0) correspond to the kinetic characteristics of the strains grown with
galactose medium at permissive temperature (28 °C). While the
kinetic behavior via center P showed no apparent change with the two
revertants in comparison with the data on the wild type strain, the
reduction of cytochrome b via center N was drastically slowed
down (Fig. 4B). The kinetics of cytochrome b oxidation with revertant S206L/N208K at 28
°C were apparently identical to those of the wild type strain (data
not shown). The impairment of bc
complex activity
was therefore correlated with the observed defect in the reduction
pathway of heme b
at the center N level. This
defect is responsible for the decrease in NADH oxidase activity and
probably for that in the specific growth rate observed with respiratory
substrate as carbon source.
Figure 4:
Temperature sensitivity of the reduction
kinetics of cytochrome b heme via the two
pathways of the Q cycle model in the wild type strain, the distal
thermosensitive revertant, and a proximal thermosensitive revertant.
Reduction obtained via center P in the presence of antimycin (20
µM) (A) and via center N in the presence of
myxothiazol (30 µM) (B). NADH (2 mM) was
used as substrate (S), and the same dithionite-reduced
cytochrome b content was present in all the experiments. The
kinetic experiments were performed at 25 °C in a rapidly stirred
reaction cuvette with mitochondria extracted from cells grown at 28
°C with galactose medium and incubated in MR
buffer at
37 °C during 0, 1 h 40 min, 3 h 15 min, and 7 h. The reactions were
followed at 562/575 nm. a, wild type box
; b, distal thermosensitive
revertant S206L/W30C; c, proximal thermosensitive revertant
S206L/N208K
With the thermosensitive revertant
S206L/N208K (the activity of which was abolished after incubating
mitochondria for about 4 h at 37 °C), the kinetics of cytochrome b reduction by the reverse electron transfer from quinol to
cytochrome b through the center N pathway were
drastically affected by the temperature, as regards both the apparent
reduction rate and the amount of substrate reducible cytochrome b (Fig. 4B) The cytochrome b reduction
kinetics through center P were also affected by the temperature in this
revertant, as shown in Fig. 4A, but to a lesser extent
than through center N: after incubating the mitochondria for 3 h, these
kinetics showed only moderate difference with that of the wild type,
while electron transfer through center N reduction pathway was nil.
Another less thermosensitive revertant which has a t
of 4 h 10 min (Fig. 2), namely S206L/W30C, was also
studied in detail and it emerged that cytochrome b reduction
by center P pathway shows almost no change during thermoinactivation up
to 7 h, in comparison with the wild type strain (Fig. 4A). This is in accordance with the spectrum in Fig. 3C. On the other hand, under similar conditions,
the reduction kinetics decreased strongly by the center N pathway (Fig. 4B). Hence, while both strains are severely
affected through the center N pathway, mutations affecting the more
thermosensitive revertant S206L/N208K, has in addition long distance
repercussions on the reduction kinetics by the center P pathway when
mitochondria were incubated at nonpermissive temperature; this resulted
in more drastic effects. The kinetics characteristics of cytochrome b oxidation by decylubiquinone via the center N pathway (in
the presence of myxothiazol) show no change with the box
strain, but are slowly affected with the
thermosensitive revertant S206L/N208K during incubation at 37 °C:
after 6 h, the apparent oxidation rate of cytochrome b
heme is about half-decreased (data not shown).
Reduction and
oxidation of cytochrome b heme by the center N
pathway are therefore both affected by incubation of this
thermosensitive revertant at nonpermissive temperature.
The activity of NADH-ubiquinone reductase is
not greatly modified in any of the strains, while the activities of the
other three complexes were drastically diminished. Mutant S206L and
distal revertant S206L/W30C still showed some bc complex activity (about 10%) however, while the proximal
revertant S206L/N208K had completely lost both complex II and complex
III activities. The respiratory activities were found to be quite low
in all the mutated strains; the weak activities still observed with
S206L/N208K might be due to a salicylhydroxamic acid-sensitive oxidase
pathway, as previously observed in yeast(33) . The
immunoblotting data obtained using polyclonal antibody against the
purified bc
complex (Fig. 5) show that bc
complex is not assembled with the proximal
revertant S206L/N208K: the core proteins I and II are in fact greatly
decreased, as is the mature form of the Rieske iron-sulfur protein and
subunit 8. With mitochondria extracted from cells grown at 37 °C,
the FeS protein appears to be a doublet which might be referred to
partially processed and mature FeS proteins(34, 35) .
In the membranes from the thermosensitive cytochrome b mutant,
only the partially processed protein band is present (Fig. 5, lane 3). This would imply that with this mutant the
iron-sulfur protein is synthesized, imported into the mitochondria, and
partially processed but presumably not assembled into the bc1 complex. It was observed moreover on low temperature spectra that
cytochrome c
is also absent in this strain (data
not shown), while cytochrome c is still present (Table 4). The nonthermosensitive recombinant strain S206/W30C
appears to be well assembled in culture at 37 °C, as it was
therefore found to contain core proteins I and II, Rieske iron-sulfur
protein and subunit 8 (Fig. 5, lane 2), as well as the
cytochromes b and cc
observed under
spectral analysis, and to still show bc
complex
activity.
Figure 5:
Immunoblot analysis of some complex III
subunits in mitochondria isolated from cells grown at 37 °C in the
nonthermosensitive recombinant strain S206/W30C, the thermosensitive
revertant S206L/N208K and in the wild type strain. Cells were grown at
37 °C with galactose medium; total mitochondrial protein (in the
presence of protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride
and purified wild type bc complex were separated
on 12% SDS-PAGE and transferred to nitrocellulose. Mitochondrial
proteins were probed with polyclonal antibodies against wild type
purified bc
complex; as seen with the purified bc
complex (lane 1), the antibody
strongly reacted with the Core protein I (CP I) and II (CP
II), Rieske iron sulfur protein (FeS), and subunit 8 (11
kDa). Lane 1, wild type purified bc
complex (20 µg); lane 2, nonthermosensitive
recombinant strain S206/W30C mitochondria (75 µg); lane 3,
thermosensitive revertant S206L/N208K mitochondria (75 µg); lane 4, wild type mitochondria (75
µg).
All in all, these data point to the conclusion that
replacing serine 206 by leucine affects the thermostability of the bc complex. This sensitivity to temperature is
reinforced by the additional mutations N208K or N208Y, but not by the
additional mutation W30C.
Among the seven non-native respiratory-competent revertants,
derived from two respiratory growth-deficient mutants as described in
our previous paper(20) , three are affected both in their
doubling time at the cellular level and in their NADH oxidase activity
at the mitochondrial level ( Table 1and Table 2); this
study shows clearly that the only significant modification in these
revertants is located at the complex III level; more specifically, this
moderate decrease in bc complex activity was found
to be closely related to a slowing down in the kinetics of heme b
reduction through center N pathway. On the
other hand, the mutant S206L is strongly affected in both the reduction
and oxidation of heme b
at center
N(36) ; in addition the two original mutants, S206L and
frameshift 204, exhibit lower activities in the three complexes II,
III, and IV, as previously observed in several mis sense cytochrome b respiratory deficient mutants(37, 33) .
This observation is rather surprising and might be related to either a
great sensitivity of succinodehydrogenase assembly to the absence of
respiration as already noticed by de Kok et al.(38) or to a direct interaction between respiratory
complexes, in particular between complexes II and III as previously
suggested by Gwak et al. (39) , or to both phenomena.
When only complex III is modified by a mutation which decreases its activity (like with some revertants), the resulting succinate cytochrome c reductase or succinate oxidase activity is not very greatly modified, at least up to a threshold in complex III decrease, while NADH-cytochrome c reductase or NADH oxidase may amplify the variation. This is in complete agreement with the finding by Taylor et al.(40) that in patients with respiratory chain abnormalities, a partially defective complex III cannot be reliably detected by measuring succinate cytochrome c reductase activity.
The spectral shift toward the red observed
with all the revertants might reflect some modification in the
electronic surroundings of heme b, due to
mutations (including the distal W30C) which map at the center N: this
confirms the location of Trp-30 in the vicinity of this heme.
The
revertants isolated from mutant S206L with an additional mutation in
position 208 or 30 have been identified as thermosensitive strains.
Thermosensitive mutants have been described previously, which affected
the yeast bc complex, either as Rieske iron-sulfur
mutants (41) or as Core protein I mutants(42) . The
thermosensitive cytochrome b mutants show progressive loss in
the ability for cytochrome b to accept electrons from
ubiquinol when mitochondria isolated from cells grown at 28 °C were
incubated at 37 °C; this appears to have resulted mainly from a
change in the tertiary structure of cytochrome b, since the
assembly of the bc
complex and cytochrome b hemes did not seem to be modified, which suggests that the
quaternary structure of the complex is not greatly perturbed. With all
thermosensitive revertants, the kinetics of cytochrome b reduction through center N were drastically and quickly altered;
long distance repercussions may also have occurred however with the
most thermosensitive revertant S206L/N208K (Fig. 4).
With
this proximal revertant, the decrease observed in both the rates of
cytochrome b oxidation and reduction at the center N level
during incubation at 37 °C suggests that the oxidoreduction
potential of the couples Q/Qand
Q
/QH
may have changed in an opposite
manner, as it was assumed in the case of the two mutants S206L and
M221K(36) ; this would result in a larger difference between
the potentials of these two couples and the semiquinone concentration
would hence have diminished(1, 2) . The stability of
the anionic semiquinone might therefore have undergone a decrease
during thermoinactivation at 37 °C as known to occur with
cytochrome b mutants of Rhodobacter
capsulatus(43) . Incubation at nonpermissive temperature
might initially favor a local distortion of the tertiary structure of
cytochrome b, probably due to an increase in the distance
inside and/or between the helices. This might thermodynamically
destabilize the center N domain, so that the electron transfer is
blocked (since the variation in the electron transfer rate as a
function of the intercenter distance is an exponential
law(44) ) and/or the quinone binding site is affected, and both
oxidation and reduction of cytochrome b through the center N
pathway are slowed down.
Whereas the mutant S206L also exhibited sensitivity to heat treatment (Fig. 2), the strain constructed with the isolated mutation W30C did not, on the contrary, lead to a thermosensitive phenotype. These data therefore suggest that the replacement of serine 206 by leucine is responsible for the thermal sensitivity of the original mutant and revertants.
Matsumura et
al.(45) have pointed out that mutations affecting protein
stability generally have a cumulative effect. Like the thermal
stabilizing effects of two substitutions(46) , the
destabilizing effects might also be additive: mutation W30C in addition
to S206L did not increase the thermal instability, while proximal
reversions, especially N208K, in addition to S206L, did (Fig. 2). These results were strengthened by the phenotypes of
these strains grown on galactose medium at their nonpermissive
temperature (37 °C); the phenotypes of mutant S206L and revertant
S206L/W30C are almost similar and show some of the activity of an
assembled complex, while that of the revertant S206L/N208K is more
drastically perturbed and shows a nonassembled bc complex with a complete loss of activity.
Argos et al.(47) mentioned that the most significant changes leading
to thermal stability in proteins were the increase in the internal
hydrophobicity, the helix forming ability of amino acids in helices and
the increase in the sheet-forming tendency of residues in -sheets.
In particular, these authors reported that the Ser
Thr exchange
inside a
-sheet region had the ability to increase the thermal
stability; this can be compared with the present finding that replacing
serine 206 by threonine (or valine) resulted in a quite thermostable bc
complex (Fig. 2). On the basis of
computer analysis, Robert Brasseur (Université Libre de Bruxelles) proposed that Ser-206 and Asn-208 are located
in a cytochrome b region showing a high probability of
-sheet conformation. (
)This is in agreement with the
implication of these two amino acids in the thermostability of the
protein, since mutations affecting the stability are assumed to map
preferentially in folded regions (
-helice or
-sheet).
In
addition to its probable involvement in the thermostability of
cytochrome b, asparagine 208 was found to be involved in
funiculosin binding(48) . Moreover, replacing asparagine 208 by
lysine in this revertant keeping the original mutation S206L leads,
when mitochondria are incubated at nonpermissive temperature, to
concomitant loss of bc complex activity and
funiculosin binding, while the antimycin binding is conserved; these
data suggest close proximity between the funiculosin binding site and
the catalytic center N domain (Q
)(49) .
Furthermore, the three thermosensitive revertants exhibited a
qualitative change in the shift induced by the funiculosin binding;
this inhibitor causes a hypsochromic effect (blue-shift) on the
spectrum of cytochrome b
heme in the wild type
strain as well as in the mutant S206L, but it causes a bathochromic
effect (red-shift) in the three double-mutated strains. This spectral
change was attributed to the interaction between the second mutation
and S206L. These data suggest that at least part of the funiculosin
binding site is very close to the b
heme(49) , which is in agreement with the first location
of a funiculosin resistance mutation in yeast, L198F, close to
histidine 197 linking the b
heme(50) .
In conclusion, it emerges that the replacement of serine 206 by
leucine in the original mutant is involved in the drastic decrease in bc complex activity, in the shift of the spectral
maximum of the b
heme toward the red and in the
thermosensitivity of bc
complex activity; whereas,
the replacement of leucine 206 by threonine or valine is compatible
with the thermostability of the protein and leads to a quasi-wild type
phenotype at all temperatures (except for the heme b
spectral shift). The three additional mutations, in position 208
or 30, interact with leucine 206, resulting in restoration at the
permissive temperature of a pseudo-wild type phenotype, with some
decrease in complex III activity; two of these mutations, in position
208, strengthen the thermosensitivity of the strain (N208K, N208Y). The
mutations were found to mainly affect the center N domain where they
were mapped, which is consistent with the eight transmembrane helix
folding model of cytochrome b. These data as a whole indicate
that the three amino acids, serine 206, asparagine 208, and tryptophan
30, are involved in the catalytic center N; two of them, serine 206 and
asparagine 208, contribute to the thermostability of cytochrome b and are probably located in a folded region (
-sheet);
asparagine 208 is also strongly involved in funiculosin binding.