(Received for publication, April 17, 1995)
From the
Trp-142 is a highly conserved residue of the cytochrome b subunit in the bc complexes. To study the
importance of this residue in the quinol oxidation catalyzed by the bc
complex, we characterized four yeast mutants
with arginine, lysine, threonine, and serine at position 142. The
mutant W142R was isolated previously as a respiration-deficient mutant
unable to grow on nonfermentable carbon sources (Lemesle-Meunier, D.,
Brivet-Chevillotte, P., di Rago, J.-P, Slonimski, P. P., Bruel, C.,
Tron, T., and Forget, N.(1993) J. Biol. Chem. 268,
15626-15632). The mutants W142K, W142T, and W142S were obtained
here as respiration-sufficient revertants from mutant W142R. Mutant
W142R exhibited a decreased complex II turnover both in the presence
and absence of antimycin A; this suggests that the structural effect of
W142R in the bc
complex probably interferes with
the correct assembly of the succinate-ubiquinone reductase complex. The
mutations resulted in a parallel decrease in turnover number and
apparent K
, with the result that there
was no significant change in the second-order rate constant for
ubiquinol oxidation. Mutants W142K and W142T exhibited some resistance
toward myxothiazol, whereas mutant W142R showed increased sensitivity.
The cytochrome cc
reduction kinetics were found to
be severely affected in mutants W142R, W142K, and W142T. The
respiratory activities and the amounts of reduced cytochrome b measured during steady state suggest that the W142S mutation also
modified the quinol-cytochrome c
electron transfer
pathway. The cytochrome b reduction kinetics through center P
were affected when Trp-142 was replaced with arginine or lysine, but
not when it was replaced with threonine or serine. Of the four amino
acids tested at position 142, only arginine resulted in a decrease in
cytochrome b reduction through center N. These findings are
discussed in terms of the structure and function of the quinol
oxidation site and seem to indicate that Trp-142 is not critical to the
kinetic interaction of ubiquinol with the reductase, but plays an
important role in the electron transfer reactions that intervene
between ubiquinol oxidation and cytochrome c
reduction.
The ubiquinol-cytochrome c oxidoreductase (the
cytochrome bc complex; EC 1.10.2.2) is an integral
multisubunit membrane protein that is involved in energy transduction
in a wide range of organisms. All the bc
complexes
contain a minimum of three redox-active polypeptide subunits carrying
four prosthetic groups: a[2Fe-2S]iron-sulfur protein, a
one-heme center cytochrome c
, and a two-heme
center apocytochrome b, cytochrome b
, and
cytochrome b
(1, 2) . This complex
catalyzes the electron transfer from ubiquinol to cytochrome c coupled to a vectorial proton translocation through the membrane
involving a mechanism known as the modified Q
cycle(3, 4, 5, 6, 7) .
According to this mechanism, the bc
complex
transfers two electrons from ubiquinol to two molecules of cytochrome c with a concomitant ejection of two protons on the positive
side of the membrane, while two additional protons are translocated
from the negative to the positive side of the membrane per pair of
electrons transferred. This mechanism requires two quinone separate
independent reaction domains: the ubiquinol oxidation domain (QP
center) located on the positive side of the membrane, which is the
target of three groups of specific inhibitors, especially the
methoxyacrylate-type inhibitor exemplified by myxothiazol (for review,
see (8) ), and the ubiquinone reduction domain (QN center)
located on the negative side of the membrane, which is the target of
other specific inhibitors exemplified by antimycin A (for review, see (8) ). The QP center is associated with two proteins, the
iron-sulfur protein and cytochrome b through
heme b
. Electrons from ubiquinol diverge at center P: one
is transferred to the iron-sulfur protein, along with the production of
an unstable semiquinone, and subsequently to cytochrome c
and cytochrome c; the second electron
is transferred to heme b
and thereafter to heme b
and is recycled through a quinone molecule to form a stable
semiquinone anion at the quinone reductase domain. The oxidation of a
second molecule of ubiquinol at center P is required to reduce this
stable semiquinone into ubiquinol at center N. Another essential
feature of the Q cycle mechanism is that cytochrome b can be
reduced through two pathways, the center P pathway, which is the more
thermodynamically favorable, and the center N pathway when the
thermodynamically favorable route through center P is blocked.
It
has by now become generally accepted that cytochrome b is
folded into an eight-transmembrane -helix structure with a ninth
extramembranous amphipathic helix. This model has been supported by
hydrophobicity and amphipathy
calculations(9, 10, 11) , gene fusion
experiments(12) , mapping of center P and center N inhibitor
resistance
mutations(13, 14, 15, 16, 17, 18, 19, 20, 21) ,
and biochemical and biophysical studies on mutants obtained by either in vivo isolation ((22, 23, 24, 25, 26, 27, 28, 29, 30) ;
for review, see (31) ) or site-directed mutagenesis ((32, 33, 34, 35, 36) ; for
review, see (37) ).
The assumption that a highly conserved
residue performs a vital functional or structural role in the protein
is often used in site-directed mutagenesis strategies. Recent results
have shown, however, that only a few of the evolutionarily conserved
amino acids seem to be essential for bc function(34) .
We report here on the mapping,
sequencing, and biochemical study of four mutants: the inactive
cytochrome b W142R mutant(26) , the mutation of which
affects an evolutionarily conserved residue, and three functional
revertants isolated from this mutant. According to degli Esposti et
al.(38) , tryptophan at position 142 is conserved among
the 800 cytochrome b sequences known to date, except in Paramecium aurelia and Bacillus PS3. Moreover, this
position belongs to one of the most conserved regions of cytochrome b, spanning residues 131-148, which may interact with
the QP center. The mutants/revertants were investigated regarding their
effects on growth, various electron transfer activities, kinetic
interaction between the substrate and the enzyme, cytochrome c reduction, cytochrome b reduction
through both centers P and N, and resistance to inhibitors. The results
suggest that Trp-142 is important to the properties of the quinol
oxidation site, especially to the electron transfer reactions that
occur between quinol oxidation and cytochrome c
reduction.
Codon 142 belongs to a very short exon (B2) of only 14 base pairs(46) . Part of this exon, including codon 142, is involved in base pairings with intron bi2, which are crucial to the splicing of this intron (see Fig. 5 in (47) ). Not surprisingly, the mutants described above accumulated significant amounts of nonprocessed cytochrome b transcripts.
Two other amino acids, Gly-142 (GGA) and Met-142 (ATA), could have been obtained by monosubstitution at the Arg-142 codon (AGA). Given the fact that they have not been found among the high number of revertants analyzed, it seems likely that the corresponding nucleotide changes would result in a respiration-negative phenotype. However, it is difficult to ascertain whether Gly-142 and Met-142 are not compatible with the function of cytochrome b or whether the corresponding nucleotide changes impair splicing of cytochrome b pre-mRNA. Also, the serine codon AGC could have been derived by a single nucleotide change from the Arg-142 codon. Since the presence of serine at position 142 gives a functional cytochrome b, one could infer that this codon blocks the splicing of intron bi2. However, this codon is normally not used by yeast mitochondria for the synthesis of respiratory and phosphorylating complexes(48) . It is therefore not surprising that it has not been found among the revertants we analyzed.
Figure 1:
Absorption difference spectra of
mitochondria from the parental strain (KM91), mutant W142R, and
revertants W142S, W142T, and W142K. Mitochondria were isolated from
yeast cells grown on galactose medium and suspended to 2.5 mg/ml in
0.65 M sorbitol, 10 mM KHPO
,
2 mM EDTA, 0.1 mM MgCl
buffer, 0.3%
bovine serum albumin, pH 6.5. In each panel, the bottom trace shows the cytochrome c + c
reduction induced by adding 40 mM ascorbate to the
sample cuvette. The spectra were recorded after complete exhaustion of
oxygen; the reference cuvette was maintained in the oxidized state by
adding 10 µM ferricyanide. In A and C-E, the middle trace shows the reduction of
heme b
observed after adding 40 mM succinate to
the sample cuvette and 40 mM ascorbate to the reference
cuvette (succinate minus ascorbate spectrum). In B, the
succinate minus ferricyanide spectrum shows the reduction of heme
b
(see ``Results'' for explanations). The top
traces show the reduction of heme b
observed after
adding a few grains of dithionite and 40 mM fumarate to the
sample cuvette and 40 mM succinate to the reference
cuvette.
A decrease in
the synthesis of all the cytochromes is usually observed in
respiration-deficient strains, which use only ATP produced by
fermentation for their growth. In some mutants, this decrease is in
approximately the same proportion as that observed in their
counterparts in the parental strain(22) . In the results
presented here, mutant W142R exhibits a greater decrease in the reduced
heme b than in the reduced heme b
or cytochrome cc
. All the revertants recovered a level of
cytochrome synthesis similar to that in the parental strain
(80-100%).
DBH-cytochrome c reductase activity was titrated with myxothiazol and antimycin A
in the parental and revertant strains. In the presence of myxothiazol,
the I
values showed that 10- and 5-fold increases in
resistance toward this inhibitor occurred with revertants W142K and
W142T, respectively, whereas no change in myxothiazol sensitivity was
observed with revertant W142S. Mutant W142R did not exhibit any
DBH
-cytochrome c reductase activity, but exhibited
cytochrome c
reduction during a single turnover of
the bc
complex (see ``Reduction Kinetics of
the bc
Complex Cytochomes''). This finding
was used to assay the sensitivity of this mutant toward myxothiazol; a
4-fold higher sensitivity was observed in comparison with the parental
strain. The sensitivity toward antimycin was not modified by mutation
W142K, W142T, or W142S (data not shown), and it was previously observed
that mutation W142R does not affect the antimycin sensitivity
either(49) .
Reduction of cytochrome cc by succinate in the presence of KCN was carried out in
mitochondrial membranes isolated from the mutant, revertant, and
parental strains (Fig. 2). The results reveal that mutation
W142R leads to a strongly negative effect on the cytochrome cc
reduction kinetics, which is partially
corrected by the W142K reversion and almost or completely corrected by
the W142T and W142S reversions. Control tests with crude bc
complexes from W142R and W142K and the parental
strains were carried out to determine cytochrome c
reduction using DBH
as substrate; in comparison with
the parental strain, the cytochrome c
reduction
rates exhibited the same relative decrease as that observed in
mitochondrial membranes (data not shown).
Figure 2:
Reduction of cytochrome cc in mitochondrial membranes. The traces show reduction of
cytochrome cc
by 40 mM succinate (S) in the presence of KCN in mitochondrial membranes prepared
from the parental strain (KM91), mutant W142R, and its revertants,
W142S, W142T, and W142K. The membranes were suspended to 0.5 µM cytochrome b in the same buffer as described in the
legend of Fig. 1.
Cytochrome b was reduced through center P in the presence of antimycin and through center N in the presence of myxothiazol in the parental, mutant, and revertant strains (Fig. 3). Mutations W142R and W142K both affected cytochrome b reduction through center P, but the effect of mutation W142R was stronger than that of W142K; W142T and W142S had no detectable effects on cytochrome b reduction through center P (Fig. 3a). Although Trp-142 is located at center P, mutation W142R also had an effect on the kinetics of cytochrome b reduction through center N (Fig. 3b). W142K, W142T, and W142S had no detectable effect on cytochrome b reduction through center N.
Figure 3: Reduction of cytochrome b through centers P and N. Reduction of cytochrome b by succinate (S) in the presence of antimycin (a) or myxothiazol (b) was recorded at 562-575 nm. The mitochondrial membranes were prepared from the parental strain (KM91), mutant W142R, and its revertants, W142S, W142T, and W142K. The membranes were suspended to 0.5 µM cytochrome b in the same buffer as described in the legend of Fig. 1.
The cytochrome b reduction kinetics were also recorded in the absence of inhibitor in the mutant, revertant, and parental strains. Adding succinate to the parental strain mitochondrial membranes led, at steady state, to a 18% reduction of the anaerobiosis-reduced cytochrome b. In the W142S, W142T, and W142K mutant strains, 23, 33, and 58% of the anaerobiosis-reduced cytochrome b were reduced at steady state, respectively, after the addition of succinate; in the W142R mutant, cytochrome b was fully reduced under these conditions. Mutant W142R exhibited slow cytochrome b reduction kinetics, similar to those recorded in the presence of myxothiazol (data not shown).
In this study, four new cytochrome b structures were
characterized and used to analyze the functional and structural role of
the evolutionarily invariant tryptophan 142 in the QP region of the bc complex. This was done by isolating three
functional revertants from a nonfunctional cytochrome b mutant; the initial mutation, W142R, is located in the
extramembranous loop between transmembrane
-helices 3 and 4 on the
positive side of the membrane (Fig. 4)(26) . This loop,
together with the C-terminal region of helix 3, might be involved in
the center P catalytic subdomain(1, 36) . The
secondary mutations, which occur at the level of the original mutation,
change Arg-142 into lysine, threonine, and serine.
Figure 4:
Secondary structure of Saccharomyces
cerevisiae cytochrome b. Shown is the predicted diagram
of the eight-membrane-spanning -helix cytochrome b(10, 11, 30) . The four conserved
histidines, thought to be the ligands to the two heme groups, are
numbered and boxed. Hemes b
and b
are
predicted to be coordinated to histidines 82 and 183 and to histidines
96 and 197, respectively. The shaded region spanning residues
131-148 is the longest fragment, exhibiting an average 70%
identity to the other species. In this region, amino acid residues that
are loci of mutations conferring resistance toward myxothiazol and/or
modifying the bc
complex activity are indicated
with boldcircles. Tryptophan at position 142 is
located on the positive side of the inner mitochondrial membrane and is
indicated with a blackcircle.
The cytochrome b W142R mutation, which leads to a respiration-deficient
phenotype, has been shown to abolish the bc complex electron transfer activity. One rather unexpected
consequence of this mutation is the low succinate-quinone
oxidoreductase activity observed in the corresponding mutant strain (Table 1). When normalized either to the amount of protein
(milligrams) or to the amount of complex II cytochrome b, this
activity is severely affected, which indicates that electron transfer
through complex II is modified as a result of mutation W142R. This is
the first time, as far as we know, that a point mutation in the bc
complex cytochrome b, which does not
impair the subunit composition of the complex(52) , has been
found to impair the complex II specific activity. One possible
explanation for our results might be that the structural effect induced
by mutation W142R in the bc
complex interfered
with the correct assembly of the succinate-ubiquinone reductase
complex. It is noteworthy that other cytochrome b mutated
residues similarly affect both complex III and complex II specific
activities. (
)
Replacing tryptophan at position 142 with
lysine, threonine, or serine does not affect the kinetic interaction of
ubiquinol with the reductase (Table 2). Tryptophan is a large,
aromatic, and polar but neutral residue. Depending on the environment,
it can either contribute to the hydrophobicity of a protein region or
act as a hydrogen bond donor through its nitrogen free doublet. In the
reaction center, His-215 (in Rhodopseudomonas viridis) and
Thr-222 (in Rhodopseudomonas spheroides) are hydrogen-bonded
to one carbonyl of the QA molecule(53, 54) . As noted
by degli Esposti et al.(38) , there exists a relevant
homology between the conserved yeast cytochrome b peptide
WGATV and the conserved peptide H
GATV of
the chloroplast D2 subunit or its homologous bacterial reaction center
M subunit. By analogy with what occurs in the reaction center, degli
Esposti et al.(38) suggested that in yeast cytochrome b, either Trp-142 or Thr-145 might be hydrogen-bonded to
ubiquinol. The polar characteristic of tryptophan would then be
important to the binding of quinol. Our results show that the Trp-142
substitutions observed in the revertant strains introduced polar
residues that were liable to exhibit a hydrogen bond and did not affect
the kinetic interaction of ubiquinol with the reductase. Two
theoretically possible reversions from codon AGA (which codes for
arginine in the mutant) that may code for the two nonpolar residues,
methionine and glycine, were never selected. Whether this is due to the
incompatibility of those codons with the pre-mRNA splicing or to the
incompatibility of a nonpolar residue at position 142 with a functional bc
complex is unknown. Trp-142 is conserved among
all the 800 species sequenced to date, except in Bacillus PS3,
where it is replaced with the nonpolar aromatic residue phenylalanine,
and in P. aurelia, where it is replaced with isoleucine. It
will be necessary to obtain mutants W142G, W142I, W142M, and/or W142F
before we can draw any conclusion about the interference of the polar
character of residue 142 with the kinetic interaction of ubiquinol with
the reductase. This can be envisaged by isolating mutants from the
intronless cytochrome b gene strain(30) .
The other
characteristic of tryptophan, namely its aromatic structure, does not
seem to be critical to the kinetic interaction of quinol with the
reductase since none of the mutants studied have an aromatic residue at
position 142 and all exhibit k values that are
similar to those of the parental strain. Thus, neither the size nor the
shape of residue 142 seems to affect the kinetic interaction of quinol
with the reductase, contrary to what occurs in Rhodopseudomonas
capsulatus with Gly-143 (yeast numbering) (35) . Indeed,
replacing Trp-142 with amino acids of various sizes (arginine, lysine,
threonine, and serine) did not affect the k
value. On the whole, Trp-142 does not seem to be involved in the
kinetic interaction of quinol with the reductase. Thus, it is
conceivable that this amino acid is not critical to the binding of
ubiquinol, although it is highly conserved.
The overall data
obtained 1) by characterizing mutants selected for their center P
inhibitor resistance from both mitochondrial(15, 55) and bacterial (36) systems and 2) by performing
inhibitor binding studies on cytochrome b-deficient mutants
and their revertants (22, 56, 57) and on
mutants obtained by carrying out site-directed mutagenesis (32, 35) indicate that two topologically different
conserved regions of the protein are involved in the QP inhibitor
pocket. The first region includes the C-terminal region of helix 3 and
the adjacent part of loops 3 and 4; the second region is formed by
loops 5 and 6 and the N-terminal region of helix 6. Myxothiazol
resistance mutations were detected at positions 125(17) ,
129(17, 15) , 132(58) , 133(56) ,
137(15, 17, 22) , and 143(35) . In
the present study, Trp-142 emerges as a new residue involved in the
interaction of cytochrome b with myxothiazol. Moreover,
depending on which amino acid occupies position 142, the strain
exhibits either resistance or greater sensitivity toward this
inhibitor, suggesting that the various amino acids induce different
conformational states of the QP pocket, leading to a lesser or greater
binding of myxothiazol. Studies on cytochrome b inhibitor
resistance mutants of various origins have provided a great deal of
information about the residues involved in the binding of the various
inhibitors acting at centers P and N. Two types of amino acid linked to
the inhibitor-binding pockets cannot, however, be found by selection of
inhibitor resistance mutants: those with which a mutation would lead to
a partial or total loss of bc complex function and
those with which the amino acid change would lead to a greater
sensitivity toward inhibitors. The results presented here and elsewhere (22, 26, 57) show that functional analysis
of cytochrome b-deficient mutants and their revertants can
serve to pinpoint these amino acids.
The cytochrome cc reduction kinetics were found to be severely to
slightly affected by mutations W142R, W142K, and W142T (Fig. 2);
on the other hand, the same kinetics were observed in both the mutant
W142S and the parental strain. Measurement of the reduction kinetics of
cytochromes cc
and b is limited by the
lag time of the apparatus, which is of the same order of magnitude as
the reduction rate obtained with the parental strain. We cannot
therefore rule out the possibility that mutation W142S may have an
effect on the cytochrome cc
reduction kinetics
that would not be measurable under our experimental conditions. The
fact that the cytochrome cc
reduction kinetics are
modified by at least three different residues at position 142
(arginine, lysine, and threonine) shows that Trp-142 plays an important
role in the electron transfer reactions that intervene between the
ubiquinol oxidation site and cytochrome c
.
Electron transfer through center N was recorded in the presence of
myxothiazol and was found to be the same with all the strains, except
for the deficient mutant W142R (Fig. 3a). Among the
four mutations we studied, only arginine causes this phenotype,
indicating that a long distance effect is possible, but probably
requires a strong structural modification. It should be noted, however,
that mutant W142R has kept all the bc complex
subunits(52) .
Arginine or lysine at position 142 affects the kinetics of cytochrome b reduction through center P, whereas threonine or serine does not interfere with this electron transfer pathway (within the limits of our experimental conditions) (Fig. 3b). This seems to indicate that tryptophan 142 present in the wild-type cytochrome b sequence is not involved in this cytochrome b reduction pathway. Mutation W142R impairs the cytochrome b reduction kinetics through both centers P and N, but the effect on center P is stronger. This result is consistent with the center P location of amino acid 142.
The amount of
cytochrome b reduced by succinate during steady state in the
absence of inhibitor, together with the results obtained in the
cytochrome cc and cytochrome b reduction
kinetic experiments ( Fig. 2and Fig. 3), provides some
helpful information about Trp-142 and the mutant phenotypes. Since
mutation W142R abolishes respiratory activity, it is surprising that
none of the electron transfer pathways are abolished in this mutant. At
steady state, W142R cytochrome b is entirely reduced by
succinate, which means that the bc
complex can
oxidize ubiquinol at least once, but that under multiple turnovers, the
electrons may be blocked. In the catalytic switch model(59) ,
this would mean that the mutant bc
complex
switches to the b state and is locked in this conformation;
the switch cannot be triggered back to the FeS state. W142K reversion
improves the cytochrome cc
and cytochrome b reduction kinetics as compared with W142R; the bc
complex recovers 12% activity, and cytochrome b is 58%
reduced during steady state. W142T reversion brings the cytochrome b reduction kinetics back to the parental value and improves
cytochrome c
reduction as compared with W142K.
Even if only the cytochrome cc
reduction step is
affected by the W142T mutation, the amount of reduced cytochrome b present during steady state is 15% higher than in the parental
strain. Finally, based on the cytochrome cc
and
cytochrome b reduction kinetics, the W142S reversion brings
both the cytochrome cc
and cytochrome b reduction kinetics back to the parental value with, however, a 5%
more reduced cytochrome b during steady state and 20% less bc
activity observed than in the parental strain.
Mutation W142S therefore probably impairs the cytochrome cc
reduction kinetics (to an extent that was not
detectable under our experimental conditions). Reversions W142K, W142T,
and W142S gradually correct the effect of arginine on the bc
complex activity; they may induce
conformational state dynamics at center P, which may be intermediate
between the mutant b state-locked conformation and the
parental b state/FeS state fast switching conformation.
The
quinol-cytochrome c oxidoreductase complex activity depends on
the quinol affinity for its site and on the kinetic parameters of the
intra- and intermolecular electron transfer. The protein space located
between two oxidoreduction centers is generally referred to as the
intervening space. Most of the mutations that confer resistance to
inhibitors and any mutation that modifies the bc electron transfer activity will affect an amino acid located at
the quinol-binding site or in the intervening spaces or both. The
quinol-binding site, the inhibitor-binding sites, and the intervening
spaces are different functional areas that may have common topological
domains.
Glycine at position 143 (yeast numbering) has been found to
be involved in the interaction of the quinol/quinone couple with center
P and in myxothiazol binding(35) . Cysteine 133 and glycine 137
are critical to the interaction of quinol with center P and are
involved in the intervening spaces between the quinol oxidation site
and cytochrome c(56) . (
)Glycine 137 and, to a lesser extent, cysteine 133 are also
involved in myxothiazol binding(22, 56) . Four
respiration-deficient yeast mutants with impairments at positions 131,
133, 137, and 142 (26) of the cytochrome b sequence
have been isolated, and a mutation affecting the cytochrome b from R. capsulatus at position 158 (homologous to
position 143 in yeast) led to a nonphotosynthetic
phenotype(35) . All these residues belong to the longest region
of cytochrome b (spanning amino acids 131-148), with an
average 70% identity to the other known cytochrome b sequences
(calculated from the sequence alignment presented in (38) ) (Fig. 4). Four residues also located in this region (at
positions 132, 133, 137, and 143) are thought to interfere with the
center P inhibitors. This region is therefore likely to have an
important role in the structure and function of center P.
Our
results suggest that tryptophan 142 is involved in the
myxothiazol-binding site, but probably not in the interaction of quinol
with center P. It would belong to the intervening space between the
quinol-binding site and cytochrome c since its
mutation to arginine, lysine, threonine, and probably serine leads to a
decrease in the electron transfer rate between ubiquinol and cytochrome c
. Trp-142 could either be an essential component
of the structural architecture of one of the center P intervening
spaces or be directly involved in one of the electron transfer
reactions that occur at center P.