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INTRODUCTION |
Succinate dehydrogenase
(SDH),1 also known as complex
II or succinate-ubiquinone oxidoreductase, participates in the
mitochondrial electron transport by oxidizing succinate to fumarate and
transferring the electrons to ubiquinone (1-5). The mitochondrial
respiratory chain carries out a series of vectorial reactions that
generate an electrochemical potential across the inner mitochondrial
membrane, which is then used to drive the synthesis of ATP (6-9).
Membrane-bound fumarate reductases are structurally and functionally
related enzymes and are present in anaerobic organisms respiring with fumarate as the terminal electron acceptor (1-4, 10). FRD catalyzes the reduction of fumarate to succinate coupled to the oxidation of
quinol, the reverse of the reaction catalyzed by SDH. Both enzymes can
catalyze their respective reverse reactions in vitro, and in
some cases, in vivo. However, SDH and FRD are
physiologically distinct enzymes (2, 4, 11).
Generally, SDH is made up of two distinct domains: a dimeric peripheral
domain and a monomeric or a dimeric membrane-intrinsic domain. In the
yeast Saccharomyces cerevisiae, the peripheral domain, which
contains the active site for succinate oxidation, comprises the 67-kDa
Sdh1p subunit to which is covalently attached an FAD cofactor (12-16)
and the 28-kDa Sdh2p subunit, which contains three iron-sulfur clusters
(17, 18). The membrane-intrinsic domain is composed of two hydrophobic
subunits, Sdh3p and Sdh4p, of 16.7 and 16.6 kDa, respectively (19-21).
The anchor domain contains a b-type heme and the active site
for ubiquinone reduction (22-25). The anchor domain was proposed to
contain a core of four anti-parallel helices comprising helices I, II,
IV, and V (4, 26). This model is now supported by two crystal
structures (10, 27, 28).
Several lines of experimentation have shown that the membrane domain
contains the binding sites for quinone substrates. First, functional
anchor polypeptides are required for interaction with quinone
substrates (1, 3). Second, a thermodynamically stable interacting
ubisemiquinone pair has been observed in the vicinity of iron-sulfur
center 3 in SDH from Bos taurus (1, 3, 4), Neurospora
crassa (29), and some green algae (30). The stability of
ubiquinone in such a hydrophobic milieu is indicative of interaction with protein. Photoaffinity-labeling experiments (31-35), mutagenesis (36, 37), and inhibitor (1, 4, 38) studies have shown that SDH and FRD
contain at least two putative quinone-binding pockets, which are
located on the opposite sides of the membrane. The crystal structure of
the Escherichia coli FRD (27) revealed two bound quinones.
However, the crystal structure of the Wolinella succinogenes
FRD (28) shows no such bound quinone, although there are two distal
cavities in subunit C that could potentially bind quinone.
We have carried out several studies to identify functionally important
residues in the yeast SDH (22, 24, 25). Residues in the unusual
carboxyl-terminal extension of the yeast Sdh4p subunit are necessary
for maintaining a stable conformation of the anchor domain required for
quinone reduction (22, 24). We have identified residues in the yeast
Sdh3p that are involved in quinone reduction (25). The location of
these residues and inhibitor sensitivity analysis suggested that the
yeast SDH contains at least two quinone-binding sites. The present
paper extends these studies to the Sdh4p subunit and provides
additional evidence for the two quinone-binding site hypothesis. We
isolated and characterized four mutations in the SDH4 gene.
The mutants were characterized in vivo for their abilities
to support respiratory growth and in vitro for quinone
reduction, enzyme assembly and stability, and inhibitor sensitivity.
The data show that a distal quinone-binding site (QD) is
associated with the cytoplasmic side of the membrane. In addition, the
low and the high affinity inhibitor-binding sites, which may correspond
to the two quinone-binding sites, map to the opposite sides of the
membrane. A histidine residue in the matrix-facing loop between helices
V and VI is proposed to interact with the catalytic domain.
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EXPERIMENTAL PROCEDURES |
Strains, Media, and Culture Conditions--
The parental yeast
strain, MH125, and the E. coli strain, DH5
, have been
described previously (24). The SDH4 knockout strain, sdh4w2,
is an isogenic derivative of MH125 (MH125,
SDH4::TRP1) and has been described
previously (22). All the yeast media used in this study (YPD, YPG, SG,
SD, YPDG, and YPGal) and yeast culture conditions have been described
(22, 25). Plasmid loss was routinely monitored by plating out aliquots
on YPD and selectable SD media. The proportion of rho
strains in the culture was determined by mating the colonies with the
rho° strain, MS10 (mat
kar1-1 leu2-3 canR
rho0), and testing for respiratory competence of the
diploid on YPG plates. Bacterial strains were routinely grown on LB
medium at 37 °C, using ampicillin as the selectable marker.
Random Mutagenesis--
Random mutagenesis was carried out
essentially as described previously (25) with the following
modifications. A plasmid-borne SDH4 gene, pSDH4-17, was
transformed into DH5
. Cells were irradiated with ultraviolet light
of 254 nm with a dose rate of 1.4 J m
1
s
1 on LB plates in the dark to achieve 1-5%
survival. Mutagenized plasmids were isolated from the bacteria and
transformed into the SDH4 knockout strain, sdh4w2.
Transformants were tested for growth on YPDG, SG, and YPG to identify
respiratory-deficient colonies. Respiratory-deficient but
rho+ colonies were further analyzed. In order to ensure
that any observed respiratory defect is plasmid-mediated, plasmids were
rescued from yeast, amplified in DH5
, and re-transformed into the
sdh4w2 strain. Mutations were identified by sequencing the entire
SDH4 gene. Sequencing was performed by the Department of
Biochemistry Core DNA Facilities, University of Alberta (Edmonton,
Alberta, Canada). To ensure that any phenotype observed is due to
mutation in the SDH4 gene (and not to mutation of vector
sequence), the SDH4 cassette was subcloned into a
non-irradiated pRS416 vector and reintroduced into sdh4W2.
Membrane Preparation and Enzyme Assays--
YPGal-grown
stationary phase cells were harvested by centrifugation, lysed in a
French pressure cell, and submitochondrial particles prepared by
differential centrifugation as described previously (22, 24, 25).
Unless otherwise stated, the succinate-dependent reduction
of quinone was monitored spectrophotometrically at 22 °C as the
malonate-sensitive, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB)-mediated reduction of 2,6-dichlorophenol indophenol (DCPIP). The
succinate-dependent, phenazine methosulfate (PMS)-mediated reduction of DCPIP was determined as a measure of the
membrane-associated Sdh1p/Sdh2p dimer. This assay only requires that
the catalytic dimer be membrane-associated but does not require a
catalytically competent membrane domain. Quinone reduction was also
directly monitored as the reduction of DB using the wavelength pair,
280 and 325 nm, with a Hewlett Packard 8453 diode array
spectrophotometer. The absorption coefficient is 16 mM
1
cm
1. Other assays have been described (22,
24, 25).
Kinetic Analysis--
The effect of quinone on the initial
velocity of the succinate-DB reductase activity was measured by varying
the concentrations of DB at fixed saturating concentrations of
succinate and DCPIP. We evaluated the apparent kinetic parameters,
Km and Vmax, from
double-reciprocal plots as described previously (22, 24, 25).
Sensitivities to the quinone analog inhibitor,
2-sec-butyl-4,6-dinitrophenol (s-BDNP), were
determined by measuring initial velocities of succinate-DB reductase in
the presence of fixed concentrations of the inhibitor. The apparent
inhibitor constants, Ki(1) and
Ki(2) were evaluated by fitting data to the
equation for noncompetitive inhibition having two nonequivalent
Ki values (39),
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(Eq. 1)
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where y is the slope or intercept in the presence of
a fixed concentration of the inhibitor I, a is
the slope or intercept in the absence of the inhibitor, and
Ki(1) and Ki(2) are the
high affinity and the low affinity inhibition constants, respectively.
Thermal Stability--
Membrane fractions (20 mg/ml) were
divided into equal aliquots (50 µl) in 1.5-ml Eppendorf tubes and
placed on ice. Using a variable temperature block heater, each sample
was incubated for 10 min in the absence of succinate and quinone, at
temperatures between 22 and 70 °C. After incubation, the sample was
cooled to 22 °C and the succinate-DB reductase activity determined.
Residual activity was compared with the activities of unheated samples.
Miscellaneous Methods--
Preparation of mitochondrial
membranes, measurements of covalently bound flavin and protein
contents, E. coli transformation, and recombinant DNA
methods have been described (22, 24, 25).
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RESULTS |
Mutagenesis of the SDH4 Gene--
An E. coli strain
carrying the plasmid-borne SDH4 gene was mutagenized with
ultraviolet light and the mutated plasmids were reintroduced into the
yeast SDH4-deficient strain, sdh4W2. Mutagenesis in E. coli, rather than in yeast, greatly reduces the proportion of
yeast mitochondrial petite mutations isolated. About 6000 Ura+ sdh4W2 transformants were tested for respiratory
growth on YPDG and SG. Approximately 1% were impaired for respiratory
growth. We isolated and sequenced the SDH4 genes from 20 respiratory-deficient strains that were Ura+ and
rho+. Sixteen of these mutants contained frameshift
mutations that resulted in null phenotypes. Four mutants contained
single base alterations, resulting in the substitutions of Phe-69 with
Val (F69V), Ser-71 with Ala (S71A), His-99 with Leu (H99L), and Tyr-89 with a stop codon (Y89OCH). The Y89OCH mutation truncates Sdh4p by
removing the third predicted transmembrane segment, TMS-III (Fig.
1). The mutated SDH4 genes
were reintroduced into the SDH4 knockout strain, sdh4W2, and
assayed for respiratory growth on media containing the non-fermentable
carbon sources, ethanol, glycerol, or lactate. All the mutant strains
were greatly impaired for growth on these media. In order to determine
the residual respiratory abilities of the mutant strains, growth on
semisynthetic medium containing 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%
galactose was also monitored spectrophotometrically. Fig.
2 depicts the growth yields monitored as
the optical densities at 600 nm of late stationary phase cultures.
Growth of yeast strains in this medium is biphasic; an initial
fermentative growth is followed by a respiratory phase when the
fermentable carbon source is limiting (22, 40). Both the wild type and
the mutant strains show similar growth rates during the fermentative
phase (data not shown). The growth yields of the parental strain,
MH125, and the sdh4W2 strain carrying a plasmid-borne wild type
SDH4 gene, pSDH4-17, are similar. As expected, sdh4W2
achieves a growth yield of 10 ± 2% by fermentation alone. The
growth yields of the F60V, the S71A, the H99L, and the Y89OCH are
significantly reduced (40 ± 3%, 36 ± 2%, 30 ± 1%, and 26 ± 2%, respectively), but it is clear that respiratory
growth is not completely abolished. This result is consistent with our observation that the mutant strains can grow slowly on SG medium, with
colonies appearing only after 6-7 days of incubation at 30 °C. We
conclude that Phe-69, Ser-71, and His-99 are important for the
respiratory function of SDH. The Y89OCH strain still exhibits significant respiratory growth (26 ± 2%), compared with the
basal fermentative growth of sdh4W2 (10 ± 2%), indicating that
respiratory growth is possible without TMS-III.

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Fig. 1.
Topological model of the yeast Sdh4p.
Arrows indicate single amino acid substitutions, and the
bar indicates a premature termination. IMM, inner
mitochondrial membrane; IMS, intermembrane space.
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Fig. 2.
Growth of yeast strains on galactose
media. Yeast strains were grown at 30 °C on semisynthetic
liquid medium containing 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%
galactose, and the optical densities at 600 nm were measured.
Precultures were prepared on selective minimal medium containing 2%
galactose and 0.01% glucose. These were used to inoculate the main
cultures at a starting A600 of 0.1. The cultures
were allowed to reach late stationary phase (~100 h). The relative
growth yields are calculated using the final absorbance values reached
on 0.5% galactose. The yeast strains are: open
squares, MH125; closed squares,
sdh4W2/SDH4-17; open circles, F69V;
closed circles, S71A; open
triangles, H99L; closed triangles,
Y89OCH; diamonds, sdh4W2.
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Assembly of Mutants and Wild Type SDH Enzymes--
We examined the
possibility that the SDH4 mutations affected the anchoring
role of the Sdh4p subunit by determining the covalent FAD contents of
mitochondrial membranes prepared from wild type and mutant strains
(Table I). In the yeast S. cerevisiae, SDH is the major protein with covalently bound FAD and
the covalent flavin levels of mitochondrial membranes quantitatively
reflect SDH assembly (41). There is no significant difference between the covalent FAD levels of the F69V mutant and the wild type strains, MH125 and SDH4-17. The S71A membranes have slightly reduced covalent FAD levels compared with the wild type values. In contrast, the covalent FAD levels of the H99L and the Y89OCH strains are
significantly reduced, suggesting that these mutations induce
structural perturbations that impair assembly into the membrane.
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Table I
Measurements of SDH assembly in yeast mitochondrial membranes
Each value represents the mean of triplicate independent
determinations ± S.E.
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Table I also shows the succinate-PMS reductase activities of mutant and
wild type enzymes. This activity requires that the membrane domain be
competent for anchoring but not for quinone reduction (1, 3). To
compare their enzymatic activities, we determined the turnover numbers,
based on the covalent FAD contents. As expected, the succinate-PMS
reductase activities closely parallel the covalent FAD levels. The
activity of the F69V enzyme is not significantly different from the
wild type or the SDH4-17 enzymes. There is a slight reduction in the
succinate-PMS reductase activity of the S71A enzyme. These data confirm
that the primary defects in the F69V and S71A are not due to impaired assembly. In contrast, the H99L and the Y89OCH enzymes show significant reductions in their succinate-PMS reductase activities, indicating assembly defects.
Quinone Reductase Activities of the SDH4 Mutants--
We assayed
mitochondrial membranes for malonate-sensitive succinate
dependent quinone reduction, using the soluble quinone analog
DB and the reporter DCPIP (Table II). The
succinate-DB reductase activities of F69V, S71A, H99L, and Y89OCH are
severely reduced (30%, 26%, 20%, and 18% of the wild type turnover
number, respectively). Succinate-DB reductase activity is not
detectable in sdh4W2. These values are consistent with the residual
levels of respiratory growth seen on limiting galactose (Fig. 2).
We also measured the malonate-sensitive,
succinate-dependent cytochrome c reductase and
the succinate-oxidase activities of mutant and wild type mitochondrial
membranes (Table II). The former assay depends on complexes II and III
of the respiratory chain, whereas the latter assay requires complexes
II-IV. Both assays rely on the reduction of endogenous quinone and are
more stringent assays of the integrity of the SDH anchor polypeptides.
The succinate-cytochrome c reductase and the succinate
oxidase activities of the mutant enzymes parallel the succinate-DB
reductase activities, confirming that these mutant enzymes are impaired
for quinone reduction. Neither activity is detectable in the sdh4W2
membranes. To rule out the possibility that the SDH4
mutations have pleiotropic effects on other respiratory enzymes of the
mitochondrial inner membrane, we measured the NADH oxidase and the
glycerol-1-phosphate cytochrome c reductase activities
(Table II). As shown in Table II, the SDH4 mutations do not
affect these activities; the respiratory deficiencies are limited to
SDH.
Kinetics of Exogenous Quinone Reduction--
In our previous
studies (22, 24), we observed that some defects in quinone reduction
could be partially rescued by preincubation with quinone. We incubated
the mutant membranes with a 10-fold excess of DB (0.5 mM)
at 22 °C for 5 min and determined the succinate-DB reductase
activity (data not shown). The activities of the F69V and the S71A
membranes could be stimulated by 25% and 20%, respectively, indicating lower affinities of these enzymes for quinone. The H89L and
the Y89OCH membranes showed only slight stimulation in their
succinate-DB reductase activities (10% and 8%, respectively). Longer
incubations could not restore these activities to wild type levels.
Stimulation of the succinate-DB reductase activities was not due to
succinate activation, since the incubation was done in the absence of
this substrate. The stimulation did not result from the higher quinone
concentration used because membranes pre-incubated with 50 µM DB (the concentration used for the non-stimulated assay), showed similar levels of stimulation (data not shown). Pre-incubation with DB has no effect on the activities of the wild
type, the SDH4-17, or the sdh4w2 enzyme activities.
We measured the apparent kinetic parameters, Km and
Vmax, using the reporter DCPIP in a coupled
enzyme assay that measures the malonate-sensitive,
succinate-dependent reduction of DB. Mitochondrial
membranes containing mutant and wild type enzymes were pre-incubated
with varying concentrations of DB at 22 °C for 5 min, and the
reactions were initiated by the addition of succinate and DCPIP. The
apparent Michaelis constants were calculated and are summarized in
Table III. The apparent
K
of the F69V and the S71A
enzymes are increased 3-4-fold, whereas the Km for
the H99L is increased 2-fold. Deletion of the Sdh4p TMS-III (Y89OCH)
leads to the largest increase in the apparent
K
(5-fold). To compare the
catalytic efficiencies of the enzymes, we expressed the apparent
Vmax as maximal turnover numbers,
kcat, based on covalent flavin contents. All the
mutant enzymes show significant reductions in their
kcat values, with the Y89OCH enzyme exhibiting
the greatest decrease. However, we noted that the increase in
Km values did not lead to a proportional decrease in
kcat. For example, although the
Km values for the F69V and the S71A mutant enzymes
are increased almost 4-fold, there is only a 2-fold decrease in their
kcat values. The Michaelis-Menten equation (39)
may be rewritten as v = kcat/Km × [E][S] (42). The ratio
kcat/Km (the rate constant
for the reaction of free enzyme, E, with free substrate,
S, to give products) is an indicator of catalytic
efficiency. The catalytic efficiencies of the F69V, S71A, and H99L
enzymes (1.4 × 102, 1 × 102, and
1.3 × 102 µmol min
1,
respectively) are about an order of magnitude lower than that of the
wild type enzyme (1 × 103 µmol
min
1), whereas that of the Y89OCH (4.1 × 10 µmol min
1) is about 2 orders of
magnitude lower. We conclude from these data that the Sdh4p mutations
markedly affect the interaction of SDH with its substrate, quinone.
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Table III
The apparent Michaelis constants for DB reduction
Kinetic parameters were determined by varying the concentrations of DB
at fixed concentrations of succinate and DCPIP. Km
and Vmax were calculated from a nonlinear regression
fit to the Michaelis equation using initial estimates from the
Lineweaver-Burk plots. Each value represents the mean of triplicate
independent determinations ± S.E.
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Thermal Stability Profiles of the SDH4 Mutants--
There are many
possible explanations for the observed effects of the Sdh4p mutations
on the Km values. These include a small perturbation
of the quinone-binding site or a more global perturbation that is
propagated to the remainder of the enzyme. An example of the latter is
a mutation that weakens the association of the catalytic dimer with the
anchor polypeptides and impairs the transfer of electrons necessary for
quinone reduction. We determined the thermal stability profiles of
mutant and wild type enzymes by incubating mitochondrial membranes at
temperatures from 25 °C to 70 °C and measuring the succinate-DB
reductase activities (Fig.
3A). All enzymes are stable up
to 40 °C. At higher temperatures, the H99L and the Y89OCH enzymes
are unstable, whereas the F69V and the S71A enzymes exhibit thermal
denaturation profiles that are comparable to that of the wild type
enzyme. We also measured the succinate-PMS reductase activities of the
heated membranes (Fig. 3B). Interestingly, the F69V and the
S71A mutant enzymes are more stable for this activity, whereas the H99L
and the Y89OCH enzymes are labile. These differential effects of
thermal inactivation on the two enzyme activities suggest that the
structural perturbations produced by the F69V and the S71A mutations
are more localized to the membrane domain, the site of quinone
reduction, whereas the H99L and the Y89OCH mutations induce structural
perturbations that are propagated to the catalytic dimer. Since
preincubation with quinone stimulates the activities of the mutant
enzymes (data not shown), we also tested whether quinone could
stabilize the enzymes at elevated temperatures. As shown in Fig.
3C, preincubation with DB restores the stability of the F69V
and the S71A mutant enzymes to wild type levels, strongly suggesting
that these residues are close to a quinone-binding site. In contrast,
preincubation with DB has no detectable effect on the profiles of the
other mutant enzymes, suggesting that impaired quinone reduction may not be the primary defects in these enzymes. Similarly, preincubation with DB has no effect on the thermal denaturation profile of wild type
enzyme.

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Fig. 3.
Thermal stability profiles of succinate-DB
reductase activities of mutant and wild type enzymes. Values
represent the means of triplicate determinations. Activities are
expressed as percentages of turnover numbers observed at 25 °C.
A, DB reduction was monitored spectrophotometrically at the
wavelength pair of 280 and 325 nm after incubating mitochondrial
membranes at the indicated temperatures for 10 min. B,
succinate-PMS/DCPIP reductase activities were monitored
spectrophotometrically at 600 nm after incubating mitochondrial
membranes at the indicated temperatures for 10 min. C, as in
A, but with the addition of excess DB during the
incubations. The yeast strains are: open squares,
MH125; closed squares, sdh4W2/SDH4-17;
open circles, F69V; closed
circles, S71A; open triangles, H99L;
closed triangles, Y89OCH.
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Sensitivities of SDH4 Mutants to a Quinone Analog
Inhibitor--
The 4,6-dinitrophenol derivatives are quinone analogs
(43) that inhibit electron transfer in many respiratory proteins (44), including the bovine heart SDH and the E. coli FRD (38). We have previously shown that s-BDNP inhibits the yeast SDH
with hyperbolic, noncompetitive kinetics (22, 24, 25). It is likely
that a mutation that alters a quinone-binding site will also alter the
sensitivity of that enzyme to a quinone analog inhibitor. Consistent
with our previous observations (22, 24, 25), s-BDNP inhibits
the yeast SDH in a non-competitive (Fig. 4A) and non-linear (Fig.
4B) manner. Accordingly, we evaluated the apparent high
affinity and low affinity inhibitor constants (Ki(1) and Ki(2),
respectively) as described previously (22, 25). The apparent
Ki(1) and Ki(2) values
for the wild type enzyme (MH125 and SDH4-17) differ by an order of
magnitude (Table IV). The apparent
Ki(1) for the F69V and the S71A mutant enzymes
show only a minimal increase compared with the wild type values,
whereas their Ki(2) values are increased 3-fold.
In contrast, both the apparent Ki(1) and
Ki(2) values for the H99L and the Y89OCH mutant
enzymes are considerably increased.

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Fig. 4.
Inhibition of quinone reduction by
2-sec-butyl-4,6-dinitrophenol. A,
double-reciprocal plots showing non-competitive inhibition of
succinate-DB reductase activity of the wild type enzyme by the
inhibitor. The inhibitor concentrations are: 0 mM
(closed squares), 0.05 mM
(triangles), 0.1 mM (open
squares), 0.2 mM (closed
circles), and 0.4 mM (open
circles). B, secondary re-plots of intercepts
(reciprocal maximal velocities) against inhibitor concentration
(MH125, open circles; SDH4-17, filled
circles; F69V, open squares; S71A,
filled squares; H99L, open
diamonds; Y89OCH, filled diamonds).
The data were fitted to Equation 1 by non-linear least squares using
the quasi-Newton algorithm.
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DISCUSSION |
The anchor polypeptides of SDHs and FRDs show considerable
variability in cofactor composition and primary structure (1, 3, 4, 10,
26, 45). It is likely that this variability contributes to the unique
properties and functions of these enzymes in different biological
systems. Intensive searches for quinone-binding sites in SDHs (31, 32,
46, 47) and FRDs (36, 37) have been carried out. In addition, the
crystal structures of the E. coli and the W. succinogenes FRDs have recently been described (27, 28), revealing
considerable structural similarity at the tertiary level. The membrane
domains, as expected, contain the most dissimilar regions. The E. coli FRD contains two bound quinones without heme, whereas the
W. succinogenes FRD has two bound hemes without quinone. The
yeast SDH differs from these two cases, having a single heme and two
quinone-binding sites.
As part of our ongoing effort to identify functionally important
residues in the anchor polypeptides of the yeast SDH (22, 24, 25), we
have randomly mutagenized the SDH4 gene. Several lines of
experimental evidence presented in this study show that the two amino
acid residues Phe-69 and Ser-71 are important in the formation of a
quinone-binding site in SDH. First, the F69V and the S71A mutations
cause a severe impairment of respiratory growth (Fig. 2). However,
residual respiratory activity remains, indicating that the mutant SDH
enzymes are assembled and partially functional in vivo.
Second, the two mutants have near normal levels of covalent flavin and
of succinate-PMS reductase activity (Table I). These results
demonstrate that the mutations do not significantly affect the
anchoring function of Sdh4p and that the mutant enzymes are assembled
into the membrane in normal amounts. Third, enzyme activities requiring
quinone reduction (succinate-DB reductase, succinate-cytochrome
c reductase, and succinate oxidase) are significantly reduced in the two mutants. Fourth, incubation of the two mutant enzymes at elevated temperatures has only a minor effect on the stability of succinate-DB reductase activity (Fig. 3A).
Stability can be restored by preincubation with DB (Fig.
3C). Furthermore, the mutations do not affect the thermal
denaturation profile of the succinate-PMS reductase activity (Fig.
3B). These data strongly suggest that the F69V and the S71A
mutations specifically affect quinone binding in the membrane domain.
Fifth, the mutations decrease the affinities of the enzymes for quinone
substrate (Table III). The significant decrease in catalytic efficiency
(kcat/Km) strongly favors
this interpretation. Sixth, the kinetics of s-BDNP inhibition (Table IV) show that only the low affinity inhibitor-binding site, Ki(2), is affected. This inhibitor-binding
site likely corresponds to a quinone-binding site (38, 44). Finally, close proximity of the residues Phe-69 and Ser-71 strongly argues for
their involvement in a common function, which we propose is the
formation of a quinone-binding site in the vicinity of the loop
connecting the Sdh4p transmembrane segments I and II.
The observed effects of the H99L mutation are explicable in terms of a
larger structural perturbation of the Sdh4p subunit that is propagated
to the catalytic domain. The H99L mutation leads to a reduced covalent
FAD content and a reduced turnover number for succinate-PMS reductase
activity, strongly suggesting that enzyme assembly is compromised. This
conclusion is further strengthened by the enzyme's thermal stability
profiles. The H99L mutant is unstable for both the succinate-DB
reductase (Fig. 3A) and the succinate-PMS reductase (Fig.
3B) activities. In a previous study (25), we proposed that
Asp-117 in the Sdh3p subunit, is at the interface of the peripheral and
the membrane domains. The Sdh4p H99L mutation mimics many of the
effects of the Sdh3p D117V mutation, suggesting that they may perform a
similar role. The H99L mutation leads to a 2-fold increase in the
Km for DB reduction; this is the smallest effect on
Km observed for all the mutants examined in this
study. Since this mutation affects enzyme assembly, we do not consider
that the elevated Km reflects a direct effect on the
quinone-binding site. Similarly, the impaired assembly of the H99L
enzyme precludes a simple interpretation of the s-BDNP
inhibition data. However, it is interesting to note that the H99L
mutation has equal effects on the Ki(1) and the
Ki(2) values, consistent with the mutation
having more global effects on the Sdh4p subunit.
Deletion of Sdh4p subunit TMS-III does not lead to a complete loss of
respiratory growth and the truncated enzyme still possesses significant
quinone reductase activity in vitro. This suggests that
TMS-III is not required for catalysis but rather plays an important
role in maintaining the structural integrity of the Sdh4p subunit. The
mutation causes the greatest reduction in catalysis as well as in
thermal stability. This conclusion is consistent with our previous
observations on the role of the Sdh4p COOH-terminal extension (22). It
is also consistent with the location of TMS-III of the E. coli FRD D subunit outside the helix bundle that forms the
quinone-binding pockets (10, 27, 28).
Based on the results obtained in this and in previous studies (22, 24,
25), we propose a two-site model for quinone-protein interaction (Fig.
5). Mutations whose effects appear to be
limited to quinone binding are located on opposite sides of the
membrane. The Sdh4p residues Phe-69 and Ser-71 are on TMS-II, toward
the cytoplasmic side of the membrane, whereas the functionally
important residues, His-113, Trp-116, and His-106, in the Sdh3p subunit are clustered toward the matrix side. Apart from their asymmetric locations, two of the residues also have differential effects on
s-BDNP binding. The F69V and S71A mutations in the Sdh4p
subunit affect mainly the low affinity constant,
Ki(2), whereas the H106Y and the H113Q mutations
in the Sdh3p subunit affect mainly the high affinity inhibition
constant, Ki(1). In addition, deletion of the
Sdh4p carboxyl terminus exerts major effects on
Ki(2), and Lys-132 was identified as a
functionally important residue in this region. These observations
strongly argue for the presence of two spatially distinct
quinone-binding sites in the yeast SDH, similar to the E. coli FRD structure (27, 36, 37). The effects of the Sdh3p W116R
mutation on inhibitor binding could not be evaluated due to low DB
reductase activities but its location is consistent with an effect on
QP. Similarly, the Sdh3p mutation F103V specifically
affected the enzyme's affinity for quinone but inhibitor studies were
not performed. Its location near the center is consistent with a role
in either QP or QD. Further investigation is
required to clarify this. Taken together, the data suggest that the
high affinity inhibitor-binding site (Ki(1)) is
equivalent to the QP site whereas the low affinity
inhibitor-binding site (Ki(2)) is equivalent to
the QD site.

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Fig. 5.
A model for quinone-binding sites in the
yeast SDH. The topological model for the anchor polypeptides was
developed after hydropathy analysis using the Kyte-Doolittle algorithm
(54). TMS-I, -II, -IV, and -V are proposed to form a bundle as in the
E. coli and the W. succinogenes FRDs (4, 27, 28).
The two quinone-binding sites are designated QP and
QD (using the nomenclature developed for the E. coli FRD) (27). Residues in ovals are proposed to
mediate quinone binding, whereas residues in rectangles are
more likely involved in intersubunit interactions.
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