Three Classes of Inhibitors Share a Common Binding Domain in
Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)*
Jürgen G.
Okun
,
Peter
Lümmen§, and
Ulrich
Brandt
¶
From the
Universitätsklinikum Frankfurt,
Institut für Biochemie I, D-60590 Frankfurt am Main and
§ Hoechst Schering AgrEvo GmbH, Biochemical Research, H872N,
D-65926 Frankfurt am Main, Federal Republic of Germany
 |
ABSTRACT |
We have developed two independent methods to
measure equilibrium binding of inhibitors to membrane-bound and
partially purified NADH:ubiquinone oxidoreductase (complex I) to
characterize the binding sites for the great variety of hydrophobic
compounds acting on this large and complicated enzyme. Taking advantage
of a partial quench of fluorescence upon binding of the fenazaquin-type
inhibitor 2-decyl-4-quinazolinyl amine to complex I in bovine
submitochondrial particles, we determined a Kd of
17 ± 3 nM and one binding site per complex I. Equilibrium binding studies with [3H]dihydrorotenone and
the aminopyrimidine [3H]AE F119209
(4(cis-4-[3H]isopropyl
cyclohexylamino)-5-chloro-6-ethyl pyrimidine) using partially purified
complex I from Musca domestica exhibited little unspecific
binding and allowed reliable determination of dissociation constants.
Competition experiments consistently demonstrated that all tested
hydrophobic inhibitors of complex I share a common binding domain with
partially overlapping sites. Although the rotenone site overlaps with
both the piericidin A and the capsaicin site, the latter two sites do
not overlap. This is in contrast to the interpretation of enzyme
kinetics that have previously been used to define three classes of
complex I inhibitors. The existence of only one large inhibitor binding
pocket in the hydrophobic part of complex I is discussed in the light
of possible mechanisms of proton translocation.
 |
INTRODUCTION |
The proton-pumping NADH:ubiquinone oxidoreductase (EC
1.6.99.3, complex I) is the first membrane-bound electron transport complex of the mitochondrial respiratory chain. Electron transfer from
NADH to ubiquinone is coupled to the translocation of two protons per
electron across the inner mitochondrial membrane (1, 2). Thereby,
complex I accounts for up to 40% of the proton-translocating capacity
of the respiratory chain.
Complex I is present in the mitochondria of most eukaryotic organisms
and many bacteria. In mammals, it consists of 43 different subunits
with a molecular mass of ~1,000 kDa (3). The homologous procaryotic complex I has a minimal number of 14 different subunits with a total molecular mass of ~500 kDa (4, 5).
Electron microscopic analysis of the Neurospora crassa (6),
Escherichia coli (7), and bovine complex I (8) indicates an
L-shaped structure with two domains arranged perpendicular to each
other that are called peripheral and membrane arm (9). In mitochondrial
complex I, seven nuclear-encoded proteins with strong homology to their
bacterial counterparts form the peripheral part (10, 11). These
subunits carry the NADH binding site and the redox groups, namely
noncovalently bound FMN and the iron-sulfur centers N-1 to N-5 (12).
The membrane arm contains the remaining seven subunits homologous to
the bacterial complex, which are encoded by the mitochondrial genome in eucaryotes.
Despite recent progress in structural knowledge, little is known about
the electron pathway, the proton translocation mechanism, and the
binding sites and mode of action of the large number of specific
inhibitors of complex I. However, it seems inevitable to conclude from
the available evidence that the proton translocating machinery resides
largely in the membrane part, although all known prosthetic groups have
been assigned to the peripheral part of the enzyme (13). This has
revived earlier ideas (14) that a mechanism similar to the proton
motive ubiquinone cycle operating in the cytochrome
bc1 complex (15) confers proton translocation in
complex I (13, 16). These hypothetical mechanisms inherently predict
that the hydrophobic part of complex I carries two or three
independently operating reaction sites for ubiquinone.
Many structurally diverse hydrophobic compounds have been described to
inhibit complex I and are considered to interfere with ubiquinone
reduction (12, 17, 18). Kinetic studies suggest that these inhibitors
can be grouped into two (19) or even three (20) classes, represented by
piericidin A (class I/A-type), rotenone (class II/B-type), and
capsaicin (C-type), respectively. It remains unclear, however, whether
these classes in fact reflect three distinct inhibitor and quinone
binding sites. Two different semiquinone species have been reported by
EPR spectroscopy during the steady state reaction of complex I (21),
but there is still some controversy whether these reflect two
ubiquinones or two forms of the same ubiquinone (22). The problem with
the large number of studies employing Michaelis-Menten type kinetics
(19, 23-27) is that the physical properties of the substrate, the
inhibitors, and the membrane-bound enzyme as well as the complexity of
the underlying catalytic mechanism make interpretation of these data difficult and ambiguous. Especially, because of their amphiphilic properties, ubiquinone and the inhibitors tend to accumulate in the
small hydrophobic membrane phase so that the actual target site
concentrations are very difficult to determine. This would be essential
to calculate meaningful kinetic parameters.
Therefore, we have developed two independent approaches to investigate
equilibrium inhibitor binding to complex I. This allowed us to test
directly if representative complex I inhibitors interact with each
other at their cognate binding sites and how these binding sites
relate to each other.
 |
EXPERIMENTAL PROCEDURES |
Inhibitors--
All inhibitors were used as ethanolic stock
solutions. The synthetic capsaicin analogue CC
441 (28) and
5'-
-epirotenone (29) were kind gifts from H. Miyoshi, Kyoto; DQA
(SAN 549 (30)), fenazaquin, pyrimidifen, and fenpyroximate 4(cis-4-tert-butylcyclohexylamino)5-chloro-6-ethylpyrimidine
(AEF117223) were obtained from AgrEvo, Frankfurt; Kresoxim-Methyl
Brio® was a kind gift from BASF, Ludwigshafen, Germany;
piericidin A was a kind gift from A. Dupuis, Grenoble; rolliniastatin-1
and rolliniastatin-2 were kind gifts from M. Degli Esposti, Clayton, Australia. [isopropyl-3H]Dihydrorotenone, 1.89 TBq
mmol
1, was synthesized by Amersham Pharmacia Biotech.
4(cis-4-[3H]Isopropyl
cyclohexylamino)5-chloro-6-ethyl pyrimidine ([3H]AE
F119209), 2.06 TBq mmol
1, was synthesized by Roussel
Uclaf, Romainville, France. All other chemicals were purchased from
Sigma or Carl Roth GmbH & Co. (Karlsruhe) in analytical quality.
Preparation of Bovine Submitochondrial
Particles--
Mitochondria were isolated as described by Smith (31).
Bovine submitochondrial particles (SMP) were prepared essentially as
described by Thierbach and Reichenbach (32). Mitochondria were diluted
in 250 mM sucrose, 10 mM potassium phosphate,
10 mM Tris/HCl, 2 mM EGTA, 2 mM
MgCl2, pH 7.4, to a protein concentration of ~10 mg/ml.
Batches of about 25 ml were treated 10 times for 15 s with a
Branson sonifier 250 (Branson, Danbury, CT) at maximum output energy in
an ice bath. The sonicated suspension was centrifuged at 10,000 × g for 10 min, and the supernatant was centrifuged at
100,000 × g for 45 min at 4 °C. The pellet was
resuspended in 1 mM EDTA, 1 mM
MgCl2, 75 mM sodium phosphate, pH 7.4, and could be stored for months in liquid nitrogen. The content of cytochrome c oxidase was determined by the reduced minus
oxidized spectrum at 605-630 nm (
605-630 nm = 24.0 mM
1 × cm
1).
Preparation of Housefly SMP--
20 g of housefly (Musca
domestica) thoraces were homogenized at 4 °C in 150 ml of 154 mM KCl, 1 mM EDTA, adjusted to pH 7.4, following the procedure of Nedergaard and Cannon (33). After filtration
through two layers of cheesecloth, the homogenate was centrifuged for
10 min at 500 × g. The pellet was discarded, and the
supernatant was centrifuged for 10 min at 3000 × g.
The pellet was resuspended in 20 mM Tris/HCl, pH 8.0, 100 mM KCl, 1.0 mM EDTA, and the protein
concentration was adjusted to 10 mg/ml.
Solubilization and Partial Purification of Housefly Complex
I--
Housefly SMP suspensions were solubilized by the addition of
4% (w/v) dodecylmaltoside and 150 mM KCl for 30 min at
4 °C and centrifuged at 100,000 ×g for 60 min at 4 °C. The
supernatant was partly delipidated on a Sephacryl S200 column (26 × 600 mm) in 20 mM Tris/HCl, pH 8.0, 100 mM
NaCl, 0.1% (w/v) CHAPS at 4 °C. Fractions of the void volume
showing n-decylubiquinone-dependent NADH
oxidation were pooled and further purified on a Q-Sepharose column
(26 × 100 mm) equilibrated with the same buffer. Proteins were
eluted with a linear gradient of 0.1-1.0 M NaCl in 20 mM Tris/HCl, pH 8.0, 0.1% (w/v) CHAPS. Fractions
exhibiting rotenone-sensitive NADH:ubiquinone oxidoreductase activity
were pooled and stored in aliquots at
80 °C.
Determination of Catalytic Activity--
We used
n-nonylubiquinone (NBQ) as a substrate for the determination
of NADH:ubiquinone activity of SMP, which has been reported as one of
the best ubiquinone-10 analogues for this purpose (25, 34). NBQ was
prepared essentially following the protocol of Wan et al.
(35). Steady state activity was recorded in a Shimadzu UV-300
spectrophotometer as NADH oxidation at 340-400 nm (
340-400
nm = 6.10 mM
1 × cm
1)
using a thermostatted cuvette (30 °C) with a final volume of 1 ml.
100 µM NADH and 50 µg of SMP were added to buffer
containing 50 mM Tris/HCl, pH 7.4, 5 µM
Kresoxim-Methyl Brio® and 2 mM KCN. The
catalytic reaction was started by the addition of 60 µM
NBQ. Inhibitors were added to the cuvette before the addition of NBQ.
Michaelis-Menten parameters were determined by varying the
concentration of NADH or NBQ.
Fluorescence Measurements--
Fluorescence spectra were
recorded on a SPEX Fluorolog 212 fluorometer attached to an AT-type
personal computer. The fluorescence quench titrations (FQT) were
performed and analyzed as described earlier (36) by directly fitting
the data to a formula derived directly from the standard binding
equation.2 DQA (
291
nm = 8.14 mM
1 × cm
1 in
ethanol) was automatically added to a stirred cuvette in 1-µl steps
from a 15 µM stock solution in ethanol using a Hamilton Microlab M dispensor equipped with a 50-µl syringe. For FQT
measurements, bovine SMP were diluted in N2-saturated
buffer (2 mM KCN, 1 mM EDTA, 1 mM
MgCl2, 2.5 µM Kresoxim-Methyl
Brio®, 75 mM sodium phosphate, pH 7.4) to 2.5 mg of protein/ml, corresponding to ~1.5 µM cytochrome
c oxidase. Based on a complex I to cytochrome c
oxidase ratio of 1:10 in bovine SMP (37, 38), the complex I
concentration was estimated at 0.15 µM or 0.06 nmol/mg of protein.
Indirect determination of the dissociation constant
Kd for the binding of 5'-
-epirotenone by
fluorescence quench titration was performed based on the standard
binding equation (36). The data were analyzed using the Psiplot
software package version 4.61 (Poly Software International).
Radioligand Binding Assays--
Specific binding of two
radiolabeled inhibitors [3H]dihydrorotenone (39-41) and
[3H]AE F119209 to the solubilized and partially purified
NADH:ubiquinone oxidoreductase from housefly flight muscle mitochondria
(18) was measured as follows.
For saturation binding experiments, 3 µg of protein in 20 mM Tris/HCl, pH 8.0, 0.25% (w/v) CHAPS radioligand
concentrations ranging from 0.4 to 100 nM were incubated at
22 °C in a sample volume of 100 µl. In competition experiments,
the radioligand concentration was fixed at 6.5 nM, and
variable concentrations of competing ligands were added. Unspecific
binding was determined using 10 µM unlabeled rotenone or
AE F119209, respectively. Methanol at a final concentration of 5%
(v/v) in the assay mix was used to mediate the dissolution of
radioligands and other inhibitors. After 20 min, 300 µl of 10 mg/ml
dextran-coated charcoal (Sigma) in 20 mM Tris/HCl, pH 7.2, were mixed in thoroughly. The charcoal was sedimented by centrifugation
at 13,000 × g for 3 min. Protein-bound radioligand was
measured in the supernatant by liquid scintillation counting. Data were
analyzed by standard algorithms with either the EBDA (Biosoft, UK) or
the SigmaPlot (Jandel Scientific) software package.
The dissociation constant of complex I inhibitors that competed with
equilibrium binding of radiolabeled AE F119209 and dihydrorotenone was
determined by measuring the amount of bound radioligand in the presence
of increasing concentrations of unlabeled inhibitor. From the resulting
logistic plots, apparent B50 values were determined as the
concentration of competing inhibitor required to displace 50% of the
radioligand. The apparent dissociation constants Kd were calculated according to the Cheng-Prusoff equation (42, 43):
Kd = B50/1 + [L]/Kd[L], where
Kd[L] is the dissociation
constant of the radioligand, and [L] is the concentration of free radioligand.
 |
RESULTS |
Kinetic Constants and I50 Values from Steady State
Kinetics--
To test for the activity of complex I in our SMP
preparation, we determined the Michaelis-Menten parameters for NADH and
NBQ. The Km values were 3.9 ± 0.5 µM for NADH and 2.3 ± 0.2 µM for NBQ.
Vmax was 1.16 ± 0.03 µmol of NADH × min
1 × mg
1 of protein. These values are
comparable with those reported by others (3, 25, 44, 45). No increase
in steady state activity was observed by applying the "activation"
procedure described in Burbaev et al. (46).
I50 values were determined as the final concentration of
inhibitor required to reduce the NADH oxidation rate to 50% of the uninhibited rate. The data listed in Table
I are in good agreement with published
values (17, 28, 47). An I50 value of 6 nM was
determined for DQA, the fenazaquin-type inhibitor used in the FQT
binding assay. DQA is an inhibitor specific for complex I, as it had no
effect on the activity of succinate dehydrogenase or cytochrome
bc1 complex (data not shown).
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Table I
I50 values and competition binding analysis of complex I
inhibitors
The inhibitors are grouped according to the classification by Friedrich
et al. (19) and Degli Esposti and Ghelli (20). I50
values were determined as described under "Experimental
Procedures." Displacement was tested by adding 0.3 µM
(2-fold molar excess) of inhibitor before FQT. Under these conditions,
DQA binding was prevented by all inhibitors tested except by
5'- -epirotenone and CC 44. In the case of 5'- -epirotenone, an
increase in apparent Kd for DQA with increasing
concentrations of competing inhibitor was observed and used to
indirectly calculate the Kd for this inhibitor. In
the case of CC 44, concentrations up to 10 µM did not
affect the DQA titrations, but higher concentrations resulted in
unspecific distortions of the titration (see text for further details).
Competition experiments with [3H]dihydrorotenone or
[3H]AE F119209 were performed as described. ND, not
determined.
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|
Analysis of DQA Binding to Bovine Complex I by Fluorescence Quench
Titration--
Fig. 1 shows the
fluorescence spectra of DQA in aqueous solution. The excitation maximum
at 316 nm, and the emission maximum at 360 nm were used to follow DQA
binding to complex I. The typical titration given in Fig.
2 shows that the fluorescence of DQA was partially quenched when bound to the enzyme. According to the numerical
fit of the data, fluorescence was quenched by 62 ± 3% upon
binding, and the Kd was 17 ± 3 nM
(n = 15). The concentration of binding sites was found
to be 0.15 ± 0.02 µM, which fits perfectly with one
binding site per complex I and a ratio between cytochrome c
oxidase and complex I of 1:10 (37, 38). Neither activation of bovine
SMP at 30 °C for 90 min as described in Burbaev et al.
(46) nor addition of 1% bovine serum albumin or 10 µM
Kresoxim-Methyl Brio® or 3% ethanol or 1 mM
N-ethylmaleimide had any effect on the Kd
or the number of binding sites for DQA (data not shown).

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Fig. 1.
Fluorescence spectra of DQA. The spectra
of 10 µM DQA were recorded in 1 mM EDTA, 1 mM MgCl2, 0.1% dodecylmaltoside, 75 mM sodium phosphate, pH 7.4. The background spectra
resulting from the buffer were subtracted.  , excitation spectrum
(emission at 360 nm); - - - - - -, emission spectrum (excitation at 316 nm).
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Fig. 2.
Mutual displacement of DQA by different
complex I inhibitors. The experimental conditions were as
described under "Experimental Procedures" and in the legend of
Table I. , bovine SMP, the line represents the least
squares fit, calculated as described in Brandt and von Jagow (36). ,
bovine SMP, preincubated with 0.3 µM piericidin A.
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|
When SMP were preincubated with 0.3 µM piericidin A (Fig.
2) rotenone, rolliniastatin-1, or rolliniastatin-2, the fluorescence of
added DQA was not quenched, indicating displacement of DQA from its
binding site. The results for all tested complex I inhibitors are
summarized in Table I. Even the rather weak binding rotenone analogue
5'-
-epirotenone (I50 = 11 µM) shifted the
apparent Kd for DQA when added at concentrations
between 1 and 100 µM. From these data a
Kd of 6 ± 2 µM
(n = 7) for 5'-
-epirotenone was calculated.
Only the capsaicin derivative CC 44 (28) did not affect the FQT
titration up to a concentration of 10 µM, indicating that it failed to specifically displace DQA. At higher concentrations of CC
44, the titrations were distorted and could not be fitted to the
equation. However, this could not be because of specific displacement,
as from an I50 of 80 nM for CC 44 (cf. Table I) one can predict that 10 µM CC 44 should have had a dramatic effect on the apparent Kd
for DQA. This is also illustrated by the fact that at 10 µM even the two orders of magnitude weaker-binding 5'-
-epirotenone had a significant effect.
Binding of AEF119209 and Dihydrorotenone to Partially Purified
Musca Complex I--
Binding of the tritiated aminopyrimidine AE
F119209 (Fig. 3) to the partially
purified housefly complex I was found to be specific (90-95% specific
binding) and saturable with an apparent dissociation constant of 9 nM as determined by Scatchard transformation of the data
(Fig. 4). The maximum number of binding
sites Bmax was 0.1 nmol/mg. Dihydrorotenone also
exhibited saturable binding with an apparent Kd of
30 nM and a Bmax of around 0.1 nmol/mg. The Scatchard plots (Fig. 4, inset) and Hill plots
(not shown) indicated a homogeneous population of a single binding site
for either ligand. Careful analysis of several independent experiments
also gave no indication for two binding sites for dihydrorotenone or
the aminopyrimidine, as it was not possible to fit the data to two
components in any meaningful way.

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Fig. 4.
Radioligand binding analysis of
dihydrorotenone and AE F119209 to Musca complex
I. Binding data obtained for both radiolabeled complex I
inhibitors to solubilized enzyme are plotted directly and in Scatchard
representation (inset). , AE F119209.
Kd = 9 nM; boundmax = 100 pmol/mg. , dihydrorotenone. Kd = 29 nM; boundmax = 110 pmol/mg. See text for
details.
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To study whether dihydrorotenone and the class I inhibitors bound
competitively, the saturation binding of labeled dihydrorotenone was
measured in the presence or absence of 10 nM of the class I
inhibitor piericidin A. Scatchard analysis of the equilibrium binding
data (Fig. 5) indicated that the apparent
Bmax of the radioligand was not changed when
piericidin A was present, i.e. the binding was competitive
with respect to the radioligand. The same result was obtained when the
aminopyridine inhibitor AE F117233 was used as a competitor for
dihydrorotenone (data not shown).

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Fig. 5.
Scatchard analysis of dihydrorotenone binding
in the presence of piericidin A. Saturation binding was determined
as described in the presence ( ) or absence ( ) of 10 nM piericidin A.
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|
Representatives of each class of complex I inhibitors were tested for
their capacity to compete with a fixed concentration of the
radioligands AE F119209 or dihydrorotenone under equilibrium binding
conditions. The Kd values calculated from these competition experiments were in good agreement with the relative I50 values determined by titration of the steady state rate
(Table I). With one important exception, both radioligands competed with all tested inhibitors and gave very similar Kd
values. The capsaicinoid CC 44 competed with dihydrorotenone but did
not affect binding of AE F119209 even at a concentration of 10 µM.
 |
DISCUSSION |
The large number of structurally different compounds that have
been described to specifically inhibit ubiquinone reduction by
proton-translocating NADH:ubiquinone oxidoreductase (17) are
potentially very useful to probe the mechanism of this most complicated
enzyme of the respiratory chain. These compounds are also of
considerable interest as lead structures for the development of
insecticides and acaricides (18). However, even the number of
independent binding sites is still controversial. The major problem has
been that most inhibitor studies with complex I were based on the
interpretation of data from steady state kinetics. This approach can
only generate indirect evidence that is difficult to validate because
of the complexity of the enzyme and experimental complications inherent
to the steady state kinetics of complex I (44). Several reports on
direct binding studies using radioligands (27, 48) and competition
experiments with a limited selection of inhibitors (41) have been
published. However, these studies were performed with membrane-bound
complex I and suffered from a high degree of nonspecific binding,
e.g. several washes with bovine serum albumin were necessary
to distinguish between specific and nonspecific binding (48), and
saturation of the binding sites was not achieved, preventing
unambiguous interpretation.
Here we report results from two independent approaches to study
equilibrium binding of hydrophobic inhibitors to membrane-bound and
partially purified complex I. Both methods were not affected by
nonspecific binding effects and gave consistent and reliable results.
We found no influence on our FQT measurements by a number of treatments
including activation of complex I (46) and addition of bovine serum
albumin or the thiol reagent N-ethylmaleimide, which were
claimed to affect inhibitor binding (48, 50).
To check whether the classification into two (19) or three (20)
inhibitor classes represented by piericidin A (class I/A-type), rotenone (class II/B-type), and capsaicin (C-type) in fact reflects two
or even three independent binding sites, we have performed direct
competition experiments with a representative selection of inhibitors.
The data obtained with both methods consistently indicated that all
tested hydrophobic inhibitors of complex I share a common binding
domain with partially overlapping sites (cf. Table I). As
illustrated in Fig. 6, the rotenone site
(class II/B-type) overlaps with both the piericidin A site (class
I/A-type) and the capsaicin site (C-type), but binding of the latter
two types of inhibitors does not interfere with each other.

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Fig. 6.
Schematic representation of the inhibitor
binding domain of complex I. The binding sites for the three
classes of hydrophobic complex I inhibitors as deduced from equilibrium
binding studies are depicted to illustrate their relative arrangement
in a common binding pocket.
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Overlapping binding sites for class I and class II inhibitors
have also been suggested from recent results by Darrouzet and Dupuis
(51), who have reported a point mutation in complex I from
Rhodobacter capsulatus that confers resistance to piericidin A and exhibits cross-resistance to rotenone. The idea of a fairly large
ubiquinone binding domain also fits well with recently published data
showing that this pocket is sufficiently spacious to accommodate rather
bulky exogenous ubiquinones (52).
The observation that some, but not all complex I inhibitors also
inhibit bacterial glucose:ubiquinone oxidoreductase (19) can be
interpreted in terms of structural similarity of its ubiquinone reactive site to part of the complex I binding pocket. However, some of
the conclusions based on enzyme kinetics claiming independent inhibitor
binding sites have to be considered as taking the interpretation of
this indirect approach too far (45).
We have also found no indications from our equilibrium binding data
that there is more than one binding site for piericidin A or
rotenone-type inhibitors per complex I as has been concluded indirectly
from kinetic studies. (17, 26, 53). The number of binding sites we
could identify matched exactly the amount of inhibitor needed to
completely block the activity of complex I (38), and in all cases of
competitive binding, inhibitors were always displaced completely. Thus,
if there where two inhibitor binding sites per complex I, they would
have to be indistinguishable in terms of ligand affinity. In the
absence of compelling evidence in favor of such unusual binding site
heterogeneity, we consider this option as highly unlikely.
It should be noted that in the light of our results, the grouping of
complex I inhibitors into three distinct classes, which we have still
used to be consistent with the literature, seems somewhat arbitrary,
e.g., although kinetic data seem to indicate that the
binding sites for piericidin A, DQA, and the aminopyridines are somehow
related and largely overlapping, the structural differences between
these three compounds suggest that the sites are not identical.
It should be noted that the emerging picture of a fairly large
ubiquinone binding pocket with several binding sites for structurally diverse inhibitors in the membrane part of complex I (Fig. 6) is very
similar to the now well documented situation (by x-ray crystal
structures) in the QB site of the bacterial reaction center (54) and in center P of the cytochrome bc1
complex (55-57).
Taken together, we cannot entirely rule out reversible binding of more
than one ubiquinone to complex I at this point. But considering the
huge array of structurally diverse high affinity inhibitors known to
inhibit ubiquinone reduction completely (17) that we have shown to
interact with each other at their cognate binding sites, there is no
indication for this. The observation of two distinct semiquinone
species by EPR during steady state of complex I can still result from
two ubiquinone molecules, one of which is the substrate exchanging with
the membrane, whereas the other is tightly bound to the complex acting
as a prosthetic group. This situation would be reminiscent to
QB and QA in the bacterial reaction center
(58).
If there is in fact only one substrate binding site, this seems
difficult to reconcile with the mechanistic models of the reverse
ubiquinone-cycle-type that have been put forward recently (13, 16).
Such ligand conduction reaction schemes require at least two such
sites, one for ubiquinol oxidation and one for ubiquinone reduction.
However, the redox-gated ligand conduction mechanism (13) can be
modified to a localized mechanism by replacing two substrate sites with
a single tightly bound ubiquinone. The modified mechanism is based on
the same general mechanistic principles, still employs the
redox-dependent protonation and deprotonation of
ubiquinone, and features one tightly bound and one substrate ubiquinone
(49).
 |
ACKNOWLEDGEMENTS |
We thank Franz J. Streb, Wolfgang Maurer, and
Raimund Saar for excellent technical assistance in performing the
experiments. We are indebted to Hideto Myoshi and Volker Zickermann
for carefully reading the manuscript and for helpful comments.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant BR 1633/1, SFB 472, and Graduiertenkolleg 145/12.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed:
Universitätsklinkum Frankfurt, Institut für Biochemie I,
ZBC Theodor-Stern-Kai 7, Haus 25B, D-60590 Frankfurt am Main, Germany.
Tel.: +49 69 6301 6926; Fax: +49 69 6301 6970; E-mail:
brandt{at}zbc.klinik.uni-frankfurt.de.
The abbreviations used are:
CC 44, 4-(p-tert-butylphenoxy)benzoic
acid-3,4-dimethoxybenzylamide; AE F117233, 4(cis-4-tert-butylcyclohexylamino)5-chloro-6-ethyl
pyrimidine; AE F119209, 4-(cis-4-[3H]isopropyl
cyclohexylamino)5-chloro-6-ethyl pyrimidine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DQA, 2-decyl-4-quinazolinyl amine; FQT, fluorescence quench titration; NBQ, n-nonylubiquinone; SMP, submitochondrial particles.
2
The formula used to analyze FQT is as follows. The
observed fluorescence Fobs during FQT is
given by
|
(Eq. 1)
|
with
|
(Eq. 2)
|
and
|
(Eq. 3)
|
where fbound and
ffree are the specific fluorescence of the bound
and free inhibitor, [Itot],
[Ibound], and [Ifree]
are the concentrations of total, bound, and free inhibitor,
[Etot] is the total concentration of enzyme,
and ns is the number of binding sites.
 |
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