(Received for publication, April 3, 1995; and in revised form, August 1, 1995)
From the
Bovine heart bc complex is reversibly
inhibited by zinc ions with an inhibition constant K
of 10
M at pH
7.0. Binding
of zinc is at least a factor of 10 tighter than binding of any other
metal ion tested. Essentially complete inhibition of
ubihydroquinone:cytochrome c oxidoreductase activity is
observed at concentrations of [Zn
] > 5
µM. Zinc does not affect the K
for the substrates, ubihydroquinone or cytochrome c, but zinc inhibits reduction of the cytochromes by
ubihydroquinone through the Q
center. A radioactive binding
assay using
Zn revealed one high affinity binding site per bc
complex with K
10
M at pH = 7.0 and
3-4 additional low affinity binding sites (K
> 2
10
M). Zinc
binding does not depend on the redox state of the high potential chain
(iron-sulfur protein and cytochrome c
). Zinc binds
3 times tighter to Fe-S-depleted bc
complex
indicating that the zinc binding site is not on the
``Rieske'' iron-sulfur protein in contrast to a recent report
by Lorusso et al. (Lorusso, M., Cocco, T., Sardanella, A. M.,
Minuto, M., Bonomi, F., and Papa, S. (1991) Eur. J. Biochem. 197, 555-561). Zinc binds to a site which has the same
affinity for zinc as for protons. We conclude that the zinc binding
site is close to a protonatable group of the bc
complex with pK
= 7.2 which
has not been identified previously. We propose that this group is part
of the proton channel at the hydroquinone oxidation center of the bc
complex.
Inhibitors have been indispensable tools for elucidating the
reactions of the ubiquitous bc complexes (for a
review, see (1) ). Almost all of these inhibitors are aromatic
organic compounds where at least part of the structure has some
structural relationship to the substrate, ubiquinone. The single
notable exception are zinc ions which have first been reported by
Skulachev et al.(2) to inhibit mitochondrial
respiration in micromolar concentrations. Subsequent studies (3, 4, 5) have established that the primary
site of zinc inhibition in bovine heart mitochondria is the bc
complex.
The knowledge about the bc complex has largely increased over the past 20
years, including the structure of the redox centers and the peptide
composition of the mammalian 11-subunit complex. The
``Q-cycle'' mechanism has been established for the sequence
of electron and proton transfers (see (6) ). In the light of
this body of information, we have reinvestigated inhibition by zinc to
obtain information about the specific interaction between zinc ions and
the bc
complex and to use this information to get
insight into mechanistic details of the electron and proton transfer
reactions.
Metal salts were obtained either as chloride or as nitrate salts from Fluka. Cytochrome c (horse heart) was from Sigma, prepared without trichloroacetic acid.
Bovine heart mitochondria
were prepared according to Smith(7) . bc complex was prepared as described by Schägger et al.(8) with the following modification: the buffer
for the final Sepharose CL-6B gel filtration column was 0.05% Triton
X-100, 100 mM NaNO
, 20 mM Pipes, (
)pH = 7.2 (no NaN
). Fe-S-deficient bc
complex + antimycin was prepared following
the procedure given by Schägger et
al.(9) . After complete elution of the iron-sulfur
protein, Fe-S-deficient bc
complex +
antimycin was eluted with 0.05% Triton X-100, 150 mM sodium
phosphate, pH 7.2, and applied to a gel filtration column (Sepharose
CL-6B, Pharmacia) equilibrated with 0.05% Triton X-100, 100 mM NaNO
, 20 mM Pipes, pH 7.2, and run with the
same buffer. The fractions containing Fe-S-deficient bc
complex + antimycin were mixed with 10% (w/v) glycerol and
frozen in liquid nitrogen.
Buffer for kinetic measurements was
prepared as follows: 200 mM sucrose, 50 mM Pipes
(from a partially neutralized stock solution), and 1 mM NaN were dissolved in tridistilled water. The solution
was purified over a Chelex 100 ion exchange resin column (analytical
grade, Bio-Rad). 20 mM HNO
, 1 mM
Ca(NO
)
, and 0.024% (0.5 mM) dodecyl
maltoside (Boehringer) were added, and the pH was adjusted with 2 M NaOH (purified over Chelex 100 and stored in a plastic bottle).
Calcium was added to prevent unspecific binding since it did not show
any inhibition even at 10 mM concentration. For the
measurement of the pH dependence, the buffer contained 25 mM Pipes and 25 mM Epps. All buffers were stored in plastic
bottles.
NBH:cytochrome c reductase activity was measured
at ambient temperature in 1-cm cuvettes, stored in EDTA solution,
washed extensively with NaOH, and finally rinsed with water. 10 µl
of cytochrome c stock solution (5 mM in 50 mM Pipes, pH 7.0) were diluted into 1 ml of buffer. 5 µl of bc complex (diluted to 1 µM cytochrome b in buffer containing 30% glycerol) and metal
ion solution were added. Reductase reaction was started by addition of
NBH in dimethyl sulfoxide (final concentration 45 µM).
Michaelis-Menten kinetics was performed as above in stirred 2-ml
cuvettes thermostatted at T = 24 °C. For cytochrome
reduction, NBH was added to bc complex (2
µM cytochrome b in kinetics buffer) in a stirred
2-ml cuvette to a final concentration of 27 µM. Reduction
kinetics was measured at pH 6.2 where electron transfer from
hydroquinone to bc
complex is slower than at pH 7.
The radioactive binding assay was performed in 4.5-ml centrifuge
tubes, washed first with EDTA solution, extensively with NaOH, and
finally with water. A ZnCl
stock solution (50
µM, specific activity 195 kBq/ml, diluted from Amersham
ZAS.1 batch 63) was diluted into 2.5 ml of Chelex 100 purified buffer
containing 50 mM Pipes, 20 mM HNO
, and 1
mM Ca(NO
)
. bc
complex was added to a final concentration of 0.4 µM cytochrome b (approximately 70 µl). The bc
complex was sedimented by centrifugation (Rotor
Ti 70.1, 35,000 rpm, 3 h). 2 ml of the supernatant were withdrawn to
measure free
Zn. The remaining supernatant was discarded,
and the tubes were centrifuged again (20,000 rpm, 30 min). The
supernatant was removed and the pellet was incubated overnight in 50
µl of 10% Triton X-100, 1 M NaCl. The dissolved pellet was
diluted with 625 µl of buffer containing 0.5% Triton X-100, 200
mM NaCl, 20 mM Mops and finally with another 625
µl of water. Thus, the final volume was exactly one-half of the
original assay solution. 1 ml (water added to a final volume of 2 ml)
was used to measure bound
Zn in a Beckman Gamma 5500
counter.
bc concentration was determined from
reduced-oxidized difference spectra using
= 28.5 mM
cm
. EPR spectroscopy was done with a Bruker
200 D spectrometer equipped with cryogenics, peripheral equipment, and
data acquisition/manipulation facilities as described
previously(10) .
Fig. 1shows inhibition of NBH:cytochrome c oxidoreductase activity of isolated bc complex by Zn
ions. The K
could be obtained by fitting the Zn
dependence
with a single homogeneous inhibition site:
Figure 1:
Complete inhibition of isolated bc complex by zinc ions. Cytochrome c reduction by NBH was measured at different zinc concentrations.
The line was fitted using K
= 0.17
µM (pH 7.0). At [Zn
] > 5
µM, inhibition is essentially complete. The inset shows a Dixon plot (1/v versus [Zn
]).
At pH 7.0, a K of 1
10
was obtained. A single inhibition site is also observed by
plotting the linearized form
At [Zn] > 5 µM, no
steady state electron transfer activity was detectable.
Figure 2:
Binding of Zn
to bc
complex. bc
concentration, 0.2 µM; pH 7.2. The line was fitted
to the data points using two independent types of binding sites. High
affinity site: 1.2 Zn
/bc
, K
= 0.13 µM. Low
affinity site: 3.3 Zn
/bc
, K
= 2.3 µM. The inset shows a Scatchard plot of the data points; the curve shows the transformed line from the direct nonlinear fit, while
the line represents the high affinity binding site only (see
text).
The K of the high affinity
binding site was essentially identical with the inhibition constant K
under all conditions tested. Therefore, we
conclude that inhibition is caused by binding of a single zinc ion per
cytochrome c
with a dissociation constant of
approximately 10
M. The additional multiple
low affinity binding sites are not related to the inhibition of
catalytic activity.
Note that the high affinity binding constant cannot be obtained by using a linear fit of the Scatchard plot (see Fig. 2, inset). A line representing only high affinity binding does not match the data points even at low zinc concentrations since approximately 15% of the first zinc ions bind to low affinity sites.
Figure 3:
Inhibition of cytochrome reduction through
the Q center by zinc ions. Top, cytochrome b reduction monitored at 562-575 nm; bottom,
cytochrome c
reduction monitored at 550-540
nm. bc
complex was diluted to 2 µMb in a stirred cuvette, and reduction was started by the
addition of 27 µM NBH as indicated. Ant, 29
µM antimycin; Ant+Zn, 29 µM antimycin + 100 µM Zn
; Ant+MS, 29 µM antimycin + 18
µM MOA-stilbene (double kill, i.e. complete
inhibition of b reduction), pH
6.2.
Figure 4:
Michaelis-Menten kinetics of bc complex partially inhibited by zinc ions. A, cytochrome c kinetics ([NBH] = 42.5 µM); B, NBH kinetics ([cytochrome c] = 50
µM).
, [Zn
] = 0;
, [Zn
] = 0.075
µM;
, [Zn
] = 0.2
µM, pH 7.2. The lines were drawn using the V
and K
values
obtained from a nonlinear fit to the Michaelis-Menten
equation.
Binding of the Q center inhibitor,
stigmatellin, which interacts with both the iron-sulfur protein and
cytochrome b(14) , was determined by red shift
titration of reduced bc
complex(15) . The K
could be determined exactly due to the high
affinity; for bc
complex both in the absence and
presence of 100 µM Zn
, K
values below 20 nM were obtained. We conclude that zinc
does not affect the binding of other Q
center inhibitors.
Figure 5:
pH
dependence of inhibition of bc complex by zinc
ions. A:
, K
values
obtained from inhibition kinetics;
, K
values obtained from binding assays. The bold line was fitted to the data points using the competitive binding model
(see text) giving K
(high pH)
= 0.07 µM; pK
= 7.2. The dotted and the dashed lines represent calculated curves for the noncompetitive binding model
using K
= 0.07
µM; pK
= 7.2; K
/K
= 100 and 1000, respectively. B, same data as
before. The curves were calculated for fully competitive inhibition
using three different pK values of 6.6, 7.2, and 7.6 (from left to right).
The data have been analyzed to obtain the dissociation
constants for both Zn and protons; the dissociation
constant for protons, K
, is generally expressed as
pK
value (-log K
). Two
different models have been considered: competitive and noncompetitive
binding. Competitive binding occurs if zinc and protons displace each
other from the same binding site, while noncompetitive binding requires
different interacting binding sites so that proton binding to one site
will influence the K
for zinc at a site nearby.
For competitive binding, the apparent K`
for zinc
ions as a function of pH is obtained as
i.e. K` is independent of pH at pH
pK
and depends linearly on
[H
] for pH pK
. For
noncompetitive binding, the apparent K`
is
where K and K
are the zinc dissociation constants
at high and low pH, respectively (see Appendix). Negative cooperativity
becomes indistinguishable from direct competition at high ratios of K
/K
and the equation reduces to .
The equations for
both competitive and noncompetitive binding fit the data points if the
zinc dissociation constant at low pH (K) is at least 3 orders of magnitude
higher than that at high pH (K
), i.e. K
/K
1000 (Fig. 5A). From these fits, a K
(high pH) of 0.07 µM and a pK
of 7.2 were obtained. In Fig. 5A, we have also constructed the curve for
noncompetitive binding using K
/K
= 100 and the same values for K
and pK
in order to show that the curve for
noncompetitive binding does not match the data points if the ratio K
/K
is 100 (or even less).
Since it was not possible to measure
either zinc binding or zinc inhibition at pH < 5.5, we cannot
distinguish between competitive and noncompetitive binding models, if K/K
1000. However, in both cases, there is strong negative
cooperativity between zinc and proton binding; on protonation, the zinc
dissociation constant increases by more than 2 orders of magnitude and
upon zinc binding, the pK
of the proton binding
site is lowered by more than 2 pH units from pK
= 7.2 to below 5.
The activity of the bc complex follows a different pH dependence (Fig. 6).
Activity increases with pH up to a pH optimum of 8.5 and decreases
again. The increase of the activity can be modelled with a single
pK
value of 6.6 where the protonated form is
assumed to be completely inactive. The pH dependence of zinc binding
cannot be modelled using the pK
of 6.6 (Fig. 5B).
Figure 6:
pH dependence of electron transfer
activity of bc complex. The line was fitted giving
pK
= 6.6. Above pH 8.5, activity
decreases.
The same dissociation constant K for zinc was obtained in all three experiments:
without cytochrome c added, with oxidized and with reduced
cytochrome c added (data not shown). This shows that zinc
binding is redox independent. Therefore, the group where protons bind
with negative cooperativity to zinc must have a redox independent
pK
value of 7.2.
Since Fe-S-depleted bc complex is prepared in the presence of antimycin(9) , we
first tested whether antimycin affects zinc binding. The K
observed with and without saturating
concentrations of antimycin was identical (Fig. 7). The K
observed for the Fe-S-depleted bc
complex was 3 times lower than for whole bc
complex, i.e. binding is three 3 times tighter (Fig. 7). The K
showed the same pH
dependence as whole bc
complex (data not shown).
We conclude that zinc does not bind to the iron-sulfur protein but to
residues in the vicinity of the iron-sulfur protein.
Figure 7:
Effect of the iron-sulfur protein on
binding of zinc ions to bc complex (Scatchard
plot).
- - -
, bc
complex (K
= 0.11
µM);
, bc
complex + antimycin (K
= 0.14 µM);
-
,
Fe-S-deficient bc
complex + antimycin (K
= 0.03 µM), pH
7.0. The curves show the transformed lines from the direct
nonlinear fit, while the lines represent the high affinity
binding sites only (see text).
Binding of zinc is completely reversible. Studies using o-phenanthroline showed that reactivation requires 3 µmol
of o-phenanthroline/µmol of Zn added.
Therefore, reactivation involves removal of zinc from the bc
complex and complexation as
Zn(phen)
. Binding of Hg
and Cu
was not fully reversible, probably due
to the oxidation of disulfide groups by both ions.
In isolated
bovine mitochondria, the zinc content has been determined as
76-104 µg of Zn/g of protein =
1.2-1.6 µmol of Zn
/g of
protein(20) . Using the bc
concentration
in bovinemitochondria isolated according to Smith(7) , 0.25
µmol of bc
/g of protein, the zinc content is
obtained as 5-6 Zn
/bc
. At
this ratio, approximately 25% inhibition of the activity of the bc
complex was observed. We therefore conclude
that zinc inhibition may occur at physiological zinc concentrations.
If zinc ions were
added together with organic Q center inhibitors, no
additional effect was observed while zinc plus antimycin lead to a
reduction of the rate of cytochrome c
and b reduction through the Q
center. This clearly shows
that zinc inhibits at the Q
center. However, it is less
effective than organic Q
center inhibitors since it does
not block single turnover through the Q
center completely
even if added at high concentration (1000 times K
). A residual activity of less than 1
s
is sufficient for the reduction kinetics shown in Fig. 3but is not detected under standard activity assay
conditions.
While this work was
in progress, Lorusso et al.(16) reported reversible
inhibition of the Q center, binding of zinc to the
iron-sulfur protein, 42% decrease of the Rieske EPR signal upon zinc
binding, and 50% inhibition of electron flow at saturating zinc
concentrations. However, there are some problems with the experimental
conditions used in the work of Lorusso et al.(16) that are likely to cause artifacts.
Lorusso et
al.(16) have measured cytochrome reductase activity and
its inhibition in buffer containing 1 mM KCN. Cyanide is a
strong complexing agent and forms the complex
Zn(CN) with a cumulative binding
constant
= 5
10
M
. Thus, at a cyanide concentration
of 1 mM, a total zinc concentration in the micromolar range
would lead to a free zinc concentration below the nanomolar level. The
zinc effect observed in (16) can therefore be explained only
if a major fraction of cyanide is complexed with other metal ions. It
is likely that the half-inhibition observed by Lorusso et al.(16) is due to a redistribution of metal ions between
various binding sites, including cyanide and bc
complex. We have used azide which has very low metal complex
formation constants.
The uninhibited reductase activity given in (16) corresponds to a turnover number of 5-8
s for durohydroquinone as substrate using the
specific bc
content reported in (21) . 50%
inhibited activity corresponds to a turnover number of 2-4
s
. We have obtained turnover numbers in excess of
350 s
using nonylubihydroquinone as substrate.
Zinc does not bind to the iron-sulfur protein since Fe-S-depleted bc complex, which can be reconstituted with
iron-sulfur protein and does still bind MOA inhibitors to the Q
center, binds zinc with 3 times higher affinity than bc
complex. In addition, we have not observed any
effect of saturating zinc concentrations on the EPR of the iron-sulfur
cluster.
bc complex will bind protons and zinc with
approximately equal affinity (K
(Zn
)/K
(H
)
1). Due to the strong negative cooperativity, zinc and protons
displace each other from their binding site(s). On zinc binding, the
pK
of the proton binding site drops from 7.2 to
below 5. However, this proton binding site is not related to the group
determining the pH dependence of the activity of the bc
complex, which depends on deprotonation of a group with
pK
= 6.6 (Fig. 6)(21, 22) . The pK
of the zinc binding site is also not identical with the redox
dependent pK
of the oxidized Rieske iron-sulfur
protein (pK
= 7.7) (23, 24, 25, 26) . Therefore, we
conclude that we have identified another functionally important
protonatable group of the bc
complex.
Hydroquinone
oxidation requires the release of two protons from the Q center to the aqueous environment. The quinone reaction pocket
itself must be shielded from the environment in order to prevent side
reactions of the reaction intermediates with water or oxygen.
Therefore, proton release will require a proton-conducting pathway to
the aqueous phase (see (6) ). The pK
= 7.2 of the newly identified protonatable group is
optimally suited for proton conduction at neutral pH. Since binding of
zinc shifts the pK
value of the proton binding
site from 7.2 to below 5, channelling of protons is inhibited by
binding of zinc. Electron transfer is then inhibited by preventing
release of the protons liberated in the redox reaction.
The four dissociation constants are:
The total enzyme concentration, E, is given
by
The total fraction of enzyme with bound zinc,
([EZn] +
[EHZn])/E, is given by
By comparing the binding equation without protons,
we obtain the binding equation for ([EZn]
+ [EHZn])/Ewith protons by replacing K
with an apparent K`
= f(pH)
For K
, binding
of zinc becomes competitive with proton binding. In this case, reduces to
The same result is obtained if the K` for
competitive binding is calculated directly.