Amoeba mitochondria possess a respiratory chain
with two quinol-oxidizing pathways: the cytochrome pathway and the
cyanide-resistant alternative oxidase pathway. The ADP/O method, based
on the non-phosphorylating property of alternative oxidase, was used to
determine contributions of both pathways in overall state 3 respiration
in the presence of GMP (an activator of the alternative oxidase in
amoeba) and succinate as oxidizable substrate. This method involves
pair measurements of ADP/O ratios plus and minus benzohydroxamate (an
inhibitor of the alternative oxidase). The requirements of the method
are listed and verified. When overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a
non-penetrating inhibitor of succinate uptake), the quinone reduction
level declined. At the same time, the alternative pathway contribution
decreased sharply and became negligible when quinone redox state was
lower than 50%, whereas the cytochrome pathway contribution first
increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This
study is the first attempt to examine the steady-state kinetics of the
two quinol-oxidizing pathways when both are active and to describe
electron partitioning between them when the steady-state rate of the
quinone-reducing pathway is varied.
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INTRODUCTION |
The mitochondrial respiratory chain of the amoeba
Acanthamoeba castellanii possesses, like that of plant
mitochondria (1), the following: (i) a cyanide- and antimycin-resistant
alternative oxidase (AOX)1 in
addition to the conventional cytochrome c oxidase (2, 3); (ii) the rotenone-insensitive external NADH dehydrogenase, located on
the outer surface of the inner mitochondrial membrane (4, 5); and (iii)
two internal NADH dehydrogenases, the rotenone-sensitive complex I and
the non-electrogenic rotenone-insensitive dehydrogenase (6).
As in mitochondria from most higher plants, many fungi, and
protozoa (7), the alternative pathway of amoeba mitochondria branches
from the main respiratory chain at the level of quinone (Q), and
electron flux through AOX is not coupled to the generation of
protonmotive force and ADP phosphorylation (2). The cyanide-resistant respiration of amoeba mitochondria is strongly stimulated by purine nucleoside 5'-monophosphates: AMP, GMP (with the highest efficiency), and IMP (2, 8, 9). A similar effect of purine mononucleotides on the
activity of the alternative pathway was observed in other microorganisms: Euglena gracilis (10), Moniliella
tomentosa (11), Neurospora crassa (12),
Paramecium tetraurelia (13), and Hansenula
anomala (14). In contrast, the alternative oxidase of higher plant
mitochondria is stimulated by
-keto acids, like pyruvate (7, 15,
16), but not by purine mononucleotides, whereas the
-keto acids do
not stimulate the alternative respiration in amoeba mitochondria (17).
These properties emphasize an important difference at the level of
allosteric regulation between both types of AOX. However, monoclonal
antibodies developed against Sauromatum guttatum cross-react
with the AOX protein of amoeba mitochondria (18), as they do in a wide
range of thermogenic and non-thermogenic plant species, some fungi, and
trypanosomes (7, 16, 19), indicating that this protein is well
conserved throughout the species.
Regulation of electron distribution between the energy-conserving
cytochrome pathway and the redox potential-dissipating alternative pathway is obviously of the utmost importance for the energy economy of
the cell. Several levels of control of AOX activity are known in plant
mitochondria (for reviews, see Refs. 7, 16, 19, and 20): (a)
the amount of the oxidase protein present in the mitochondria (gene
expression); (b) the redox status of the protein (post-translational modification); (c) the presence of
allosteric regulators (allostery); and (d) Q concentration
in the inner mitochondrial membrane and the redox state of quinone
(Qred/Qtot) (substrate-product availability).
With the exception of the regulation by change in redox state of the
protein, all these levels have been found for amoeba AOX (18, 21).
Precise understanding of the interplay and quantitative analysis of
these various levels of regulation can be reached only by measurements
of the actual activities of the two branching oxidases during
steady-state respiration. Such a study depends on the ability to
measure the respective contributions of both pathways in total
respiration. These determinations are hampered by the fact that both
oxidative pathways have the same substrate and product and that the use
of specific inhibitors must be considered as unsuitable because an
inhibition of one of the pathways inevitably affects electron flux
via the other pathway (22, 23).
A kinetic approach has been developed (24) taking into account the
interplay between quinol-oxidizing and quinone-reducing steady-state
electron fluxes and the redox poise of the quinone pool. This method is
based on the homogeneous quinone pool hypothesis and on a strict
specificity of the inhibitors of each oxidative pathway that are used
to measure the activity of one pathway when the other is inhibited. It
allows the prediction of the real contribution of each pathway at a
given total steady-state respiratory rate. Another method, considered
to be the only method that exists at present for making quantitative
measurements of AOX activity (23), has been developed for measuring
relative contributions even in vivo. This method is based on
the differential oxygen isotope discrimination between the cytochrome
and alternative oxidase pathways (25, 26). Calibration used in this
method requires the use of inhibitor for each respiratory pathway and
assumes that change in the total respiration rate does not change the discrimination factor.
Because the alternative pathway does not contribute to ATP synthesis
with succinate (in the presence of rotenone) as oxidizable substrate,
comparison of the ADP/O ratio with and without an inhibitor of AOX
(e.g. hydroxamic acids) allows the estimation of the
contribution of this pathway in total mitochondrial respiration. A
method based on ADP/O ratio determination proposed a long time ago (27,
28) has not been widely used, although it is one of the best as it avoids the use of the rates of electron transport of both pathways in
the presence of inhibitors (cyanide and hydroxamic acids), and it does
not require a strict specificity of the inhibitors of both pathways.
Moreover, the ADP/O ratio method is not linked to the homogeneous
quinone pool hypothesis. This method is claimed to have a limited range
of application (29). Of course, it cannot be applied under state 4 conditions and could hardly be used with intact tissues. However, this
method could be applied in permeabilized cells and in isolated
mitochondria in state 3 respiration, even if an AOX inhibitor does not
decrease total state 3 respiration. In this study, we have successfully
developed the ADP/O method to determine the actual contributions of
both the cytochrome and alternative pathways in isolated amoeba
mitochondria and to describe how these contributions change when the
steady-state rate of the quinone-reducing pathway is varied.
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MATERIALS AND METHODS |
Cell Culture and Mitochondrial Isolation--
The soil amoeba
A. castellanii (strain Neff) was cultured as described by
Jarmuszkiewicz et al. (18). Trophozoites of amoeba were
collected 22-24 h following inoculation at the middle exponential phase (at a density of ~2-4 × 106 cells/ml).
Mitochondria were isolated and purified on a self-generating Percoll
gradient (31%) as described before (18).
Assay Procedures--
Oxygen uptake was measured
polarographically using a Rank Brothers oxygen electrode in 3 ml of
standard incubation medium (25 °C) containing 120 mM
KCl, 20 mM Tris-HCl (pH 7.4), 3 mM
KH2PO4, 8 mM MgCl2, and
0.2% (w/v) bovine serum albumin with 1.5-1.8 mg of mitochondrial
protein. The membrane potential of mitochondria (
) was measured
simultaneously with oxygen uptake using a
tetraphenylphosphonium-specific electrode according to Kamo et
al. (30). For calculation of 
, the matrix volume of amoeba
mitochondria was assumed as 2.0 µl × mg
1 protein.
Measurements of 
, performed in the presence of 8 µM tetraphenylphosphonium, allowed the fine control of the duration of
state 3 respiration. The ADP/O ratio was determined by the ADP pulse
method, as illustrated in Fig. 1. The oxidizable substrate was
succinate (10 mM) in the presence of rotenone (15 µM) to block electron input from complex I. Increasing
concentrations of n-butyl malonate from 2 to 20 mM were added to the incubation medium to decrease
steady-state 3 respiration. A rather large amount of added ADP
(480-520 nmol) was chosen to ensure an accurate determination of the
steady-state O2 consumption in state 3. The total amount of
oxygen consumed during state 3 respiration was used to calculate the
ADP/O ratio (see Fig. 1). A prepulse of ADP (200 nmol) was always
applied before the main pulse to ensure that a true state 4 had been
achieved and to activate succinate dehydrogenase by the produced ATP.
To fully activate the alternative pathway, GMP, the allosteric effector
of AOX in amoeba mitochondria, was added to the incubation medium at a
concentration of 0.6 mM (K0.5 = 20 µM) (9). Measurements of the ADP/O ratio were performed in the absence or presence of 1.5 mM benzohydroxamate
(BHAM), a concentration sufficient to totally inhibit the
KCN-insensitive respiration in amoeba mitochondria.
The redox state of quinone in steady-state respiration was determined
by an extraction technique according to Van den Bergen et
al. (24). For calibration of the peaks, commercial Q-9 (Sigma) was
used. Protein was estimated by the biuret method (31) with bovine serum
albumin (fraction V) as a standard.
Chemicals--
Tetraphenylphosphonium was obtained from Fluka,
and n-butyl malonate was from Aldrich. All other chemicals
were purchased from Sigma.
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RESULTS |
The method proposed in this paper is based on the ADP/O ratio
determination for complexes III and IV in the presence and absence of
BHAM, a specific inhibitor of the alternative oxidase, taking into
account that the alternative pathway, when supplied with electrons by
succinate as oxidizable substrate (+rotenone), is not
energy-conserving. The method consists in measuring the ADP/O ratios
and the rate of state 3 respiration during ADP pulses (Fig. 1). To describe how the contribution of
each pathway changes with variations in the overall state 3 respiration
(without BHAM), n-butyl malonate, a non-penetrating
competitive inhibitor of succinate uptake, is used to decrease the rate
of the quinone-reducing pathway. If v3 = vcyt + valt, where
v3 is the rate of state 3 respiration, vcyt is the contribution of the cytochrome
pathway in electron flux, and valt is the
contribution of the alternative pathway in electron flux, and if
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(Eq. 1)
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where ADP/O is the ADP/O ratio in the absence of BHAM (when both
respiratory pathways are active), (ADP/O)cyt is the ADP/O ratio in the presence of BHAM (when only the cytochrome pathway is
active), and Ocyt and Oalt are the amounts of
oxygen taken up related to the activities of the cytochrome and
alternative pathways, respectively, then Equations 2 and 3 follow.
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(Eq. 2)
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(Eq. 3)
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Fig. 1.
ADP/O ratio determination. Assay
conditions were as described under "Materials and Methods."
Mitochondria (mito) were incubated in the presence of 10 mM succinate (succ), 200 nmol of ADP (prepulse),
15 µM rotenone (rot), 0.6 mM GMP,
and 1.5 mM BHAM. The ADP pulse amounted to 520 nmol.
Afterward, respiration was uncoupled by 1 µM carbonyl
cyanide p-trifluoromethoxyphenylhydrazone.
Numbers on the trace refer to O2 consumption
rates in nmol of oxygen × min 1 × mg 1
protein. Membrane potential changes are presented in mV. RC,
respiratory control.
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Requirements--
The proposed ADP/O method is valid only if (i)
the ADP/O ratio in the presence of cyanide is equal to zero; (ii) BHAM
does not induce a proton leak (has no uncoupling effect) and thereby does not affect 
; (iii) the ADP/O ratio in the presence of BHAM is independent of the state 3 respiratory rates (within the applied range); and (iv) isolated mitochondria are well coupled and stable during the experimental procedure.
It is well known that the electron transport from ubiquinol to
O2 via the alternative pathway does not lead to the
synthesis of ATP (for review, see Ref. 32). The non-electrogenic
character of the alternative pathway, when supplied with electrons from complex II, was confirmed under conditions applied in this study (i.e. in the presence of the allosteric activator of the
pathway (GMP) and rotenone). After the addition of KCN that excluded
the activity of the cytochrome pathway (entire collapse of 
), no effect of either ADP or carbonyl cyanide
p-trifluoromethoxyphenylhydrazone on the respiratory rate is
observed (data not shown). Acceleration of the respiration caused by
GMP, entirely sensitive to BHAM, is due exclusively to the stimulation
of the electron flux through the alternative pathway. Thus, the ADP/O
ratio in the presence of cyanide is certainly equal to zero.
Among the tested inhibitors of the alternative oxidase, only BHAM meets
the second requirement of the method and, at the maximal concentration
used (1.5 mM), does not decrease 
generated by the
cytochrome pathway during oxidation of succinate and also decreases the
O2 uptake in both state 4 and state 3 respiration (data not
shown). In amoeba mitochondria, the other inhibitors (1.5 mM salicylhydroxamate and 100 µM
n-propyl gallate) display a slight uncoupling effect on

, decreasing it by ~2-5
mV.2 Therefore, for further
investigations, BHAM was chosen as the most suitable inhibitor of AOX.
Moreover, there is no residual respiration in amoeba mitochondria in
the presence of 1.5 mM BHAM and 1 mM KCN.
The constancy of the (ADP/O)cyt ratio (third requirement),
when the operation of the alternative pathway was excluded by BHAM, was
checked for state 3 respiration, ranging from 65 to 230 nmol of
oxygen × min
1 × mg
1 protein (Fig.
2A). This range comprises data
from 15 mitochondrial preparations with various rates of state 3 respiration gradually decreased by increasing concentrations of
n-butyl malonate. The (ADP/O)cyt ratios obtained
(1.41 ± 0.06 (S.D., n = 22) in the absence of GMP
and 1.41 ± 0.07 (S.D., n = 76) in the presence of
GMP) display the constancy required by the method, as they fluctuate
randomly without any correlation with either v3
or the respiratory control ratio (Fig. 2, A and
B). This result is of the utmost importance regarding the
validity of the calculation of
proposed above (Equation 1) and
suggests that native proton leak has a negligible influence on the
ADP/O ratios within the investigated range of
v3. Moreover, membrane potential measured during
state 3 respiration triggered by ADP pulse in the presence of BHAM
reveals a constant level of 
for the whole range of state 3 respiration (155.5 ± 5.5 mV, S.D., n = 56)
(Fig. 2C). This constancy is also important in the
interpretation of proton leak contribution in state 3 respiration (see "Discussion").

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Fig. 2.
(ADP/O)cyt ratio
versus state 3 respiration (A) and
versus respiratory control ratio (B) and
membrane potential versus state 3 respiration
(C). Assay conditions were as described for Fig. 1.
Oxidation of succinate in the presence of BHAM was gradually decreased
by increasing concentrations of n-butyl malonate (0.5-18
mM). A and B, plus and minus GMP;
C, plus GMP. Data deal with 15 experiments with different
rates of uninhibited state 3 respiration. The mean value of
(ADP/O)cyt in the presence of BHAM (±GMP) is 1.41 ± 0.07 (S.D., n = 98). The mean value of  is
156 ± 6 mV (S.D., n = 56).
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The ATPase activity has also been proven not to interfere with state 4 respiration in the presence of BHAM, as the addition of oligomycin does
not affect the respiratory rates (as an example, 68 ± 4 and
70 ± 2 nmol of oxygen × min
1 × mg
1 protein without and with oligomycin, respectively).
On the other hand, the addition of oligomycin in state 3 decreases the
respiratory rate exactly to that observed in state 4 (from 144 ± 6 to 68 ± 3 nmol of oxygen × min
1 × mg
1 protein). Finally, mitochondria isolated from amoeba
are characterized by a considerable stability. They exhibit a good
coupling of the oxidative phosphorylation, which is not significantly
affected within the duration of an experiment (up to 10 h). Thus,
all considered conditions required to determine the respective
contributions of both respiratory pathways in total respiration were
positively verified for amoeba mitochondria.
Contribution of Both Respiratory Pathways in Total
Respiration--
Table I shows an
example of the ADP/O ratios obtained from a single mitochondrial
preparation in the presence and absence of BHAM, used for calculation
of the contributions of both pathways in total respiration. The rates
of state 3 respiration with succinate (in the presence of rotenone and
GMP and in the absence of BHAM) are decreased with increasing
concentrations of n-butyl malonate from the control value of
179-173 to 103 nmol of oxygen × min
1 × mg
1 mitochondrial protein. At the same time, the ADP/O
ratios increase gradually from 0.93-0.94 to 1.43, approaching the
value observed in the presence of BHAM ((ADP/O)cyt). It
must be noted that the (ADP/O)cyt ratio is almost constant
whatever v3 is (mean ± S.D. for this
experiment = 1.44 ± 0.04, n = 7). These
results indicate that the changes in the ADP/O ratios determined in the
absence of BHAM are due to the change in the activity of the
alternative pathway, which gradually decreases together with the
lowering of electron transfer from complex II to quinone.
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Table I
Evolution of the ADP/O ratio (±BHAM) when state 3 respiration is
decreased by n-butyl malonate
Assay conditions were as described for Fig. 1. Oxidation of succinate
(in the presence of rotenone and GMP) was decreased by increasing
concentrations of n-butyl malonate (0.6-16 mM).
ADP/O ratios were determined in the presence and absence of 1.5 mM BHAM for the given concentrations of n-butyl
malonate. Values of O2 uptake in state 3 respiration are in
nmol of oxygen × min 1 × mg 1 protein.
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From the pair measurements of ADP/O ratios in the presence and absence
of BHAM, at various state 3 respiration rates, the contributions of
both pathways have been calculated (according to Equations 1-3) for
series of mitochondrial preparations, which, in the absence of
n-butyl malonate, present two different rates of state 3 respiration: 180 and 212 nmol of oxygen × min
1 × mg
1 mitochondrial protein (Fig.
3A). When the calculated
vcyt and valt
contributions are plotted as a function of v3,
two groups of results are observed (groups A and B in Fig.
3A). Each group exhibits a similar behavior. Inhibition of
v3 by increasing concentrations of
n-butyl malonate is accompanied by a sharp decrease in the valt contribution, whereas
vcyt first slightly increases, passes through a
maximum, and then clearly decreases when valt
becomes very small. When results of groups A and B are divided by the respective values of v3 in the absence of
n-butyl malonate (contributions and
v3 are then expressed in percent), both groups
belong to the same vcyt and
valt curves (Fig. 3B) that exhibit
the same profiles as curves in Fig. 3A.

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Fig. 3.
Contributions of the cytochrome pathway
(vcyt) and of the alternative pathway
(valt) in state 3 respiration (A)
and normalized contributions of the cytochrome and alternative pathways
versus normalized state 3 respiration (B).
A, assay conditions were as described for Fig. 1.
Calculations of vcyt and
valt were performed according to Equations 1-3.
Oxidation of succinate (in the presence of rotenone and GMP) was
decreased by increasing concentrations of n-butyl malonate
(0.5-18 mM). Contributions (vcyt
( and ) and valt ( and )) and state
3 respiration are expressed in nmol of oxygen × min 1 × mg 1 protein. Points of groups A
( and ) and B ( and ) were obtained from two sets of
experiments exhibiting similar respiratory rates in the absence of
n-butyl malonate (180 and 212 nmol of oxygen × min 1 × mg 1 protein, respectively).
B, normalized contributions of both pathways are expressed
in percent and were obtained from the data in A as described
under "Results." Triangles correspond to group A, and
circles correspond to group B in A.
BM, n-butyl malonate.
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The three experiments belonging to group B (Fig. 3A) include
also determination of Q reduction level. It was previously found that
the endogenous quinone in amoeba mitochondria is Q-9 (21). As shown in
Fig. 4, reproducible results are obtained
in the three experiments when v3 is decreased by
n-butyl malonate from 212 to 100 nmol of oxygen × min
1 × mg
1 protein (see Fig.
3A). When the two contributions (vcyt
and valt) calculated for the decreasing rate of
state 3 respiration are presented as a function of
Qred/Qtot (Fig. 4), it can be observed that 1)
the Q reduction level decreases from 65 to 38%; 2)
valt decreases sharply and vanishes when
Qred/Qtot is lower than 53%; and 3)
vcyt first slightly increases, reaching a
maximum (~160 nmol of oxygen × min
1 × mg
1 protein) when the Qred/Qtot
value is ~58%, and then decreases. The quinone reduction level
obtained in the absence of substrate (mean value of 22%) and upon
anaerobiosis and complete inhibition of the quinol-oxidizing pathways
(mean value 83%) suggests the presence of the inactive
Q-H2 and Q pools as observed with some plant mitochondria
(24). In amoeba mitochondria, the inactive pools would constitute 22%
(Q-H2) and 17% (Q) of the total quinone pool size, and the
active pool could be ~60% of the total membrane quinone.

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Fig. 4.
Dependence of vcyt,
valt, and v3
(inset) on quinone reduction level. Assay conditions
were as described under "Materials and Methods." Calculations of
vcyt and valt were
performed according to Equations 1-3. The three symbols ( / ,
/ , and / ) correspond to the three experiments of group B in
Fig. 3A. The asterisks correspond to values of
Qred/Qtot obtained in the absence of substrate
(mean value = 22%) or upon anaerobiosis and in the presence of 1 mM KCN and 1.5 mM BHAM (mean value = 83%).
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DISCUSSION |
The (ADP/O)cyt Ratio and the ADP/O Method--
It is
obvious that determination of the cytochrome and alternative pathway
contributions using the ADP/O ratios requires an (ADP/O)cyt
value independent of vcyt (i.e.
electron flux through the cytochrome pathway) when the electron supply
by dehydrogenase is modified. Such independence was not a priori
expected because it has been discredited by several examples in the
literature. In yeast mitochondria where complex I is missing, the ADP/O
ratio increases when the respiratory rate decreases below 100 nmol of oxygen × min
1 × mg
1 protein at a
constant protonmotive force, indicating a change in the mechanistic
stoichiometry of one of the proton pumps (33). However, when in these
mitochondria the respiratory rates measured with various substrates of
internal dehydrogenases are higher than 100 nmol of oxygen × min
1 × mg
1 protein, a constant ADP/O ratio
of 1.5 is observed. Other results (34) suggest similar flux efficiency
dependence in potato tuber mitochondria (where the alternative oxidase
activity is missing). Nevertheless, when in these mitochondria the
succinate-dependent state 3 respiratory rate (in the
absence of rotenone) is decreased by malonate, an inhibition by 75%
does not change the ADP/O ratio (~1.4). Thus, it seems possible that
no flux efficiency dependence occurs for succinate-sustained
respiration through the cytochrome pathway when electron input is
sufficient.
The results reported here show that the (ADP/O)cyt value
determined for succinate oxidation (plus rotenone) by the ADP pulse method in the presence of BHAM is constant for respiratory rates between 65 and 230 nmol of oxygen × min
1 × mg
1 of mitochondrial protein (Fig. 2A).
Therefore, there is no flux efficiency dependence within this range of
respiratory rates. The vcyt values (Fig. 3) were
obtained within the respiratory rate range characterized by
(ADP/O)cyt constancy as required for the ADP/O method. Of
course, to calculate vcyt, one must assume that
the (ADP/O)cyt value that characterizes the efficiency of the energy-conserving system of the cytochrome pathway is not modified
by the AOX activity and is independent of the presence or absence of
BHAM. Such a hypothesis is unavoidable regardless of the method used
(including the one based on oxygen isotope discrimination (25, 26, 35)
in which discrimination values are used instead of ADP/O ratios to
estimate the partitioning of electron flow into both quinol-oxidizing
pathways).
The constancy of (ADP/O)cyt observed with isolated amoeba
mitochondria (Fig. 2, A and B) indicates that the
proton pumps' stoichiometries remain essentially constant within the
range of respiratory rates used and that the influence of the proton
leak is either negligible or constant. However, a constant influence of
the proton leak on the (ADP/O)cyt value would require that the proton leak remain in a constant ratio with
vcyt, and consequently, 
is expected to
vary in the same direction as vcyt. It has been observed that in state 3 respiration in the presence of BHAM, 
does not correlate significantly with this respiratory rate (Fig.
2C), but remains almost constant (156 ± 6 mV, S.D.,
n = 56). It can be concluded that the proton leak is
likely to be negligible in state 3 respiration. In relation with this
conclusion, it should be noted that the (ADP/O)cyt value
(1.41 ± 0.07, S.D., n = 8) is very close to the
theoretical value (1.46 ± 0.04, S.E.) calculated for zero proton
leak in rat liver mitochondria by Van Dam et al. (36) (see
also Ref. 37).
It has also been observed that the state 4 respiratory rates in the
presence of BHAM, sustained mainly by the proton leak (as they are
insensitive to oligomycin), are not small compared with the respiratory
rates in state 3 (respiratory control ratio ranges from 2.3 to 1.5;
Fig. 2B) and that 
in state 4 (174 ± 5 mV, S.D.,
n = 42) is only 18 mV higher than in state 3 respiration. If the proton leak is negligible in state 3 respiration,
these observations suggest the existence of an acute "non-ohmic"
apparent conductance of the inner mitochondrial membrane of amoeba.
Contributions of the Two Pathways--
This study is the first
direct approach that has examined the steady-state kinetics of the two
quinol-oxidizing pathways when both are active. Indeed, ADP/O ratio
determination in the presence and absence of BHAM has made possible the
description of the behavior of the steady-state contribution of both
quinol-oxidizing pathways under state 3 conditions when the rate of
succinate transport is inhibited by different concentrations of
n-butyl malonate. Furthermore, it has been verified that the
total respiratory rate and the quinone reduction level evolve in the
same direction (Fig. 4, inset). When the two contributions
are presented as a function of the total respiratory rate (Fig. 3) or
as a function of Qred/Qtot (Fig. 4), similar
curves are obtained. The mitochondrial preparations behave
reproducibly despite quantitative differences when contributions are
plotted versus v3 (Fig. 3A); these
differences disappear when normalized rates are considered (Fig.
3B).
In amoeba mitochondria, the AOX activity stimulated by GMP is
significant in the absence of n-butyl malonate (equal to
40% of the total respiratory rate), but decreases sharply and becomes negligible at 40% inhibition of the total respiratory rate (Fig. 3B). In contrast to the cytochrome pathway, the alternative
pathway is not engaged when the reduction level of the active pool of quinone is lower than 50% (Fig. 4). In plant mitochondria, when the
cytochrome pathway is blocked by a specific inhibitor, the respiratory
rate mediated by AOX exhibits, in most cases, a threshold-like relationship with the Q redox state (for review, see Ref. 32). A
typical example is that of soybean cotyledon mitochondria (38), for
which a mechanism with a two-step reduction of the alternative oxidase
by quinol has been proposed (39, 40). In conclusion, it appears that in
isolated amoeba mitochondria, the kinetic behavior of AOX (when both
pathways are active) is similar to that observed with plant
mitochondria when the cytochrome pathway is blocked.
The steady-state kinetics of the cytochrome pathway presents an
unexpected soft maximum (Figs. 3 and 4). The cytochrome pathway activity has been reported to be proportional to the steady-state reduction level of Q (for a discussion of such kinetics, see Ref. 41),
first in uncoupled submitochondrial particles from beef heart
mitochondria by Kröger and Klingenberg (42) and then also under
state 3 and state 4 conditions in plant mitochondria (for reviews, see
Refs. 20 and 32). The peculiar behavior of the cytochrome pathway in
the amoeba A. castellanii (i.e. the observed
maximum in vcyt) may be due to 1) a special
feature of the cytochrome pathway activity when
Qred/Qtot is varied together with a possible
modification of the cytochrome c redox state imposed by the
steady-state conditions within the cytochrome pathway or 2) variation
in the concentration of a putative inner effector of the cytochrome
pathway antagonizing the substrate effect of Qred/Qtot variations.
The first proposal might be met in the following way. The protonmotive
Q cycle (43) is generally accepted as the mechanism by which complex
III (cytochrome bc1 complex) links the redox reaction between Q-H2 and oxidized cytochrome c
to the proton translocation from the matrix to the cytosol. In this
mechanism, the catalytic cycle involves oxidation of two
Q-H2 molecules at an enzymatic site localized on the
cytosolic side of the complex (center o) and the reduction
of one Q molecule on the matrix side (center i). It can be
supposed that for both reactions, Q and Q-H2 belong to the
active Q pool of the mitochondrial membrane (44). Then, the
steady-state activity requires the presence of the oxidized form of
quinone in the bulk pool. A situation in which the steady-state
Qred/Qtot ratio should be exactly equal to 1 (fully reduced quinone) would lead to zero activity, and this implies
that the activity of complex III as a function of Qred/Qtot exhibits a maximum. However, if the
Q-binding site of center i is saturated even at a low Q
level (binding site with a very high affinity for Q), the maximum will
be situated at a Qred/Qtot value very close to
1, and it could be not detectable. Indeed, in such a case, the reaction
occurring at site i does not need to be explicitly taken
into account (e.g. Ref. 45), so a steady-state kinetic
analysis could be performed considering the global reaction
Qred + cyt cox + E
Qox + cyt cred + E (where cyt c is oxidized or reduced cytochrome c and
E is complex III), which follows a ping-pong mechanism (46,
47). It can be speculated that the kinetic constants of complex III in
amoeba mitochondria may be such that the mechanistic scheme does not
simplify in this way (i.e. that the quinone reduction at
site i is interfering), so that a maximum of activity can be
observed in the steady-state kinetics of the cytochrome pathway. If
this explanation is correct, the maximum in vcyt
must exist in whatever way the electrons are supplied to the Q pool.
Disappearance of the maximum with another reducing substrate would
favor the second proposal, which assumes the existence of a new factor
of regulation of the cytochrome pathway in amoeba mitochondria.
Therefore, further studies are needed to for a straightforward
interpretation of this peculiar behavior of the cytochrome pathway in
amoeba.
It can be concluded that the ADP/O method is valid for direct
determination of activities of the cytochrome and alternative pathways
in isolated mitochondria during steady-state phosphorylating respiration. Moreover, the approach described in this paper has led to
the first detailed examination of the steady-state kinetics of the two
quinol-oxidizing pathways when both are active and to the description
of electron partitioning between them when the steady-state rate of the
quinone-reducing pathway is decreased.