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INTRODUCTION |
In addition to a cytochrome c oxidase, plant
mitochondria contain a cyanide- and antimycin-resistant alternative
oxidase that catalyzes the reduction of molecular oxygen to water upon
oxidation of ubiquinol (1-5). Alternative oxidase activity is
regulated by the reduction level of the
Q-pool1 (6-9) that is a
result of the kinetic interplay between the quinone-reducing and
quinol-oxidizing enzymes (9). Additional regulatory factors include the
mitochondrial concentration of
-keto acids (10) and the redox status
of the sulfhydryl/disulfide system (11). The isolation of cDNA
clones from several plant and fungal species (for review see Ref. 2)
has considerably improved our understanding of the molecular nature of
the oxidase. Scrutiny of cDNA-deduced amino acid sequences has
resulted in a model of the catalytic site of the protein that has been
postulated to contain a coupled binuclear iron center (12, 13). Recent work in our laboratory has resulted in the functional expression of the
alternative oxidase (from Sauromatum guttatum) in the yeast Schizosaccharomyces pombe (14), a system that has been used to study structure-function relations of the alternative oxidase by
site-directed mutagenesis (15).
The plant alternative oxidase is non-protonmotive (16, 17), suggesting
that any contribution of the enzyme to the overall respiratory activity
would affect the efficiency of mitochondrial energy metabolism in
plants. Although constitutive over-expression or silencing of the
alternative oxidase protein in tobacco and potato has been shown to
affect mitochondrial respiration (18, 19), it has not been shown
to alter the characteristics of plant growth. The functional expression
of the alternative oxidase in S. pombe (14) has raised the
question as to whether or not the mitochondrial respiratory kinetics
and growth of this yeast are affected by the constitutive presence of
the oxidase.
Relatively few studies have been performed to characterize the
mitochondrial respiratory kinetics of S. pombe (20, 21). However, the presence of at least two substrate dehydrogenases is
evident as both added NADH and succinate are readily oxidized in
isolated mitochondria (20, 21). The exhibited respiratory activity is
fully sensitive to antimycin and myxothiazol and is linearly dependent
upon the Q-redox poise (20), suggesting that the respiratory chain does
not branch at the level of the Q-pool.
Heterologous expression of the plant alternative oxidase establishes
cyanide-insensitive mitochondrial respiratory activity in S. pombe (14) and thereby introduces an additional oxygen-reducing pathway. Because of the presence of two oxidases and an external-NADH dehydrogenase, the mitochondrial respiratory chain in this yeast system
is comparable with those found in plants. In transformed S. pombe cells, functional expression of the alternative oxidase may
be fully repressed or induced by including or excluding thiamine in the
growth medium, respectively (14). This system therefore provides an
excellent model to study effects of the presence of the plant
alternative oxidase on growth and mitochondrial respiration.
In the present paper, growth physiology experiments suggest that the
functional expression of the alternative oxidase inhibits the growth of
S. pombe cells, both in terms of growth yield and rate.
Kinetic analysis of respiration strongly indicates that these effects
on growth are mitochondrial in origin since they reveal that the
non-protonmotive alternative oxidase actively competes for reducing
equivalents with the cytochrome pathway, contributing up to 24% to the
total mitochondrial respiratory activity. MCA reveals that the
alternative oxidase has a considerable degree of control on overall
electron flux (CJ = 0.22) at the expense of the
cytochrome pathway. The calculated control coefficients indicate that
the cytochrome route and the alternative oxidase exert comparable
control on the relative fluxes through the respective quinol-oxidizing
pathways. Both of these observations confirm the potential of the
alternative oxidase to divert reducing equivalents away from the
cytochrome pathway, thereby lowering the overall efficiency of energy conservation.
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EXPERIMENTAL PROCEDURES |
Strains and Growth Conditions--
The S. pombe
strain used was sp.011 (ade6-704, leu1-32,
ura4-D18, h
). Minimal media and
growth conditions for both growth and kinetic experiments were as
described by Murray et al. (22) with the additional
inclusion of 2.75 µM FeCl3 and lack of
Mn2+ and the presence or absence of 0.5 mM
thiamine. Growth experiments were performed in minimal medium
containing 2% glucose or 3% glycerol with 0.1% glucose.
Yeast Transformation--
The construction of plasmid pREP1/AOX
that contains the alternative oxidase coding sequence under control of
the nmt1 promoter has been previously reported by Albury
et al. (14). S. pombe cells were transformed
using a modified lithium acetate method (23).
Isolation of Mitochondria--
S. pombe cells were
aerobically grown overnight in 1-liter minimal medium cultures to a
density of approximately 5 × 107 cells
ml
1 (mid-logarithmic phase). Mitochondrial isolation was
based on a protocol previously described by Moore et al.
(20) but without incubation of cells with 0.5 M
-mercaptoethanol nor the subsequent KCl washes and purification of
mitochondria on Percoll gradients. Spheroplasts were prepared
essentially as described (20) but with modified concentrations: cells
(0.5-1.5 × 109 ml
1) were incubated at
30 °C for 15 min with 1 mg ml
1 Zymolyase and for a
further 45 min following the addition of 1 mg ml
1 lysing enzymes.
Measurement of Respiratory Kinetics--
Respiratory activity
and reduction level of the Q-pool were simultaneously measured
voltametrically in a specially constructed chamber (University of
Sussex) housing a Rank oxygen electrode and glassy carbon and platinum
electrodes connected to a Ag/AgCl reference electrode similar to that
described by Moore et al. (6). Mitochondria (0.5-1.0 mg)
were incubated in 2.2-ml reaction medium that contained 0.3 M mannitol, 1 mM MgCl2, 5 mM K2PO4 buffer, 10 mM
KCl, 20 mM MOPS (pH 7.2), and 1 µM Q-1. Other
chemicals specific to the experiment were added as described in the
legends of Figs. 2 and 3.
Growth Experiments--
Batch cultures (150-ml minimal medium
with or without 0.5 mM thiamine and as carbon source either
2% glucose or 3% glycerol with 0.1% glucose) were inoculated from
agar plates with transformed (pREP1/AOX) S. pombe cells and
grown aerobically in an incubator/shaker at 30 °C. Light microscopy
revealed that the morphology of cells with and without alternative
oxidase was comparable, and therefore light scattering was considered a
suitable way to determine cell numbers. At set times, aliquots (2 ml)
were taken and diluted, and the optical density was read at 595 nm
(A595). Determination of a standard curve
revealed that the scattering of light at 595 nm is linearly dependent
upon the counted number of S. pombe cells ml
1
up to A595 values of 0.5.
Assays and Reagents--
The concentration of mitochondrial
protein was estimated using the bicinchoninic acid method with bovine
serum albumin as a standard (24). Chemicals were, unless stated
otherwise, obtained from Sigma (Poole, Dorset, UK) or ICN Biomedicals,
Inc. (Aurora, OH).
Kinetic Modeling and Metabolic Control
Analysis--
Mitochondria isolated from S. pombe cells
expressing the alternative oxidase contain a respiratory network that
is comparable with those found in plant systems. A relatively easy to
use model to describe and predict respiratory kinetics within plant
mitochondria has been postulated by Van den Bergen and colleagues (9).
We have used this approach, in which both quinone-reducing and
quinol-oxidizing enzymes are assumed to exhibit reversible
Michaelis-Menten kinetics with respect to Q/QH2 (sum of
substrate and product is constant), to model the kinetic S. pombe data presented in this paper. Because this model accounts
for total electron flux from respiratory substrate to oxygen, it
effectively describes the kinetic behavior of the entire electron
transfer system and therefore provides a good starting point to study
mitochondrial electron transfer in terms of metabolic control analysis
(see Refs. 25 and 26).
The electron transfer system in S. pombe mitochondria was
studied using a "top-down" approach (26-29), being divided into
two quinone-reducing blocks (succinate dehydrogenase with dicarboxylate carrier and external-NADH dehydrogenase) and two quinol-oxidizing blocks (cytochrome pathway and alternative oxidase), all of which are
linked by the Q-pool. Hyperbolic fits (9) describing the experimentally
derived kinetics of the individual blocks were used to calculate steady
state reduction levels of the Q-pool and to derive elasticities of the
respective enzyme blocks with respect to QH2 (27, 28). Flux
and flux ratio control coefficients were calculated from the elasticity
coefficients using a matrix-based method according to Westerhoff and
Kell (30).
 |
RESULTS |
The Effect of Alternative Oxidase Expression on Growth
Physiology--
To investigate whether or not growth characteristics
of batch-cultured S. pombe were affected by the expression
of the non-protonmotive alternative oxidase, growth curves were
determined using cells transformed with pREP1/AOX grown on either a
fermentable or non-fermentable carbon source in the presence or absence
of thiamine (Fig. 1). The results
presented in Fig. 1A indicate that growth of S. pombe on glucose is not affected by the presence of the
alternative oxidase since the growth yield in the absence or the
presence of the oxidase is the same (~1.3 × 108
cells ml
1). However, a semi-logarithmic representation of
these data (Fig. 1B) reveals a small difference in growth
rate during log phase with cells expressing the oxidase exhibiting a
12% lower rate than control cells (0.167 instead of 0.190 h
1). The decrease in growth rate during (early)
exponential phase is more pronounced (18%) when cells are grown on
glycerol (from 0.131 to 0.108 h
1, Fig. 1D).
The growth curve presented in Fig. 1C clearly shows that
under the latter conditions, when the yeast has to rely predominantly on mitochondrial respiration to produce ATP, the expression of the
alternative oxidase has an additional effect on the growth yield that
is lowered by 20% (from 1.14 × 108 to 9.12 × 107 cells ml
1).

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Fig. 1.
Effect of plant alternative oxidase
expression on growth characteristics of S. pombe.
Cells transformed with pREP1/AOX were grown in the presence or absence
of 0.5 mM thiamine in minimal media with 2% glucose
(A and B) or 3% glycerol with 0.1% glucose
(C and D) as the sole carbon source. As described
under "Experimental Procedures," cell densities were calculated
from A595 values (4-5 for each data point) that
varied less than 3%. Typical experiments (repeated at least twice) are
presented showing growth curves of cells in which expression of the
alternative oxidase was repressed ( ) or induced ( ).
Dotted data points in the semi-logarithmic plots were fitted
using linear equations. Numbers refer to growth yield (C) or
maximum specific growth rate (B and D).
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To confirm that these effects on growth yield and rate could be
specifically attributed to the expression of the alternative oxidase
and were not because of a thiamine limitation, similar growth studies
were performed using cells transformed with pREP1 only (i.e.
a plasmid lacking the alternative oxidase gene). No differences could
be observed between cells grown in the absence or the presence of
thiamine (data not shown).
The Effect of Alternative Oxidase Expression on Mitochondrial
Respiratory Kinetics--
The results presented above indicate that
the growth physiology of S. pombe is negatively affected by
the expression of a single gene encoding the plant alternative oxidase
(Fig. 1). These results strongly suggest that the observed effects on
growth might be because of an effect on mitochondrial respiration. We
therefore investigated respiratory kinetics in mitochondria isolated
from cells in which the expression of the alternative oxidase was
either induced or repressed.
The dependence of electron flow through quinol-oxidizing pathways upon
the reduction level of the Q-pool was determined by titrating
respiratory activity with malonate in a variety of respiratory states
(6-9); the results of these experiments are summarized in Fig.
2. In cells not expressing the
alternative oxidase, electron flow through the cytochrome pathway under
state 4 conditions is approximately linearly dependent upon the Q-redox
poise (Fig. 2A). Addition of ADP induces a subtle change in
this kinetic behavior that is reflected by stimulated electron transfer
rates at high (>80%) Q-reduction levels only. Uncoupling with CCCP
results in a more significant increase in oxygen uptake rates than in
state 3, but again, this occurs exclusively at these high reduction levels of the Q-pool (Fig. 2A). Consequently, in S. pombe mitochondria the kinetic behavior of the cytochrome pathway
with respect to Q/QH2 under both state 3 and uncoupled
conditions is concave. These kinetics are different from those found in
any plant system studied to date (cf. Ref. 31).

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Fig. 2.
Effect of alternative oxidase expression on
kinetics of quinol-oxidizing enzymes in S. pombe
mitochondria. The dependence of electron flow through
quinol-oxidizing pathways upon the reduction level of the Q-pool was
determined in mitochondria lacking (A) or containing
(B) the alternative oxidase by titrating the oxidation of 9 mM succinate with 0-9 mM malonate in the
presence of 0.2 mM ATP and 9 mM glutamate.
Titrations to determine the total Q-oxidizing kinetics in these two
types of mitochondria i.e. cytochrome pathway alone
(A) and sum of cytochrome + alternative pathway
(B) were performed under state 4 ( ), state 3 ( , 1 mM ADP), and uncoupled conditions ( , 1 µM
CCCP). To reveal the kinetics of the alternative oxidase alone,
titrations were performed in the presence of 1.7 µM
antimycin A ( ). The malonate titrations were obtained from six
different mitochondrial preparations, and each set of kinetics is a
summation of data from two to four separate respiratory traces. The
data shown in the insets represent steady states unaffected
by malonate and are averages of 5-26 respiratory traces from ten
separate mitochondrial preparations. Steady states upon oxidation of 9 mM succinate were achieved with substrate alone (1) or in
the cumulative presence of 0.2 mM ATP (2), 9 mM
glutamate (3), 1 mM ADP (4), and 1 µM CCCP
(5). Steady states upon oxidation of 1.8 mM NADH were
reached with substrate only (6) or in the presence of 1 mM
ADP (7), 1 µM CCCP (8), or 1.7 µM antimycin
A (9). Curves were obtained by modeling the data mathematically as
described under "Experimental Procedures" and represent total
Q-oxidizing kinetics under state 4 (solid line through ),
state 3 (dotted line through ), and uncoupled conditions
(dot/dashed line through ) or the alternative oxidase
kinetics alone (solid line through ). Cytochrome pathway
kinetics in mitochondria lacking (solid lines) or containing
(dashed lines) the alternative oxidase are shown in
panel C. Solid lines are modeled data from
panel A; dashed lines were obtained by
subtracting the modeled alternative oxidase kinetics from the modeled
total Q-oxidizing kinetics shown in panel B.
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The data depicted in Fig. 2B represent experiments performed
using mitochondria possessing the alternative oxidase. Fig.
2B indicates that electron transfer through the alternative
oxidase commences when the Q-pool is approximately 20% reduced and
that this flow increases disproportionally upon an increasing Q-redox poise. Activity at such a low Q-reduction level suggests that the
relative affinity of the alternative oxidase for QH2 is
high compared with that found in plants (when the alternative oxidase has not been activated) and indicates that the oxidase has the potential to compete for electrons with the cytochrome pathway (10). A
direct comparison of cytochrome pathway kinetics (Fig. 2C)
reveals that electron flow through the cytochrome route under state 4 and state 3 conditions is diminished by expression of the alternative
oxidase, particularly at Q-reduction levels higher than 30%, whereas
electron transfer under uncoupled conditions is not affected
significantly at any Q-reduction level. This is illustrated by the
kinetics of the overall oxygen uptake which show that, under state 4 and state 3 conditions, oxygen uptake rates are higher at comparable
reduction levels in mitochondria lacking the alternative oxidase (Fig.
2A) than in mitochondria possessing the enzyme (Fig.
2B). Under uncoupled conditions, the kinetics are comparable
in both types.
The kinetics of quinol-oxidizing pathways should be independent of the
source of reducing equivalents i.e. the type of substrate. The data represented by the insets of Fig. 2, A
and B were obtained during succinate-oxidation in the
absence of malonate and also include steady states achieved upon
oxidation of added NADH under different energetic conditions (see
steady states 6-9). It is clear that the latter steady states can be
readily described by the appropriate curves fitted through data
obtained by malonate titrations. This indicates that the
quinol-oxidizing pathways are indeed kinetically independent of the
employed quinone-reducing enzyme(s), both in cells with and without
alternative oxidase. A second point to be addressed with respect to the
insets of Fig. 2 concerns succinate dehydrogenase which, as
is the case in many mammalian (32) and plant mitochondria (33),
requires activation. In S. pombe, activation is ensured by
including ATP and glutamate (to remove any bound oxaloacetate by
transamination) in the reaction mixture (20). From the
insets of Fig. 2, it can be seen that the steady states
reached in the absence of glutamate and/or ATP (see steady states 1 and
2) fit well on the curves describing the overall state 4 kinetics of
Q-oxidizing routes, confirming that it is indeed the activation of
succinate dehydrogenase that is responsible for increased respiratory
activity and not a stimulation of the cytochrome or alternative
pathway. Finally, the steady states achieved in the presence of
antimycin A (see steady state 9) confirm that the alternative oxidase
is either absent (Fig. 2A, inset) or present
(Fig. 2B, inset), respectively, in mitochondria isolated from cells grown in medium containing or lacking thiamine. Antimycin-insensitive respiratory activity (steady state 9 in Fig.
2B, inset) was fully inhibited by octyl-gallate
(data not shown), a potent specific inhibitor of the alternative
oxidase (34).
The dependence of electron transfer through quinone-reducing enzymes
upon the reduction level of the Q-pool was determined by titrating the
oxidation rates of succinate or NADH with antimycin A (9). The data
shown in Fig. 3 reveal that the
expression of the alternative oxidase does not have a significant
effect on the kinetic behavior, under state 4 conditions, of either
succinate dehydrogenase (Fig. 3A) or the external-NADH
dehydrogenase (Fig. 3B). The insets of Fig. 3
show respiratory steady states that are unaffected by antimycin A and
were achieved during succinate- or NADH-oxidation under state 4, state
3, and uncoupled conditions in the presence or the absence of the
alternative oxidase. An effect of the protonmotive force upon kinetics
of non-protonmotive quinone-reducing pathways would not be anticipated.
It is clear, however, that addition of ADP or CCCP to mitochondria
lacking the alternative oxidase results in steady states (see steady
states 3 and 5 in insets, respectively) that cannot be
described by the fits of state 4 kinetics of succinate (Fig.
3A, inset) and external-NADH dehydrogenase (Fig.
3B, inset). The reason for the lack of fit is, at
present, unclear.

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Fig. 3.
Effect of alternative oxidase expression on
kinetics of quinone-reducing enzymes in S. pombe
mitochondria. The dependence of electron flow through
succinate dehydrogenase (A) and the external NADH
dehydrogenase (B) upon the reduction level of the Q-pool was
determined in mitochondria lacking ( ) or containing ( ) the
alternative oxidase by titrating the oxidation of 9 mM
succinate in the presence of 0.2 mM ATP and 9 mM glutamate (A) or the oxidation of 1.8 mM NADH (B) with 0-30 nM antimycin
A under state 4 conditions. The antimycin A titrations were obtained
from three different mitochondrial preparations, and each set of
kinetics is a summation of data from two to three separate respiratory
traces. The data shown in the insets represent steady states
unaffected by antimycin A and are averages of 5-24 respiratory traces
from ten separate mitochondrial preparations. Steady states were
reached upon oxidation of 9 mM succinate + 0.2 mM ATP + 9 mM glutamate (inset A) or
1.8 mM NADH (inset B) under state 4 (1 and 2), state 3 (1 mM ADP;
3 and 4), or uncoupled conditions (1 µM CCCP; 5 and 6). Steady states 1, 3, and 5 were reached in mitochondria lacking the alternative oxidase
and 2, 4, and 6 in mitochondria containing the oxidase. Combined data
in each panel were modeled as described under
"Experimental Procedures."
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Kinetic Modeling and Metabolic Control Analysis--
To quantify
the effects of the expression of the alternative oxidase on the kinetic
behavior of the overall electron transfer system in S. pombe
mitochondria, experimental data representing the kinetics of both the
quinol-oxidizing (Fig. 2) and quinone-reducing (Fig. 3) enzymes were
modeled using hyperbolic expressions as described under "Experimental
Procedures." The parameters obtained from the mathematical fits were
used to calculate Q-reduction levels under different metabolic
conditions at which the system reaches a steady state and to calculate
the relative contributions of the individual enzymes to the total
electron flow. In Fig. 4A,
fitted data from Figs. 2B, 3A, and 3B
have been combined and reveal the kinetic interplay between
quinone-reducing and quinol-oxidizing enzymes in mitochondria
containing the alternative oxidase. Respiratory steady states are
achieved when the electron flow through the reducing pathways equals
the flow through oxidizing pathways and are represented by the
intersection of the curves describing the kinetics of the respective
routes. In vivo, it is likely that multiple substrates are
oxidized. We have therefore calculated the kinetic parameters for the
combined oxidation of succinate and NADH under state 4, state 3, and
uncoupled conditions (labeled A-C in Fig. 4,
respectively) that are tabulated in Fig. 4B. Of particular
importance are the findings that the alternative oxidase contributes up
to 24% of the total electron flow under state 4 conditions and that
even under state 3 conditions
likely to reflect the in vivo
situation most closely
as much as 19% of the overall respiratory
activity is accounted for by alternative oxidase activity. Since the
alternative oxidase is a non-protonmotive enzyme (16, 17), such a high
engagement in respiration is likely to lower the overall efficiency of
energy transduction.

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Fig. 4.
Kinetic interplay between quinone-reducing
and quinol-oxidizing enzymes in S. pombe
mitochondria containing the alternative oxidase. Curves
representing the kinetics of dehydrogenases and oxidases (panel
A) were taken from Figs. 2B, 3A, and
3B; the curve representing the combined kinetics of SDH and
NADH DH was obtained by addition of the curves describing the
individual kinetics of both dehydrogenases. Parameters defining the
hyperbolic fits (not shown) were used to calculate the Q-reduction
level (Qr/Qt), the total
rate of electron transfer (V), and the contribution of the
alternative oxidase to the total electron transfer rate (AOX) upon the
combined oxidation of succinate and NADH under state 4, state 3, and
uncoupled conditions (panel B). SDH, succinate
dehydrogenase; NADH DH, external-NADH dehydrogenase;
AOX, alternative oxidase.
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The mathematical expressions that we have used to describe the
experimentally determined kinetic dependence of each of the respiratory
enzymes on the reduction level of the Q-pool (Figs. 2 and 3) provide a
good basis to evaluate the effect of alternative oxidase expression on
the kinetic behavior of the overall mitochondrial electron transfer
system using MCA. The elasticity of the respective enzymes with respect
to the Q-redox poise can be directly calculated from the rate equation
of the enzyme, its derivative function, and the steady state reduction
level of the Q-pool. Control coefficients can then be calculated from
the elasticity coefficients using a matrix-based approach (30).
The results presented in Fig. 5
illustrate how the position of the respiratory steady state reached
upon the combined state 4 oxidation of succinate and NADH is affected
by the presence of the alternative oxidase (Fig. 5A).
Furthermore, the data quantify how control in these two steady states
is distributed among the individual respiratory enzymes (Fig.
5B). Although intuitively it is expected that the additional
presence of a quinol-oxidizing enzyme should result in an increased
flux control (CJ) exerted by the dehydrogenases,
it can be seen from Fig. 5B that control on total electron
flux exerted by the combined activities of succinate and external-NADH
dehydrogenase is not significantly affected by the expression of the
alternative oxidase (remains ~10%). Interestingly, control exerted
by the alternative oxidase on total electron flux is approximately
22%. This appears to be fully at the expense of the control formerly
exerted by the cytochrome pathway. The relatively powerful position the
alternative oxidase appropriates within the electron transfer system is
further illustrated by the degree of control exerted by this oxidase on
the ratio of fluxes through both pathways. The negative control exerted by the alternative oxidase on the flux-ratio of electrons through quinol-oxidizing routes is comparable with the positive control exerted
on this flux-ratio by the cytochrome pathway
(Crca is
0.964 compared with 1.115, Fig.
5B).

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Fig. 5.
Effect of alternative oxidase expression on
distribution of control under state 4 conditions in S. pombe
mitochondria oxidizing succinate and NADH. Curves
representing state 4 kinetics of quinol-oxidizing pathways were taken
from Fig. 2, A and B; the curve representing the
combined kinetics of SDH and NADH DH was obtained by addition of curves
taken from Fig. 3, A and B (panel A).
Flux and flux ratio control coefficients (CJ and
Crca, respectively) of the respiratory enzymes
(panel B) were calculated as described under "Experimental
Procedures." Flux ratio refers to the quotient of the electron
transfer rate through the cytochrome pathway and the alternative
oxidase. SDH, succinate dehydrogenase; NADH DH,
external-NADH dehydrogenase; Cyt. pathway, cytochrome
pathway; Alt. oxidase = aox, alternative
oxidase.
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DISCUSSION |
The results described in this paper show that expression of the
plant alternative oxidase inhibits the glycerol-dependent growth of the yeast S. pombe (Fig. 1, C and
D). It is unlikely that this inhibition is an artifactual
effect of heterologous expression since growth on glucose is
considerably less affected (Fig. 1, A and B).
This paper is, to our knowledge, the first to report a phenotypic
effect of alternative oxidase expression. Previously, plants have been
genetically modified to yield a higher level of, in vitro
active, alternative oxidase protein (18, 19). However, these plants
were not phenotypically affected, possibly because of restricted
engagement of the oxidase in vivo. The negative effect on
S. pombe growth suggests that, in this system, the
alternative oxidase is not merely expressed (cf. Ref. 14)
but also contributes in vivo to the overall respiratory activity.
Analysis of mitochondrial respiratory kinetics strongly indicates that
the alternative oxidase has indeed the potential to be engaged during
S. pombe respiration. This is particularly evident from the
alternative oxidase kinetics with respect to the Q-redox poise that
suggest a relatively high affinity of the enzyme for QH2
relative to Q (Fig. 2B). In plants, alternative oxidase
activity is stimulated by pyruvate, mainly at low Q-redox levels (35). This apparently increased relative affinity for QH2 enables
the enzyme to compete for reducing equivalents with the cytochrome pathway (10). When the alternative oxidase kinetics in S. pombe are considered in relation to the kinetics of the other
enzymes interacting with the Q-pool, the potential of the oxidase to
actively compete for reducing equivalents is confirmed. MCA shows that the alternative oxidase exerts ~22% of the total control on overall electron flux during state 4 conditions, which is at the expense of the
cytochrome path (Fig. 5). Furthermore, control of the alternative oxidase on the relative electron fluxes through the
QH2-oxidizing pathways is almost equal (in absolute terms)
to that of the cytochrome pathway (Fig. 5). Kinetic modeling of the
interaction between mitochondrial respiratory enzymes predicts that, in
transformed S. pombe, ~20% of the total respiratory
activity is accounted for by the alternative oxidase (Fig. 4).
From Fig. 2C it is apparent that the presence of a kinetically
competent alternative oxidase results in decreased electron flow
through the cytochrome pathway both under state 4 (at any Q-redox
poise) and state 3 (at Q-redox levels > 30%) conditions. Since
the uncoupled cytochrome pathway kinetics are not significantly affected, particularly at Q-reduction levels above 40% (Fig. 2), it is
unlikely that the decreased state 4 and 3 activities are because of
reduced expression levels of any one of the components of the
cytochrome pathway. Instead, the competitive action of the alternative
oxidase is likely to restrict the amount of substrate (i.e.
QH2) available for the cytochrome
bc1 complex. Interestingly, not only the
cytochrome pathway but also the total oxidative state 4 and 3 activities are affected by alternative oxidase expression (cf. Fig. 2, A and B). This suggests
that the decrease in cytochrome pathway activity is not fully
compensated for by alternative oxidase activity. This may be explained
by the different capacities of the respective pathways.
The electron turnover capacity of the cytochrome pathway is measured as
the specific oxygen consumption rate in mitochondria lacking the
alternative oxidase (Fig. 2A). Under comparable conditions, the electron turnover capacity of the alternative pathway is measured as the specific antimycin-resistant oxygen uptake rate (Fig.
2B). It is evident that the total electron turnover capacity
of the alternative oxidase is considerably lower than that of the
cytochrome pathway under any energetic condition and at any Q-reduction
level. This might be because of differences in protein levels and/or might be related to the minimum Vmax values of
the two pathways. The reported minimum Vmax of
the alternative oxidase is 186 electrons s
1 (34), which
is approximately half the value reported for the yeast cytochrome
bc1 complex (36) and one-eighth of that reported for yeast cytochrome c oxidase (37). It is therefore
tempting to speculate that alternative oxidase expression confers a
respiratory pathway in S. pombe that transfers fewer
electrons per second than the endogenous cytochrome pathway. When the
availability of QH2 for the latter pathway is restricted by
the competitive action of the alternative oxidase, the overall rate of
electron turnover is decreased.
The kinetic data shown in Figs. 2 and 3 are reasonably well described
by the mathematical model developed by Van den Bergen and co-workers
(9, 27, 29). This model, which assumes homogeneity of the Q-pool, has
been successfully used to model mitochondrial respiratory kinetics in
several plant systems (9, 31). S. pombe is therefore
comparable with plants in that the core of its respiratory chain is
formed by one homogeneous Q-pool that connects dehydrogenases with
oxidases. However, the two systems differ in the way in which the
kinetics of the cytochrome pathway are regulated by the protonmotive
force. In plant mitochondria, a reduced protonmotive force results in
strongly increased cytochrome pathway activity at any Q-reduction level
(6, 7, 9, 10, 31). In contrast, cytochrome pathway activity in S. pombe is stimulated by a decreased protonmotive force only at high
reduction levels of the Q-pool (Fig. 2A).
Although the effect of the protonmotive force in S. pombe
mitochondria is not clearly understood, it appears that the electron transfer rate of the cytochrome pathway is increased upon uncoupling while the relative affinity for QH2 is decreased. This may
explain why the cytochrome pathway kinetics are less affected by
alternative oxidase expression under uncoupled conditions than under
state 4 or state 3 conditions. Although the mathematical fits do not differ significantly (Fig. 2C), the data in Fig. 2,
A and B show that, at low Q-reduction levels
(<40%), uncoupled oxygen uptake rates are higher in the absence than
in the presence of the alternative oxidase. This suggests that the
oxidase is only able to compete for reducing equivalents at a
relatively low Q-redox poise. At higher Q-reduction levels, (>40%)
cytochrome pathway activity is increased to such a degree that the
contribution of the alternative oxidase to the overall rate becomes insignificant.
In conclusion, the results presented in this paper are the first to
report a phenotypic effect because of the expression of the plant
alternative oxidase. We suggest that the mechanisms which down-regulate
the engagement of the alternative oxidase under physiological
conditions in plants are non-operative in S. pombe.
Consequently, the alternative oxidase is capable of contributing
considerably to respiration under a variety of physiological conditions. Interestingly, such a contribution results in diminished mitochondrial oxygen consumption rates and, significantly, in a
decreased overall efficiency of energy conservation. This has a
distinct detrimental effect on yeast growth, particularly when cells
are cultured on a non-fermentable carbon source.