Cytochrome Pathway Research Articles

The dependence receptor DCC defines a new mechanism for caspase activation

The DCC (Deleted in Colorectal Cancer) gene has been found to be deleted in more than 50% of primary colorectal cancers and in numerous other carcinomas, although the role of DCC as a tumor suppressor remains controversial. DCC is also a receptor for netrin-1, a lamininrelated molecule involved in axon guidance. Recently, we reported that DCC is actually a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand, but inhibiting apoptosis in the presence of ligand. Here, we show that DCC drives apoptosis through a mechanism requiring caspase-9 activation. Unexpectedly, if DCC kills through caspase-9, it does not go through the classic ‘mitochondria pathway’ since Bcl-2 and an inhibitor of cytochrome c release, cyclosporin A, are unable to block CCC-induced ceil death. Of interest, DCC intera& with both caspase3 and caspase-9 suggesting that DCC could play the role of an initiator complex for caspase activation. Moreover, in vitro translated DCC drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c. Hence, DCC defines the first pathway for cytochrome c-independent-and thus mitochondria-independentactivation of caspase-9.

Respiratory metabolism during cold storage of apple fruit. II. Alternative oxidase is induced at the climacteric

Changes in mitochondrial respiration were investigated during cold storage of apple fruit (Malus domestica Borkh. cv. Reinette du Canada). Postharvest calcium treatment of the apples had no effect on the aspects of mitochondrial metabolism studied, suggesting that the role of calcium in fruit ripening is not related to respiration. A transient increase in mitochondrial oxidative capacity occurred during storage around day 80 after harvest. This climacteric burst was associated with an enhanced capacity of the alternative pathway, but not of the cytochrome pathway. The major factor determining this rise in alternative pathway capacity at the climacteric was the transient increase in the amount of alternative oxidase (AOX) protein. Antibodies raised against the Sauromatum guttatum enzyme recognized a reduced form product of ampproximately 37 kDa and a 67‐kDa band corresponding to the oxidized form of the protein. During cold storage of the apple fruit, alternative pathway capacity correlated with the levels of the AOX and was ampparently not dependent on major changes in the oxidation state of the enzyme. This is the first work to report that the respiratory climacteric and the induction of AOX are tightly and temporally linked.

Effect of the methoxyiminoacetamide fungicide, SSF129, on respiratory activity inBotrytis cinerea

(E)-2-Methoxyimino-N-methyl-2-[2-(2,5-dimethylphenoxymethyl)phenyl]acetamide (SSF129) has been developed as a broad-spectrum systemic fungicide for control of cereal and fruit diseases. This compound inhibited NADH-oxidation by submitochondrial particles from mycelial cells of Botrytis cinerea, with an EC50 value of 14.5 nM, due to blockage of electron transport through the cytochrome bc1 complex in the mitochondrial respiratory chain. However, SSF129 did not suppress, but rather increased, oxygen consumption by mycelial cells of the fungus. This was because mycelial cells contain an alternative oxidase protein and the cells have the ability to rapidly switch electron flux from the main cytochrome pathway to the alternative pathway on blockage of the former by SSF129. The alternative pathway of the mycelia seems not to be operative when the cytochrome pathway is functional. Naturally occurring flavonoids inhibited the alternative oxidase of the mycelial cells in a dose-dependent manner, with EC50 values of 68.4 µM for flavone and 63.7 µM for flavanone. These observations suggested that plant components play an important role in control of gray mould by SSF129. © 1999 Society of Chemical Industry

Effects of water stress on development, operation and gene expression of cyanide-resistant respiratory pathway in wheat

Osmotic dehydration of wheat seedlings in -0.5 MPa polyethylene glycol (PEG) solutions for 24, 48 and 72 h resulted in mild, moderate and severe water stress respectively in leaves, but only caused mild water stress in roots as reflected by the changes in relative water content (RWC). In response to the above water stress conditions, leaf total respiratory rate (V(t)) decreased progressively, and the alternative pathway (AP) capacity (V(alt)) and its actual operation activity (rhoV(alt)) decreased more severely. Water stress also led to continuous reduction in cytochrome pathway (CP) activity ((rho' V (cyt)) and different changes in the contribution of rhoV(alt) and rho'V(cyt) to V(t), in leaves, with rhoV(alt)/ V(t), decreasing and rho'V(cyt)/V(t) increasing. The change pattern of root V(t) was similar to that of its RWC, while root V(alt) and rhoV(alt) were found to decrease during the first 24 h of stress and thereafter recover to a level close to that of the control (0 h). These data indicate that the alternative pathway is sensitive to water stress and can adapt mild water stress. The results of northern hybridization using an alternative oxidase gene (Aox) as probe revealed that there was a good correlation between V(alt) (also rhoV(alt)) and Aox mRNA levels in both leaves and roots, suggesting that water stress affects the development and operation of AP respiration through affecting the expression of the alternative oxidase gene.

Ligand Exchange during Unfolding of Cytochrome c

The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), were also identified during the folding reaction. In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain.

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea (Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O2 evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO2 (1.0 mM NaHCO3) than that at limiting CO2 (0.1 mM NaHCO3). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO2. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO2 than at optimal CO2. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO2 (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.

Functional Expression of the Plant Alternative Oxidase Affects Growth of the Yeast Schizosaccharomyces pombe

We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.

Involvement of the alternative oxidase in cellular energy production in the wheat ‘take all’ fungus,Gaeumannomyces graminisvartritici

This summary describes a functional role for the alternative respiratory pathway of the wheat ‘take-all’ fungus, Gaeumannomyces graminis var tritici (Ggt), which utilises the alternative oxidase as its terminal oxidase. Unlike other plant and fungal alternative oxidases, the alternative oxidase of Ggt is both constitutively expressed and active. We have demonstrated that for cellular respiration, and therefore for ATP synthesis, to occur both the cytochrome and alternative pathways must remain active. © 1999 Society of Chemical Industry

Involvement of the alternative oxidase in cellular energy production in the wheat ‘take all’ fungus, Gaeumannomyces graminis var tritici

This summary describes a functional role for the alternative respiratory pathway of the wheat ‘take-all’ fungus, Gaeumannomyces graminis var tritici (Ggt), which utilises the alternative oxidase as its terminal oxidase. Unlike other plant and fungal alternative oxidases, the alternative oxidase of Ggt is both constitutively expressed and active. We have demonstrated that for cellular respiration, and therefore for ATP synthesis, to occur both the cytochrome and alternative pathways must remain active. © 1999 Society of Chemical Industry

Prolongation of photosynthetic induction as a consequence of interference with mitochondrial oxidative metabolism in mesophyll protoplasts of the pea ( Pisum sativum L.)

The restriction of mitochondrial oxidative metabolism by metabolic inhibitors caused a prolongation of induction of photosynthetic O 2 evolution in mesophyll protoplasts of the pea ( Pisum sativum L.) at both optimal and limiting CO 2. There was a significant increase in the lag period of photosynthesis in the presence of oligomycin (an inhibitor of oxidative phosphorylation) or antimycin A (an inhibitor of cytochrome pathway), while the increase in lag was marginal in presence of salicylhydroxamic acid (SHAM) or propyl gallate (inhibitors of alternative oxidase (AOX) pathway). There was a pronounced decrease in steady-state levels of ribulose-1,5-bisphosphate (RuBP) in the presence of oligomycin or antimycin A, but not in the presence of SHAM or propyl gallate. Our results suggest that the restriction of the cytochrome pathway of mitochondrial electron transport or oxidative phosphorylation, leads to a prolongation of the photosynthetic induction period and a marked decrease in the steady state levels of RuBP.

Respiratory activities in chloramphenicol‐treated tobacco cells

Chloramphenicol (CAP) inhibited tobacco cell growth as shown by a reduction (34%) of cell mass 4 days after treatment. The rates of cell respiration were slightly higher than control under coupled conditions. However, CAP‐treated cells showed a decreased maximal capacity of the cytochrome pathway (48%) and an increased maximal capacity of alternative path (56%) 4 days after treatment. In purified mitochondria, the rates of NADH or malate oxidation under state 4 conditions were not significantly changed by CAP treatment. However, the state 3 rates were 34–40% lower in CAP‐treated than in control mitochondria. Succinate oxidation decreased by 31–46% under both state 4 and state 3 conditions after CAP treatment. The activities of complexes I, III, and IV, which contain mitochondrially encoded subunits, decreased by about 50% in CAP‐treated mitochondria. There was also a decrease in the contents of mitochondrial cytochromes. Unexpectedly, the activities of complex II and the matrix‐facing rotenone‐insensitive NADH dehydrogenase, which are thought to be nuclear‐encoded, also declined. The activities of external NADH dehydrogenase, NAD‐linked malic enzyme, and fumarase remained unchanged after CAP treatment. There was a slight increase in the activity and protein level of alternative oxidase. An electrochemical gradient across the mitochondrial membranes was observed by Rhodamine 123 staining in CAP‐treated cells. However, the morphology of most of the mitochondria changed from spherical to vermicular. A method for purifying a high yield of intact mitochondria from tobacco cell suspension cultures is described.

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM) (inhibitor of alternative pathway). The increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO2 (1.0 mM NaHCO3) compared to that at limiting CO2 (0.1 mM NaHCO3). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO2 than that at limiting CO2. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.

Alteration of dark respiration and reduction of phototrophic growth in a mitochondrial DNA deletion mutant of Chlamydomonas lacking cob, nd4, and the 3' end of nd5.

We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.

The role of the alternative oxidase in stabilizing the in vivo reduction state of the ubiquinone pool and the activation state of the alternative oxidase

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Qr/Qt), thereby avoiding an increase in free radical production. If the Qr/Qt were stabilized by the alternative pathway, then Qr/Qt should be less stable when the alternative pathway is blocked. Qr/Qt increased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Qr/Qt increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Qr/Qt. Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Qr/Qt on the activity of the alternative pathway in intact roots is discussed.

Effect of hypoxia on sugar accumulation, respiration, activities of amylase and starch phosphorylase, and induction of alternative oxidase and acid invertase during storage of potato tubers (Solanum tuberosum cv. Russet Burbank) at 1°C

The transfer of potato (Solanum tuberosum) tubers from 10 to 1°C was associated with an initial decline in the rate of CO2 output followed by a rapid increase reaching, within some 12 days, a peak which was about 3‐fold higher than at 10°C. Thereafter the rate of CO2 evolution declined gradually for the duration of the experiment. The specific rate of mitochondrial O2 uptake decreased initially, followed by a rise to a level similar to that of mitochondria prepared from tubers stored at 10°C. Low temperature decreased by 30% the capacity of the cytochrome pathway while it sharply increased the capacity of the alternative pathway. Sucrose was the first sugar to accumulate at 1°C, followed after a delay of 6‐7 days by glucose and fructose. Low temperature induced within 4‐5 days a rise in amylase activity which increased by 10‐fold after 30 days. The increase was reflected in only two out of four existing isoforms. In addition a novel isoform of amylase was detected later in storage. The induction and the accumulation of invertase mRNA and extractable activity followed the increase in sucrose but preceded that of hexoses. The activity of starch phosphorylase isoforms was not affected by temperature. There was a 3‐fold increase in chlorogenic acid at 1°C. Hypoxia strongly inhibited the accumulation of sugars and chlorogenic acid, the increase in the amylase activity, and the appearance of the novel isoform. Low O2 totally suppressed the induction of invertase mRNA and increased the capacity of the alternative oxidase. It did not, however, prevent the decrease in cytochrome capacity; neither did it affect the activity of starch phosphorylase isoforms.

Salinity tolerance in Triticum aestivum-Lophopyrum elongatum amphiploid and 5E disomic addition line evaluated by NaCl effects on photosynthesis and respiration

Effects of NaCl salinity on photosynthesis and respiration were studied in wheat (Triticum aestivum) cv. ‘Chinese Spring’ (abbreviated as CS) and the amphiploid and 5E disomic addition line of CS with wheatgrass (Lophopyrum elongatum), a wild facultative halophyte. Only the amphiploid showed NaCl tolerance based on seedling growth. The net photosynthetic CO2 assimilation measured by the rate of O2 evolution was decreased and chlorophyll a/b ratio increased in CS under the presence of 0.4 M NaCl, but no such changes occurred in the amphiploid and 5E disomic addition line. The amounts of total soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per fresh weight were increased in all the lines, while the relative amount of Rubisco per protein remained unchanged. Rubisco specific activity was inhibited in all the lines but Rubisco activity per fresh weight did not change. The total respiratory electron flow and the flow through the cytochrome pathway were increased in CS under the salinity condition, while no changes occurred in both amphiploid and 5E disomic addition lines. The sustained CO2 assimilation, the stability of the light-harvesting photosystem II complex and the normal level of respiratory electron flow through the cytochrome pathway attested the NaCl tolerance in the amphiploid. A positive effect of chromosome 5 on salinity tolerance was suggested by the stability of the photosystem II complex and the cytochrome pathway under the salinity condition.

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.

Suppression of a +1 T mutation by a nearby substitution in the mitochondrial cox1 gene of Chlamydomonas reinhardtii: a new type of frameshift suppression in an organelle genome.

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role.

Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term (post-translational modification, allosteric activation) regulated. Electron distribution (partitioning) between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach). Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.