The charge stoichiometry of cytochrome c oxidase in the reconstituted system.

Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Protonmotive stoichiometry of rat liver cytochrome c oxidase: determination by a new rate/pulse method

The stoichoimetry of vectorial H+ ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the----H0+/2e- ratio was found to be constant during the time-course of oxygen reduction, and thus independent of delta pH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

The mechanism of transmembrane delta muH+ generation in mitochondria by cytochrome c oxidase.

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.     

The reaction of reduced xanthine oxidase with oxygen. Kinetics of peroxide and superoxide formation

Product formation during the oxidation of xanthine oxidase has been examined directly by using cytochrome c peroxidase as a trapping agent for hydrogen peroxide and the reduction of cytochrome c as a measure of superoxide formation. When fully reduced enzyme is mixed with high concentrations of oxygen, 2 molecules of H2O2/flavin are produced rapidly, while 1 molecule of O2-/flavin is produced rapidly and another produced much more slowly. Time courses for superoxide formation and those for the absorbance changes due to enzyme oxidation were fitted successfully to the mechanism proposed earlier (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). In this scheme, each oxidative step is initiated by the very rapid and reversible formation of an oxygen.FADH2 complex (the apparent KD = 2.2 X 10(-4) M at 20 degrees C, pH 8.3). In the cases of 6- and 4-electron-reduced enzyme, 2 electrons are transferred rapidly (ke = 60 s-1) to generate hydrogen peroxide and partially oxidized xanthine oxidase. In the case of the 2-electron-reduced enzyme, only 1 electron is transferred rapidly and superoxide is produced. The remaining electron remains in the iron-sulfur centers and is removed slowly by a second order process (ks = 1 X 10(4) M-1 s-1). When the pH is decreased from 9.9 to 6.2, both the apparent KD for oxygen binding and the rapid rate of electron transfer are decreased about 20-fold. This result is suggestive of uncompetitive inhibition and implies that proton binding to the enzyme-flavin active site affects primarily the rate of electron transfer, not the formation of the initial oxygen complex.     

Carbon monoxide-driven reduction of ferric heme and heme proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.     

The effect of oxygen concentration on the steady-state kinetics of the solubilized cytochrome c oxidase.

1. The steady-state kinetics of ascorbate oxidation as a function of oxygen concentration was measured with a solubilized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparation. 2. Linear double reciprocal plots were obtained at various fixed concentrations of ascrobate, cytochrome c and cytochrome aa3. 3. The results are interpreted in terms of an oxidase model similar to that put forward by Minnaert in 1961 (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23-34). 4. The Km for oxygen at infinite cytochrome c concentration is 0.95 muM and the intramolecular rate constant for the transfer of electrons from cytochrome c to cytochome aa3 is 400 s(-1). According to the model, this implies that the second order rate constant for the reaction between oxygen and the oxidase is 9.5 X 10(7)M(-1)-s(-1).     

Explaining the enigmatic K(M) for oxygen in cytochrome c oxidase: a kinetic model

We present a mathematical model for the functioning of proton-pumping cytochrome c oxidase, consisting of cyclic conversions between 26 enzyme states. The model is based on the mechanism of oxygen reduction and linked proton translocation postulated by Wikstr?m and Verkhovsky (2007). It enables the calculation of the steady-state turnover rates and enzyme-state populations as functions of the cytochrome c reduction state, oxygen concentration, membrane potential, and pH on either side of the inner mitochondrial membrane. We use the model to explain the enigmatic decrease in oxygen affinity of the enzyme that has been observed in mitochondria when the proton-motive force is increased. The importance of the 26 transitions in the mechanism of cytochrome oxidase for the functional properties of cytochrome oxidase is compared through Metabolic Control Analysis. The control of the K(M) value is distributed mainly between the steps in the mechanism that involve electrogenic proton movements, with both positive and negative contributions. Positive contributions derive from the same steps that control enzyme turnover rate in the model. Limitations and possible further applications of the model are discussed.     

Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase

Cytochrome c oxidase couples electron transfer from cytochrome c to O 2 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with O 2, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.     

Cytochrome c oxidase: charge translocation coupled to single-electron partial steps of the catalytic cycle

The paper presents a survey of time-resolved studies of charge translocation by cytochrome c oxidase coupled to transfer of the 1st, 2nd 3rd and 4th electrons in the catalytic cycle. Single-electron photoreduction experiments carried out with the A-class cytochrome c oxidases of aa(3) type from mitochondria, Rhodobacter sphaeroides and Paracoccus denitrificans as well as with the ba(3)-type oxidase from Thermus thermophilus indicate that the protonmotive mechanisms, although similar, may not be identical for different partial steps in the same enzyme species, as well as for the same single-electron transition in different oxidases. The pattern of charge translocation coupled to transfer of a single electron in the A-class oxidases confirms major predictions of the original model of proton pumping by cytochrome oxidase [Artzatbanov, V. Y., Konstantinov, A. A. and Skulachev, V.P. "Involvement of Intramitochondrial Protons in Redox Reactions of Cytochrome a." FEBS Lett. 87: 180-185]. The intermediates and partial electrogenic steps observed in the single-electron photoreduction experiments may be very different from those observed during oxidation of the fully reduced oxidase by O(2) in the "flow-flash" studies. .     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol.

A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake. 2. The rapid proton uptake associated to oxidation and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e- ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 4. Intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

The polyphasic reduction of oxygen to water by purified cytochrome c oxidase

The time course of oxygen consumption by purified cytochrome oxidase has been studied in reactions where the fully reduced enzyme was rapidly mixed with molecular oxygen. Similar to intact mitochondria (Reynafarje & Davies, Am. J. Physiol. 258, 1990), the enzyme reduces oxygen to water in a kinetically and well defined polyphasic event. The initial rates of O2 consumption depended hyperbolically on O2 concentration, with a bimolecular rate constant of near 10(7) M-1 s-1. The Vmax of O2 uptake was, however, a complex function of the concentrations of both enzyme and cytochrome c. It is concluded that the reduction of oxygen to water takes place in a cyclic process in which the oxidase undergoes redox changes at rates depending on the relative concentration of the enzyme and its 3 substrates: O2, electrons and protons. No evidence was found for impairments in the intramolecular flow of electrons per se.     

The H+/e? stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria

1. The -->H(+)/e(-) quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O(2), ferricyanide or ferricytochrome c, and from NNN'N'-tetramethyl-p-phenylenediamine+ascorbate to O(2), were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10(-3) pH unit. 3. The -->H(+)/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O(2) consumption or duroquinol oxidation. Higher -->H(+)/O quotients for succinate oxidation, obtained from polarographic measurements of O(2) consumption, resulted from underestimation of the respiratory rate. 4. The -->H(+)/2e(-) quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the -->H(+)/e(-) ratio was only 0.25 and not 0.7-1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the -->H(+)/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.     

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.     

Fungal respiration: a fusion of standard and alternative components

In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.