The activity of cytochrome‐c oxidase, the terminal enzyme of the mitochondria) respiratory chain, is known to be regulated by the substrate pressure, i.e. the ferro‐/ferricytochrome c ratio, by the oxygen concentration, and by the electrochemical proton gradient ΔμH+ across the inner mitochondrial membrane. Here we describe a further mechanism of ‘respiratory control’ via allosteric inhibition of cytochrome‐c oxidase by ATP, which binds to the matrix domain, of subunit IV. The cooperativity between cytochrome‐c‐binding sites in the dimeric enzyme complex is mediated by cardiolipin, which is essential for cooperativity of the enzyme within the lipid membrane.

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Apoptotic cell death can occur by two different pathways. Type 1 is initiated by the activation of death receptors (Fas, TNF-receptor-family) on the plasma membrane followed by activation of caspase 8. Type 2 involves changes in mitochondrial integrity initiated by various effectors like Ca(2+), reactive oxygen species (ROS), Bax, or ceramide, leading to the release of cytochrome c and activation of caspase 9. The release of cytochrome c is followed by a decrease of the mitochondrial membrane potential DeltaPsi(m). Recent publications have demonstrated, however, that induction of apoptosis by various effectors involves primarily a transient increase of DeltaPsi(m) for unknown reason. Here we propose a new mechanism for the increased DeltaPsi(m) based on experiments on the allosteric ATP-inhibition of cytochrome c oxidase at high matrix ATP/ADP ratios, which was concluded to maintain low levels of DeltaPsi(m) in vivo under relaxed conditions. This regulatory mechanism is based on the potential-dependency of the ATP synthase, which has maximal activity at DeltaPsi(m)=100-120 mV. The mechanism is turned off either through calcium-activated dephosphorylation of cytochrome c oxidase or by 3,5-diiodo-L-thyronine, palmitate, and probably other so far unknown effectors. Consequently, energy metabolism changes to an excited state. We propose that this change causes an increase in DeltaPsi(m), a condition for the formation of ROS and induction of apoptosis.

The short-term effects of thyroid hormones, which do not occur via gene expression, were postulated to be based on interaction of diiodothyronines with mitochondria. We demonstrate specific binding of labelled 3,5-diiodothyronine to subunit Va of cytochrome-c oxidase from bovine heart. 3,5-Diiodothyronine, and to a small extent triiodothyronine, but not thyroxine and thyronine, abolish the allosteric inhibition of ascorbate respiration of reconstituted cytochrome c oxidase by ATP [Arnold, S. & Kadenbach, B. (1997) Eur. J. Biochem. 249, 350Ϫ354]. This abolition of ATP-inhibition by 3,5-diiodothyronine is completely prevented by a monoclonal antibody to subunit Va. The results explain at the molecular level the short-term action of thyroid hormones on basal metabolic rate.Keywords : cytochrome-c oxidase; 3,5-diiodothyronine ; ATP/ADP ratio; subunit Va; short-term action of thyroid hormones.Thyroid hormones act in two different ways on the energy diiodothyronines are able rapidly to stimulate the mitochondrial oxidative capacity and respiration rate in human and rat (Horst metabolism of higher organisms. The long-term effects induce within days the expression of specific genes of energy metabo-et al., 1989; Lanni et al., 1992 Lanni et al., , 1993 Lanni et al., , 1994aO'Reilly and Murphy, 1992; Kvetny, 1992). Similar results have been obtained on lism via interaction of triiodothyronine with the thyroid hormone receptor (Weinberger et al., 1986;Sap et al., 1986), which forms isolated mitochondria from fish (Leary et al., 1996), indicating that such effects are not restricted to mammalian species. Morehomodimers or heterodimers allowing complex interactions with genes (Glass and Holloway, 1990; Marks et al., 1992; Cheng et over, diiodothyronines were found to stimulate directly cytochrome-c oxidase activity in rat liver homogenate (Lanni et al., al., 1994;Bogazzi et al., 1994). The short-term effects refer to stimulation, within minutes, of mitochondrial activities (for 1994b) and of the isolated enzyme (Goglia et al., 1994b). Recently the stimulatory effect of diiodothyronines on the energy review see Soboll et al., 1993; Wrutniak and Cabello, 1996). While the mechanism of stimulation of gene expression has been metabolism was shown in vivo (Lanni et al., 1996; Cimmino et al., 1996), and in a very recent report it was shown that the investigated extensively, the mechanism of short-term stimulation of respiration by thyroid hormones remained obscure. The effect of diiodothyronines on the resting metabolism was more rapid than that exerted by triiodothyronine, with 3,5-diiodothyoriginal observation that thyroid hormones uncouple oxidative phosphorylation in mitochondria (Martius and Hess, 1951; ronine seeming to show a clearer effect .However, in spite of the presence of specific binding sites for Lardy and Feldott, 1951) was later withdrawn (Tata et al., 1962; Kadenbach, 1966a, b). Nevertheless, specific binding sites for diiodothyronines in rat liver mitochondria (Lanni et at., 1994a ; Goglia et al., 1994a) t...

Recently the signal transduction function for oxidative phosphorylation was found to be second order in ADP

Life of higher organisms is essentially dependent on the efficient synthesis of ATP by oxidative phosphorylation in mitochondria. An important and as yet unsolved question of energy metabolism is how are the variable rates of ATP synthesis at maximal work load during exercise or mental work and at rest or during sleep regulated. This article reviews our present knowledge on the structure of bacterial and eukaryotic cytochrome c oxidases and correlates it with recent results on the regulatory functions of nuclear-coded subunits of the eukaryotic enzyme, which are absent from the bacterial enzyme. A new molecular hypothesis on the physiological regulation of oxidative phosphorylation is proposed, assuming a hormonally controlled dynamic equilibrium in vivo between two states of energy metabolism, a relaxed state with low ROS (reactive oxygen species) formation, and an excited state with elevated formation of ROS, which are known to accelerate aging and to cause degenerative diseases and cancer. The hypothesis is based on the allosteric ATP inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios ("second mechanism of respiratory control"), which is switched on by cAMP-dependent phosphorylation and switched off by calcium-induced dephosphorylation of the enzyme.

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.

According to the chemosmotic hypothesis, ATP is synthesized in mitochondria, bacteria and chloroplasts via the proton motive force v vp, the energy-rich intermediate of electron transport and photosynthetic phosphorylation. The general applicability of the chemosmotic hypothesis, however, was disputed until present. In particular the relationship between the rate of respiration and v vp in mitochondria was found variable, depending on the experimental conditions. Recently, a new mechanism of respiratory control was found, based on binding of ATP or ADP to subunit IV of cytochrome c oxidase, which is independent of v vp and could explain many previous results contradicting the chemosmotic hypothesis.z 1999 Federation of European Biochemical Societies.Key words: Respiratory control; Chemosmotic hypothesis; Cytochrome c oxidase; ATP or ADP binding site; Allosteric inhibition; Oxidative phosphorylation The ¢rst mechanism of respiratory controlRespiratory control was originally de¢ned as stimulation of respiration, i.e. oxygen consumption of isolated mitochondria by ADP (active, state 3 respiration), followed by its decrease (controlled, state 4 respiration) due to conversion of ADP into ATP [1,2]. A molecular explanation for this phenomenon was presented by the chemosmotic hypothesis [3,4] which postulates the proton motive force vp across the inner mitochondrial membrane as the energy-rich intermediate of oxidative phosphorylation (vp = v8-ZUvpH, Z = 2.303URT/F; vpUF = vW r . Three proton pumps of the respiratory chain (NADH dehydrogenase, cytochrome c reductase and cytochrome c oxidase) generate vp which is used by the proton gradient driven ATP-synthase for the synthesis of ATP from ADP and phosphate. Respiratory control is explained by the following events: uptake of ADP into mitochondria stimulates the ATP-synthase accompanied by a decrease of vp, which in consequence stimulates the activity of the three proton pumps and thus mitochondrial respiration [5]. The chemosmotic hypothesis was strongly supported by the e¡ect of uncoupler' of oxidative phosphorylation which increases the H conductance of biological membranes and dissipates vp. After the ¢rst description of 2,4-dinitrophenol as uncoupler of phosphorylation from oxidation by Loomis and Lipman in 1948 [6], numerous other compounds were identi¢ed to uncouple oxidative phosphorylation, all representing hydrophobic weak acids [7]. Results in con£ict with the chemosmotic interpretation of respiratory controlThe chemosmotic hypothesis became established after Peter Mitchell received the Nobel price in chemistry in 1978. The hypothesis was strongly supported by results obtained with bacteria [8,9] and chloroplasts [10], but various observations, in particular from studies with mitochondria, could not be explained by that theory ([11], for review see [12,13]). In 1959, Ernster and colleagues [14] described the lack of respiratory control in skeletal muscle mitochondria from a patient with hypermetabolism (the ¢rst described case of a mitochondrial myop...