Recent observations point to the role played by Zn2؉ as an inducer of neuronal death. Two Zn 2؉ targets have been identified that result in inhibition of mitochondrial respiration: the bc 1 center and, more recently, ␣-ketoglutarate dehydrogenase. Zn 2؉ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn 2؉ is imported through the Ca 2؉ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following.

The present study reveals that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of respiratory chain functioning may be observed at different absolute values of A$ on the mitochondrial membrane. This fact points out that the membrane potential is not a unique regulator in coupling of ATP-synthetase and respiratory chain activities. We found, using the double-inhibitor titration technique, that ATP-synthetase inhibition induces proportional inhibition of respiratory chain enzymes and vice versa respiratory chain inhibition induces proportional inhibition of ATP-synthetase. This effect is shown to exist only when osmolarity is close to 150-300 (mosM) (in the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. Under conditions of high osmolarity (400-600 mosM) the respiratory chain and ATP-synthetase behave as if they were coupled by bulk phase Aj&+, from the kinetic point of view.

This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Deltamusol) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation.

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A concerted function of purine nucleotide (PN) binding and fatty acid (FA) release from the uncoupling protein (UP) resulting in the maximum coupling (potential) of brown adipose tissue (BAT) mitochondria was demonstrated. The uncoupling effect of FA was studied (at 4 mM MgCI2): 17 nmol oleate per mg protein caused a slight uncoupling with 8.9 mM ATP but with ATP below 3.6 mM almost total uncoupling was achieved. This shows that the PN-controUed gate can be stabilized in the closed conformation (with 8.9 mM ATP), also when FA is bound to UP. The sensitivity of the FA effect to ATP proves that oleate directly interacts with UP. The closed conformation of the H ÷ channel of UP is then abolished by oleate when a lower free ATP concentration is maintained outside.Fatty acid uncoupling; Uncoupling protein; Brown adipose tissue mitochondria; H + channel