ATP synthase inhibition

The electrochemical potential difFetence across the membrane, once established as a tesult of proton translocation, inhibits further transport of teducing equivalents through the respiratory chain unless discharged by back-translocation of protons across the membtane through the vectorial ATP synthase. This in turn depends on availability of ADP and Pj. 

Ca2+ cycling into and out of the mitochondria leads to NAD depletion and a fall in ATP. The entry of Ca2+ into the mitochondria dissipates the potential difference across the mitochondrial membranes and so inhibits the function of ATP synthase, which relies on the charge difference across the membrane (Fig. 6.13 and 7.60). Export of Ca2+ from the mitochondrial matrix may occur and be stimulated by some chemicals. However, this will lead to repeated cycling, which damages the membrane and further compromises ATP synthesis. The export of Ca2+ also uses up ATP as a result of the Ca2+ ATPases involved. Hence ATP levels fall. 

Thus, an initial drop in ATP is followed by increases in Ca2+, which inhibits ATP synthase and increases ROS and reactive nitrogen species (RNS) formation via xanthine oxidase. These inhibit thiol-dependent Ca2+ transport. The reactive molecules can also inhibit the electron transport chain (by reacting with Fe at the active sites) and enzymes in glycolysis, notably glyceraldehyde 3-phosphate dehydrogenase, leading to further losses of ATP. The depleted ATP exacerbates the intracellular Ca2 increase as a result of reduced transport out and sequestration into the endoplasmic reticulum. 

ATP is synthesized. Addition of cyanide (CN ), which blocks electron transfer between cytochrome oxidase and 02, inhibits both respiration and ATP synthesis, (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and P, are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis. 

Now covers the role of IF 1 in the inhibition of ATP synthase during ischemia... 

Synthesis of ATP by mitochondria is inhibited by oligomycin, which binds to the OSCP subunit of ATP synthase. On the other hand, there are processes that require energy from electron transport and that are not inhibited by oligomycin. These energy-linked processes include the transport of many ions across the mitochondrial membrane (Section E) and reverse electron flow from succinate to NAD+ (Section C,2). Dinitrophenol and many other uncouplers block the reactions, but oligomycin has no effect. This fact can be rationalized by the Mitchell hypothesis if we assume that Ap can drive these processes. 

DCCD inhibits proton translocation through the F subunit of the ATP synthase. Thus, the value of A/xh increases to a point where proton translocation, and hence electron transport, becomes thermodynamically unfavorable. In addition, DCCD inactivates the ATP synthesis function of the ATP synthase. The uncoupler. 2,4-dinitrophenol, renders the inner mitochondrial membrane permeable to protons, leading to a decrease in the value of A/jih< and restoration of electron transport. However, 2,4-dinitrophenol cannot restore the activity of the DCCD-treated ATP synthase. 

Inhibition of ATP synthase (energy transfer) reduces proton flow from the inter-membrane space to the matrix, which inhibits electron flow in the respiratory chain. Oligomycin, a macrolide antibiotic, prevents phosphoryl group transfer of ATP synthase. Dicyclohexylcarbodimide (DCCD) binds to and inhibits ATP synthase. Similar to the inhibitors of Complexes I, III, and IV, energy transfer inhibitors cause accumulation of reactive electrons and generate ROS. 

The organotins (cyhexatin, fenbutatin-oxide, and azocyclotin), diafenthiuron, and organosulfurs (propargite and tetradifon) are inhibitors of mitochondrial ATP synthase. However, diafenthiuron is a proacaricide. It is metabolized to the corresponding carbodi-imide (Figure 7.33), which inhibits the ATP synthase. 

ATP synthase also can be inhibited. Oligomycin and dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled. 

A question of coupling. What is the mechanistic basis for the observation that the inhibitors of ATP synthase also lead to an inhibition of the electron-transport chain ... 

Identifying the inhibition. You are asked to determine whether a chemical is an electron-transport-chain inhibitor or an inhibitor of ATP synthase. Design an experiment to determine this. 

Add the inhibitor with and without an uncoupler, and monitor the rate of O2 consumption. If the O2 consumption increases again in the presence of inhibitor and uncoupler, the inhibitor must be inhibiting ATP synthase. If the uncoupler has no effect on the inhibition, the inhibitor is inhibiting the electron-transport chain. 

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