Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase.

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.

The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a Vmax equal to 6% of the Vmax with ATP. The Km values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F1. Both 8-N-ATP and 8-N-ADP are bound mainly to the beta subunits and at all levels of saturation the distribution of the label is 77% to the beta and 23% to the alpha subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the beta subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the alpha subunit, due to the location of the binding site at an interface between a beta and an alpha subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high Km values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.

Inhibition of energy-transducing reactions by 8-nitreno-ATP covalently bound to bovine heart submitochondrial particles: direct interaction between ATPase and redox enzymes.

The photoaffinity label 8-azido-ATP has been used to study the effect of inhibition of ATP synthase on ATP-driven reverse electron transfer from succinate to NAD+ ('reversal'), succinate- and NADH-driven ATP synthesis and ATP-Pi exchange. In reversal, where ATPase functions as primary proton pump, inactivation by covalently bound nitreno-ATP results in an inhibition that is proportional to the inactivation of ATP hydrolysis, or, consequently, with the concentration of inactivated ATP synthases. Up to 60% inactivation of the reversal rate does not lead to a decrease in delta mu H+. Inhibition of ATP synthase as secondary proton pump results in case of NADH-driven ATP synthesis in a proportional inhibition, but with succinate as substrate ATP synthesis is less than proportionally inhibited, compared with inactivation of ATP hydrolysis. Inhibition of one of the primary pumps of NADH-driven ATP synthesis, the NADH:Q oxidoreductase, with rotenone also resulted in an inhibition of the rate of ATP synthesis proportional to that of the NADH oxidation. ATP-Pi exchange is much more affected than ATP hydrolysis by photoinactivation with 8-azido-ATP. Contrary to reversal and NADH-driven ATP synthesis the rate of ATP-Pi exchange does not depend linearly, but quadratically on the concentration of active ATP synthases. The observed proportional relationships between inhibition of the primary or secondary pump and the inhibition of the overall energy-transfer reactions do not support the existence of a pool intermediate in energy-transduction reactions. However, the results are consistent with a direct transfer of energy from redox enzymes to ATP synthase and vice versa.