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.