Diazoxide, a selective opener of the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)), has been reported to enhance F(0)F(1)ATP-synthase inhibition during ischemia, but the underlying mechanisms are still unclear. Here, we demonstrate that diazoxide directly interacts with the F-1 sector of beef heart F(0)F(1)ATPsynthase markedly promoting the binding of the inhibitor protein (IF1) to beta subunit. More specifically, the treatment of soluble F-1 with one equivalent of diazoxide was sufficient to decrease the K-d of IF1-F-1 complex at low pH. Such effect was revealed only on the cycling enzyme, while no effect was observed in the absence of Mg-ATP. However, diazoxide binding occurred independently from the catalysis, as shown by the structural changes induced by the drug in not catalytically active F-1 and revealed by CD spectra. In addition, kinetic analysis of ATP hydrolysis demonstrated that diazoxide exerts a stabilising role on Mg-ADP bound in the catalytic site of the beta subunit adopting the tight conformation (beta(DP)). In accordance, a stabilising effect of Mg-ADP at the nucleotide binding domain (NBD) has been reported also for K-ATP channel. These results suggest that diazoxide binds to beta subunit at NBD, which is highly conserved in the ATP-binding cassette protein family, thus inducing nucleotide stabilisation and favouring F-1 conformation suitable for IF, binding. Finally, diazoxide also increased IF1 binding to membrane bound F1, while it did not influence the energisation-dependent IF1 release. As IF1 binding mediates the F(0)F(1)ATPsynthase inhibition, we suggest that such mechanism may contribute to cardioprotection during ischemia.
