Relevance of divalent cations to ATP-driven proton pumping in beef heart mitochondrial F0F1-ATPase.

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F0F1-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me-ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the Km of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the Km value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F0F1-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the epsilon subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.