Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were fo und to induce activation: heat, membrane energization by succinate oxidatio n, methanol, oxyanions (sulfite. phosphate, arsenate, bicarbonate) and limi ted proteolysis with trypsin. Among the oxyanions, sulfite induced the high er increase in ATPase activity. Sulfite functioned as a nonessential activa tor that slightly modified the affinity for ATP and increased notoriously t he V-max. There was a competitive effect between sulfite, bicarbonate and p hosphate for ATPase activation; their similar chemical geometry suggests th at these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decre ased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensit ive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the a ctive states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.