The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

Abstract The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E 2(K 2 )Mg–MgF 4 , an E 2∙Pi-like state capable to occlude K + (or Rb + ). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb + with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E 1– E 2 conformational transition and the kinetics of Rb + exchange between the medium and the E 2(Rb 2 )Mg–MgF 4 state. Our results show that both in the absence and in the presence of Rb + , simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E 2 conformation, presumably the E 2Mg–MgF 4 complex, that is unable to shift to E 1 upon addition of Na + . The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E 2Mg–MgF 4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb + caused by Mg 2 + . Na + -induced release of Rb + from E 2(Rb 2 )Mg–MgF 4 occurs at the same rate as from E 2(Rb 2 ) but is insensitive to ADP. The rate of Rb + occlusion into the E 2Mg–MgF 4 state is 5–8 times lower than that described for the E 2Mg–vanadate complex. Since the E 2Mg–MgF 4 and E 2Mg–vanadate complexes represent different intermediates in the E 2-P →  E 2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.