| Summary: | Na<sup>+</sup>,K<sup>+</sup>-ATPase actively extrudes three cytoplasmic Na<sup>+</sup> ions in exchange for two extracellular K<sup>+</sup> ions for each ATP hydrolyzed. The atomic structure with bound Na<sup>+</sup> identifies three Na<sup>+</sup> sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na<sup>+</sup> binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na<sup>+</sup> sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na<sup>+</sup> sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na<sup>+</sup> access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na<sup>+</sup> affinity. This indicates that Na<sup>+</sup> binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E<sub>1</sub>-like configuration, allowing Na<sup>+</sup> but not K<sup>+</sup> to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.
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