The complex ATP-Fe²⁺ serves as a specific affinity cleavage reagent in ATP-Mg²⁺ sites of Na,K-ATPase: Altered ligation of Fe²⁺ (Mg²⁺) ions accompanies the E₁P→E₂P conformational change

In the presence of ascorbate/H₂O₂, ATP-Fe²⁺ or AMP-PNP-Fe²⁺ complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP-Mg²⁺ sites. The cleavages reveal contact points of Fe²⁺ or Mg²⁺ ions. In E₁ and E₁Na conformations, two major cleavages are detected within the conserved ⁷⁰⁸TGDGVNDSPALKK sequence (at V712 and nearby), and one (E₁Na) or two (E₁) minor cleavages near V440. In media containing sodium and ATP, Fe²⁺ substitutes for Mg²⁺ in activating phosphorylation and ATP hydrolysis. In the E₁P conformation, cleavages are the same as in E₁. Fe²⁺ is not bound tightly. By contrast, in the E₂P conformation, the pattern is different. A major cleavage occurs near the conserved sequence ²¹²TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe²⁺ is bound very tightly. On E₂P hydrolysis, the Fe²⁺ dissociates. The results are consistent with E₁ mutually implies E₂ conformation-dependent movements of cytoplasmic domains and sites for P(i) and Mg²⁺ ions, inferred from previous Fe-cleavage experiments. Furthermore, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature (London) 405, 647-655]. Altered ligation of Mg²⁺ ions in E₂P may be crucial in facilitating nucleophilic attack of water on the O - P bond. Mg²⁺ ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP-Fe²⁺ or other nucleotide-Fe²⁺ complexes could be widely used to investigate nucleotide binding proteins.