The complex ATP-Fe2+ serves as a specific affinity cleavage reagent in ATP-Mg2+ sites of a 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 E-1 and E1Na conformations, two major cleavages are detected within the conserved (708)TGDGVNDSPALKK sequence (at V712 and nearby), and one (E1Na) or two (E-1) 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 E-1. Fe2+ is not bound tightly. By contrast, in the E2P conformation, the pattern is different. A major cleavage occurs near the conserved sequence (212)TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe2+ is bound very tightly. On E2P hydrolysis, the Fe2+ dissociates. The results are consistent with E-1<->E-2 conformation-dependent movements of cytoplasmic domains and sites for P-i and Mg2+ ions, inferred from previous Fe-cleavage experiments. Further-more, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura. H. & 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.