Selective Fe2+-catalyzed Oxidative Cleavage of Gastric H+,K+-ATPase

In the presence of ascorbate/H2O2, Fe2+ ions or the ATP-Fe2+ complex catalyze selective cleavage of the α subunit of gastric H+,K+-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E1 and E2 conformational states are essentially identical to those described previously for renal Na+,K+-ATPase. The cleavage pattern of H+,K+-ATPase by Fe2+ ions is consistent with the existence of two Fe2+ sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe2+ or the ATP-Fe2+ complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca2+-ATPase crystal structure (Protein Data Bank identification code 1EUL; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647–655), anE1Ca2+ conformation, and a theoretical model of Ca2+-ATPase in anE2 conformation (Protein Data Bank identification code 1FQU). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with theE1 ↔ E2 transitions, are similar in all P-type ATPases. Fe2+-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg2+, and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E1 ↔E2 transitions, and subconformations induced by H+,K+-ATPase-specific ligands such as SCH28080.