Selective Fe -catalyzed Oxidative Cleavage of Gastric H ,K -ATPase IMPLICATIONS FOR THE ENERGY TRANSDUCTION MECHANISM OF P-TYPE CATION PUMPS*

In the presence of ascorbate/H2O2, Fe 2 ions or the ATP-Fe complex catalyze selective cleavage of the 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 Fe ions is consistent with the existence of two Fe 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 Fe or the ATP-Fe 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 Ca -ATPase crystal structure (Protein Data Bank identification code 1EUL; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647–655), an E1Ca 2 conformation, and a theoretical model of Ca -ATPase in an E2 conformation (Protein Data Bank identification code 1FQU). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E17 E2 transitions, are similar in all P-type ATPases. Fe -catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg , and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E1 7 E2 transitions, and subconformations induced by H ,K -ATPase-specific ligands such as SCH28080.