Selective Fe2+-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, Fe2+ ions or the ATP-Fe2+ complex catalyze selective cleavage of the alpha subunit of gastric H+, K+-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E-1 and E-2 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 I within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and MI. 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), an E1Ca2+ conformation, and a theoretical model of Ca2+-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 E-1 <----> E-2 transitions, are similar in all P-type ATPases. Fe-2-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 E-1 <----> E2 transitions, and subconformations induced by H+, K+-ATPase-specific ligands such as SCH28080.