Investigating the energy transduction mechanism of P-type ATPases with Fe2+-catalyzed oxidative cleavage.

This paper discusses specific oxidative cleavage of renal Na(+),K(+)-ATPase and gastric H(+),K(+)-ATPase, catalyzed by bound Fe(2+) or the complex ATP-Fe(2+), and its implication for the energy transduction mechanism of P-type ATPases. The cleavage technique provides information on the spatial organization of the proteins in different conformations and, since ATP-Fe(2+) substitutes for ATP-Mg(2+) in activating ATPase activity, on Mg(2+)-ligating residues in different conformations. The experiments predict the existence of large movements of N, P, and A cytoplasmic domains accompanying E(1)-E(2) and E(1) P-E(2)P conformational transitions-open in E(1) conformations and closed in E(2) conformations. These features fit well with the Ca(2+)-ATPase crystal structures in E(1) or E(2) conformations and also provide evidence on ATP and Mg(2+) binding sites that is not available from the structures. By combining information from cleavage experiments with molecular modeling, based on the Ca(2+)-ATPase structure, features such as an N to P domain interaction in an E(1). ATP-Mg(2+) conformation can be inferred. The organization of the N, P, and A domains and the ATP and Mg(2+) binding sites in the different conformational states appears to be essentially similar for Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase. The oxidative cleavage technique may be a valuable tool to investigate long-range interactions that transduce the free energy of hydrolysis of ATP to active cation movements in P-type ATPases.