Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Incubation of Na/K-ATPase with ascorbate plus H(2)O(2) produces specific cleavage of the α subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The β subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe(2+) ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe(3+)-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe(2+) but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH⋅ radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E(2)) or media containing Rb ions [E(2)(Rb)], cleavage is much faster than in high ionic strength media (E(1)) or media containing Na ions (E(1)Na). N-terminal fragments and two C-terminal fragments (N-terminals E(214) and V(712)) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe(2+) (or Fe(3+)) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe(2+) (or Fe(3+)) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the α subunit, within the minor and major cytoplasmic loops, interact in the E(2) or E(2)(Rb) conformations but move apart in the E(1) or E(1)Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.