Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline

This paper describes a novel approach to specific oxidative cleavage of Na, K+-ATPase, mediated by Cu2+ ions and a hydrophobic phenanthroline, 4,7-di phenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H2O2. Th e cleavage produces two major fragments of the a subunit, with apparent mol ecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entran ce of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu2+-DPP complex. We infer th at M3 and MI are in proximity near the cytoplasmic surface. The yields of 9 6.5 and 76 kDa fragments are not significantly affected by ligands that sta bilize different E1 and E2 conformations. In E-2(K) and E2P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 9 6.5 and 76 kDa fragments are indistinguishable from two fragments near M3 a nd M I produced by Fe2+-catalyzed cleavage described previously [Goldshlege r, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], where as other Fe2+-catalyzed cleavage fragments in the cytoplasmic P and A domai ns are not observed with the Cu2+-DPP complex. These findings provide exper imental support for the concept of two separate Fe2+ sites. A homology mode l, with Na+, K+-ATPase residues within transmembrane segments and connectin g loops substituted into the crystal structure of Ca2+-ATPase, shows the pr oximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasm ic surface. Thus, the model strongly supports the conclusions based on clea vages mediated by the Cu2+-DPP complex (or Fe2+ at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na+, K-ATPase and Ca2+-ATPase.