Ca2+ Release from the Phosphorylated and the Unphosphorylated Sarcoplasmic Reticulum Ca2+ ATPase Results in Parallel Structural Changes

Structural changes of the sarcoplasmic reticulum Ca2+-ATPase occurring in the reaction step involving phosphoenzyme conversion and Ca2+ release (Ca2 E 1-P →E 2-P) were followed using time-resolved infrared spectroscopy in H2O and2H2O. The difference spectra measured between 1800 and 1500 cm−1 were almost identical to those of Ca2+ release from the unphosphorylated ATPase (Ca2 E 1 → E), implying that parallel structural changes occur in both steps. This suggests that characteristic structural features of the high affinity Ca2+ binding sites of Ca2 E 1 are still present in the ADP-sensitive phosphoenzyme Ca2 E 1-P. In both Ca2+release steps at least two carboxyl groups become protonated, each of them experiencing the same strength of hydrogen bonding irrespective of whether or not the Ca2+ free ATPase is phosphorylated. This suggests that the same amino acid residues are involved and that they are most likely those that participate in high affinity Ca2+ binding and H+ countertransport. We propose that during Ca2+ release from the phosphoenzyme protons from the lumenal side have access to these residues. Our results are consistent with only one pair of Ca2+ binding sites on the ATPase that serves both Ca2+ translocation and H+ countertransport.