Protonation and Hydrogen Bonding of Ca2+ Site Residues in the E2P Phosphoenzyme Intermediate of Sarcoplasmic Reticulum Ca2+-ATPase Studied by a Combination of Infrared Spectroscopy and Electrostatic Calculations

Protonation of the Ca2+ ligands of the SR Ca2+-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of CO bands of the Ca2+-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca2+ release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pKa values near 8.3: a band at 1758 cm−1 characteristic of nonhydrogen-bonded carbonyl groups, a shoulder at 1720 cm−1, and part of a band at 1710 cm−1, both characteristic of hydrogen-bonded carbonyl groups. The bands are thus assigned to H+ binding residues, some of which are involved in H+ countertransport. At pH 9, bands at 1743 and 1710 cm−1 remain which we do not attribute to Ca2+/H+ exchange. We also obtained evidence for a pH-dependent conformational change in β  -sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(TG+MgF42−), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca2+ ligands are hydrogen bonded. The carbonyl groups of Glu771, Asp800, and Glu908 were found to be hydrogen bonded and will thus contribute to the lower wave number bands. The carbonyl group of some side-chain conformations of Asp800 is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.