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

Protonation of the Ca ligands of the SR Ca-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of C1⁄4O bands of theCa-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups uponCa release. At least three of the infrared signals fromprotonated 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 , and part of a band at 1710 cm , 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 Ca/H exchange.Wealso obtainedevidence for a pH-dependent conformational change inb-sheet or turn structures of theATPase.With MCCE on the E2P analog E2(TG1MgF4 2 ), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca ligands are hydrogen bonded. The carbonyl groups of Glu, Asp, andGlu 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 Asp is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.