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

Abstract

Protonation of the Ca(2+) ligands of the SR Ca(2+)-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of C=O bands of the Ca(2+)-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca(2+) release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pK(a) 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 Ca(2+)/H(+) exchange. We also obtained evidence for a pH-dependent conformational change in beta-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(TG+MgF(4)(2-)), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca(2+) ligands are hydrogen bonded. The carbonyl groups of Glu(771), Asp(800), and Glu(908) 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(800) is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.

Conformational changes of recombinant Ca2+–ATPase studied by reaction‐induced infrared difference spectroscopy

“…This is evidence for a very similar phosphate environment in both samples. Recombinant and rabbit SERCA1a also have similar positions for the bands near 1750 and 1710 cm À1 , which indicate protonation of the Ca 2+ ligands [34]. Their similar position in both samples indicates a similar strength of hydrogen bonding to the carbonyl group of the protonated carboxyl ligands.…”

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum

“…Following Ca 2+ release, two luminal H + bind to the transport sites to neutralize the negative charge in the TM domain and stabilize a protonated, low Ca 2+ -affinity intermediate E2-P. 24–27 SERCA is then dephosphorylated 28, 29 to populate a Ca 2+ -free, protonated E2 state. 23, 30, 31 At normal intracellular pH (7.1–7.2), 32, 33 two H + spontaneously translocate from the transport sites to the cytosol, 24, 34, 35 thus completing H + transport from the lumen to the cytosol. H + release to the cytosol induces an E2-to-E1 transition of SERCA required for the next Ca 2+ -pumping cycle.…”

“…It has been suggested that proton shuttling from Glu309 to the cytosol occurs through a transitions from a high-p K a , lumen-facing orientation to a low-p K a , cytosol-facing orientation of the carboxylic moiety. 24, 34 However, microsecond MD simulations have shown that H + shuttling to the cytosol occurs primarily through a water-filled pore connecting the side chain of Glu309 with the cytosol (Figure 2). 40 Helices M1, M2 and M4 form the narrow (0.55 nm in diameter) N-terminal H + pathway.…”

Gas-Phase Interaction of Calcium (Ca2+) with Seleno Derivatives of Uracil

“…17,18 This motivated a growing interest in the study of the interaction of biochemical systems with this metal dication, both from the experimental and the theoretical viewpoints. [19][20][21][22][23][24][25][26][27][28][29][30][31] …”