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• W2010264522 abstract "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. 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. The sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; Ca2+-ATPase, Ca2-transporting ATPase (EC 3.6.1.38); caged ATP,P 3-1-(2-nitro)phenylethyladenosine 5′-triphosphate; Ca2 E 1, ATPase with two Ca2+ ions bound to the cytoplasmic high affinity sites; Ca2 E 1-P, ADP-sensitive phosphoenzyme; DM-nitrophen, 1-(2-nitro-4,5-dimethoxy-phenyl)-N,N,N′,N′-tetrakis[(oxycarbonyl) methyl]-1,2-ethanediamine; E, Ca2+ free form(s) of the ATPase; E 2-P, ADP-insensitive phosphoenzyme; FTIR, Fourier transform infrared; MOPS, 3-(N-morpholino)propanesulfonic acid. Ca2+-ATPase couples active Ca2+ transport to the hydrolysis of ATP. A simplified form of its reaction cycle is shown in Fig.1. Ca2+ is bound from the cytoplasmic side of the membrane to high affinity binding sites of the ATPase (left-hand step in Fig. 1); ATP then phosphorylates the ATPase (upper step in Fig. 1), which occludes the bound Ca2+ ions in the protein. Subsequently, the phosphoenzyme converts from the ADP-sensitive to the ADP-insensitive form, and Ca2+ is released into the SR lumen (right-hand step in Fig. 1). Hydrolytic cleavage of the phosphoenzyme completes the reaction cycle (bottom step in Fig. 1). For reviews see Refs. 1Andersen J.P. Biochim. Biophys. Acta. 1989; 988: 47-72Crossref PubMed Scopus (99) Google Scholar, 2Mintz E. Guillain F. Biosci. Rep. 1995; 15: 377-385Crossref PubMed Scopus (7) Google Scholar, 3Inesi G. Chen L. Sumbilla C. Lewis D. Kirtley M.E. Biosci. Rep. 1995; 15: 327-339Crossref PubMed Scopus (20) Google Scholar, 4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar. Effector molecule induced infrared difference spectroscopy has been used by several groups to investigate the reaction cycle of the Ca2+-ATPase (5Barth A. Mäntele W. Kreutz W. Biochim. Biophys. Acta. 1991; 1057: 115-123Crossref PubMed Scopus (68) Google Scholar, 6Barth A. Mäntele W. Kreutz W. FEBS Lett. 1990; 277: 147-150Crossref PubMed Scopus (63) Google Scholar, 7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar, 9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Troullier A. Gerwert K. Dupont Y. Biophys. J. 1996; 71: 2970-2983Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 11Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1991; 1069: 209-217Crossref PubMed Scopus (39) Google Scholar, 12Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1992; 1104: 207-214Crossref PubMed Scopus (24) Google Scholar). They disagree as to whether the structural changes induced by Ca2+ binding to the unphosphorylated ATPase are reversed upon ATPase phosphorylation Ca2 E 1 → Ca2 E 1-P (or E 1∼P) (11Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1991; 1069: 209-217Crossref PubMed Scopus (39) Google Scholar, 12Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1992; 1104: 207-214Crossref PubMed Scopus (24) Google Scholar, 13Martonosi A.N. Biochim. Biophys. Acta. 1996; 1275: 111-117Crossref PubMed Scopus (34) Google Scholar, 14Martonosi A.N. Biosci. Rep. 1995; 15: 263-281Crossref PubMed Scopus (39) Google Scholar) or in the subsequent step of phosphoenzyme conversion and Ca2+ release Ca2 E 1-P →E 2-P (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar). We have demonstrated recently that the infrared absorbance changes of these two reaction steps can be separated temporally using time-resolved FTIR spectroscopy (9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). With the high quality FTIR spectra now obtained in real time, we can unambiguously show that it is the phosphoenzyme conversion and Ca2+ release step that reverses most of the structural changes induced by Ca2+ binding to the unphosphorylated ATPase. The resulting implications for the Ca2+ transport mechanism will be discussed. Samples for time-resolved infrared spectroscopy of the Ca2 E 1-P →E 2-P reaction were prepared as described previously (9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) by removal of free water from an SR suspension in a stream of nitrogen. Samples were immediately rehydrated with 20% Me2SO in H2O or 2H2O. This method resulted in active ATPase samples (5Barth A. Mäntele W. Kreutz W. Biochim. Biophys. Acta. 1991; 1057: 115-123Crossref PubMed Scopus (68) Google Scholar). Approximate concentrations were 0.7 mm ATPase, 300 mmimidazole, pH 7.0, 1 mm CaCl2, 20 mm glutathione, 20 mm caged ATP, 0.5 mg/ml A23187, 2 mg/ml adenylate kinase, 20% Me2SO in approximately 1 μl of sample volume. Approximately 2–3 mm ATP were released per flash. Time-resolved FTIR measurements of the Ca2 E 1-P →E 2-P reaction were performed with a modified Bruker IFS 66 spectrometer as described previously (9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Difference spectra for the reaction were obtained by subtracting a spectrum recorded 3.3–11 (H2O) or 11–19 s (2H2O) after photolysis of caged ATP from a spectrum recorded between 88 and 146 s (H2O and2H2O) and were normalized as described (9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Spectra were recorded at 1 °C. Difference spectra of Ca2+ release from the unphosphorylated ATPase were obtained from experiments originally monitoring Ca2+ binding at 25 °C at pH 7.0 after the release of Ca2+ from DM-nitrophen (8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar) but were processed in a different way for a better comparison with the conversion spectra; the spectrum after Ca2+ release from DM-nitrophen was subtracted from the spectrum beforeCa2+ release. These spectra then represent the absorbance of the Ca2+-free state E minus the absorbance of Ca2 E 1 and thus the Ca2+release reaction from the unphosphorylated ATPase Ca2 E 1 → E. Infrared bands due to the photolysis reaction were subtracted as described (8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar). Difference spectra were normalized to equal protein content by normalizing spectra measured in H2O to an amide II absorbance of 0.26 (difference in absorbance between 1546 and 1492 cm−1) and spectra measured in 2H2O to an amide I absorbance of 0.47 (difference in absorbance between 1706 and 1648 cm−1). Fig. 2 shows infrared difference spectra of Ca2+ release from the unphosphorylated (Ca2 E 1 → E,dotted lines) and the phosphorylated ATPase (Ca2 E 1-P →E 2-P, solid lines) in H2O (Fig. 2 A) and 2H2O (Fig.2 B). Negative bands are characteristic for the states Ca2 E 1 and Ca2 E 1-P, positive bands for the Ca2+ free states E and E 2-P. For Ca2+ release from the phosphoenzyme we note that the spectra show the overall transition from Ca2 E 1-P to E 2-P. The term Ca2+ release therefore refers to the overall Ca2+ transfer from Ca2 E 1-P to the SR lumen. Putative intermediates in this reaction, such as a Ca2+ form of the ADP insensitive phosphoenzyme Ca2 E 2-P were not detected in our former experiments and if they exist are only short-lived intermediates (9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The spectral range shown in Fig. 2 covers the absorption region of the C=O mode of protonated carboxyl groups (1800–1700 cm−1), the νas COO− mode of unprotonated carboxyl groups (1610–1540 cm−1 in water in the presence of counterions (15Tackett J.E. Appl. Spectrosc. 1989; 43: 483-489Crossref Scopus (230) Google Scholar), and the amide I (1700–1610 cm−1) and the amide II (1580–1520 cm−1) mode of the polypeptide backbone. Other amino acid side chains may contribute to the signals below 1700 cm−1. Thus, the difference spectra are expected to reveal backbone secondary structure changes as well as perturbations of the putative Ca2+ ligating carboxyl groups (4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar). Their vibrational frequencies are sensitive to the mode by which carboxylates bind ions (15Tackett J.E. Appl. Spectrosc. 1989; 43: 483-489Crossref Scopus (230) Google Scholar, 16Deacon G.B. Phillips R.J. Coord. Chem. Rev. 1980; 33: 227-250Crossref Scopus (4841) Google Scholar, 17Nara M. Tasumi M. Tanokura M. Hiraoki T. Yazawa M. Tsutsumi A. FEBS Lett. 1994; 349: 84-88Crossref PubMed Scopus (94) Google Scholar) (i.e. unidentate, bidentate, and bridging), and their extinction coefficient is relatively high (18Venyaminov S.Y. Kalnin N.N. Biopolymers. 1990; 30: 1243-1257Crossref PubMed Scopus (603) Google Scholar, 19Chirgadze Y.N. Fedorov O.V. Trushina N.P. Biopolymers. 1975; 14: 679-694Crossref PubMed Scopus (475) Google Scholar), making them ideal reporter groups for events at the Ca2+ binding sites. Therefore, band shifts upon Ca2+ release are expected in the region of the νas COO− vibration (around 1570 cm−1). In addition, protonated carboxyl groups absorb in the 1700–1800 cm−1 region without interference by bands of other groups, which makes the assignment straightforward. Signals in the difference spectra of ATPase partial reactions have been tentatively assigned to Ca2+ release from carboxylate groups (at 1570/1554 cm−1) and protonation of at least two carboxylate groups (at 1758 and 1710 cm−1) (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 10Troullier A. Gerwert K. Dupont Y. Biophys. J. 1996; 71: 2970-2983Abstract Full Text PDF PubMed Scopus (48) Google Scholar). As seen in Fig. 2, spectra of Ca2+ release are remarkably similar irrespective of whether or not the ATPase is phosphorylated (compare solid and dotted lines in Fig. 2). However, some differences are observed between the two types of Ca2+ release spectra, which we discuss first before turning to the implications of the similarities. Differences between the Ca2 E 1-P →E 2-P and the Ca2 E 1 → E spectra are expected because release from the unphosphorylated ATPase is to the cytoplasm, whereas release from the phosphoenzyme is toward the SR lumen. Furthermore, the Ca2+ binding sites of Ca2 E 1 and Ca2 E 1-P are different in that the binding site of Ca2 E 1 is accessible from the cytoplasm, whereas the bound Ca2+ in Ca2 E 1-P is occluded and the affinity for Ca2+ may be different in the two enzyme states. Totally unrelated spectra may be expected if Ca2+ release proceeds from binding sites that are different or different in structure in Ca2 E 1 and Ca2 E 1-P, i.e. if ATPase phosphorylation moves the Ca2+ ions from one set of binding sites to another or if it changes the structure. However, there is only one major difference between the two types of release spectra, found between 1650 and 1620 cm−1, and some subtle differences at 1689 cm−1 (H2O), 1580 cm−1(2H2O) and 1555 cm−1(H2O and 2H2O). They point to structural differences between E and E 2-P as well as between Ca2 E 1 and Ca2 E 1-P, which are associated with the Ca2+ release reactions. In addition, the different conditions under which the two types of experiment were performed may also contribute to the differences: (i) A regulatory ATP molecule is bound to the two forms of the phosphoenzyme (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which are monitored after ATP release but not to the unphosphorylated ATPase, because there is no ATP present in these experiments. (ii) Experiments with the phosphorylated ATPase were done in the presence of Me2SO, A23187, and adenylate kinase, none of which were present in the experiments with unphosphorylated ATPase. The buffer used in the latter experiments was MOPS containing KCl at 25 °C, whereas imidazole without KCl at 1 °C was used in the former. Given the number of factors that could result in differences in the Ca2+ release spectra, the overall similarity of the spectra is striking. Nearly all positive and negative bands are found at approximately the same wavenumber in both types of Ca2+release spectra, and this holds for H2O and2H2O. As each of the bands corresponds to a particular change in structure and interaction, we conclude that parallel secondary structure changes and parallel alterations of amino acid side chain interactions take place in the two Ca2+release reactions Ca2 E 1-P →E 2-P and Ca2 E 1 → E. To put it differently, during enzyme turnover most of the structural changes induced by Ca2+ binding to the unphosphorylated ATPase are reversed upon phosphoenzyme conversion and Ca2+ release to the SR lumen, thus confirming our previous interpretation (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar). Buchet, Martonosi, and co-workers have reached a different conclusion (11Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1991; 1069: 209-217Crossref PubMed Scopus (39) Google Scholar, 12Buchet R. Jona I. Martonosi A. Biochim. Biophys. Acta. 1992; 1104: 207-214Crossref PubMed Scopus (24) Google Scholar, 13Martonosi A.N. Biochim. Biophys. Acta. 1996; 1275: 111-117Crossref PubMed Scopus (34) Google Scholar, 14Martonosi A.N. Biosci. Rep. 1995; 15: 263-281Crossref PubMed Scopus (39) Google Scholar) by focusing attention on a positive band at 1650 cm−1 and its negative side bands in ATP-induced difference spectra. These spectra were assigned to the Ca2 E 1 → Ca2 E 1-P transition, which is supported by the lack of the E 2-P marker bands at 1750, 1616 (shoulder), and 1552 cm−1 (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar). Buchet, Martonosi, and co-workers suggest that the bands near 1650 cm−1 in the Ca2 E 1 → Ca2 E 1-P spectra represent the reversal of structural changes induced by Ca2+ binding E → Ca2 E 1. We have shown (7Barth A. Kreutz W. Mäntele W. Biochim. Biophys. Acta. 1994; 1194: 75-91Crossref PubMed Scopus (42) Google Scholar, 9Barth A. von Germar F. Kreutz W. Mäntele W. J. Biol. Chem. 1996; 271: 30637-30646Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) that the ATP-induced 1650 cm−1 band is already present upon nucleotide binding and increases further in intensity upon phosphorylation as well as upon phosphoenzyme conversion and Ca2+ release. It is tempting to speculate that this band represents a structural transition proceeding stepwise in one direction in these consecutive reactions. Therefore, it seems difficult to assign the reversal of Ca2+ induced changes observed near 1650 cm−1 to only one of these steps. On the other hand, the intensities at 1650 cm−1 in Fig. 2 approximately match, thus arguing in favor of a reversal of the associated conformational changes in the conversion and Ca2+ release step, in line with most other changes as discussed above. The similarity of the spectra of Ca2+ release from the phosphorylated and the unphosphorylated ATPase sheds some light on the Ca2+ binding sites of the different enzyme states. Nearly all of the negative bands and minima in Fig. 2, which are characteristic for the Ca2+ loaded forms Ca2 E 1 and Ca2 E 1-P, are found in both Ca2+ release spectra. Therefore, characteristic structural features of the high affinity Ca2+ binding sites of Ca2 E 1 are also present in Ca2 E 1-P, comprising elements of secondary structure and aspects of the Ca2+ binding mode (see above). This suggests that after phosphorylation Ca2+remains bound to the same sites in Ca2 E 1-P as it was in Ca2 E 1, in agreement with site-directed mutagenesis studies (4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar), but in contrast to the model of Jencks and co-workers (20Jencks W.P. Yang T. Peisach D. Myung J. Biochemistry. 1993; 32: 7030-7034Crossref PubMed Scopus (37) Google Scholar). Following the same line of argument, structural features of the free Ca2+ binding sites of E and E 2-P are very similar. In particular, in both Ca2+ release reactions signals of at least two protonated carboxyl groups are observed (bands at 1758 and 1710 cm−1). In principle, these signals may originate from the protonation of carboxyl groups or from a change in the environment of already protonated carboxyl groups. The latter seems to be unlikely, because it would lead to a shift in the vibrational frequency and therefore to negative and positive bands in the 1700–1800 cm−1 region of the difference spectrum, of which the negative bands are not observed. Therefore, we think that at least two carboxyl groups of E and E 2-P become protonated in both Ca2+ release reactions. They are first discussed for the unphosphorylated ATPase. Ca2+ binding to the unphosphorylated ATPase E→ Ca2 E 1 is accompanied by H+ release, and the protonated residues have been associated with the Ca2+ binding sites and proton countertransport (3Inesi G. Chen L. Sumbilla C. Lewis D. Kirtley M.E. Biosci. Rep. 1995; 15: 327-339Crossref PubMed Scopus (20) Google Scholar, 21Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10961-10968Abstract Full Text PDF PubMed Google Scholar, 22Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar, 23Levy D. Seigneuret M. Bluzat A. Rigaud J.-L. J. Biol. Chem. 1990; 265: 19524-19534Abstract Full Text PDF PubMed Google Scholar, 24Inesi G. Hill T.L. Biophys. J. 1983; 44: 271-280Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 25Yu X. Hao L. Inesi G. J. Biol. Chem. 1994; 269: 16656-16661Abstract Full Text PDF PubMed Google Scholar, 26Henderson I.M.J. Khan Y.M. East J.M. Lee A.G. Biochem. J. 1994; 297: 615-624Crossref PubMed Scopus (37) Google Scholar, 27Martin R.B. FEBS Lett. 1992; 308: 59-61Crossref PubMed Scopus (8) Google Scholar) (for a contrasting view see Ref. 28Da Costa A.G. Madeira V.M.C. Biochim. Biophys. Acta. 1994; 1189: 181-188Crossref PubMed Scopus (11) Google Scholar). They are probably direct Ca2+ ligands. At pH 7.0 and 25 °C, the conditions for recording the Ca2 E 1 → E spectra, there seems to be either an equilibrium of species holding one or three protons at the Ca2+ binding sites (22Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar) or the predominant species holds two protons (27Martin R.B. FEBS Lett. 1992; 308: 59-61Crossref PubMed Scopus (8) Google Scholar). Proton ejection during enzyme turnover has been observed at 25 °C and pH 6–8 (23Levy D. Seigneuret M. Bluzat A. Rigaud J.-L. J. Biol. Chem. 1990; 265: 19524-19534Abstract Full Text PDF PubMed Google Scholar, 25Yu X. Hao L. Inesi G. J. Biol. Chem. 1994; 269: 16656-16661Abstract Full Text PDF PubMed Google Scholar). It has been proposed (29de Meis L. Vianna A. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (541) Google Scholar) that the Ca2+ binding sites become accessible for high affinity binding from the cytoplasm only after a conformational change of the Ca2+ free enzyme, which switches the accessibility from the lumen to the cytoplasm. However, several partial reactions involving the cytoplasmic high affinity sites are unaffected by 20–40 mm lumenal Ca2+ (26Henderson I.M.J. Khan Y.M. East J.M. Lee A.G. Biochem. J. 1994; 297: 615-624Crossref PubMed Scopus (37) Google Scholar, 30Myung J. Jencks W.P. FEBS Lett. 1991; 278: 35-37Crossref PubMed Scopus (13) Google Scholar, 31Myung J. Jencks W.P. Biochemistry. 1994; 33: 8775-8785Crossref PubMed Scopus (24) Google Scholar), indicating that such a transition may not occur. The Ca2+/H+ exchange at the high affinity sites for Ca2+ is expected to be detected in the infrared difference spectrum, which was recorded under similar conditions (see above). As the most straightforward interpretation of the protonation signals of carboxyl groups seen in the Ca2 E 1 → E spectra, we therefore suggest that these carboxyl groups are those that participate in the Ca2+/H+ exchange and tentatively assign them to residues within the high affinity binding sites. The high affinity binding sites are thought to be located in the transmembrane region of the Ca2+-ATPase (4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar) and to be in a hydrophobic environment (32Pick U. Racker E. Biochemistry. 1979; 18: 108-113Crossref PubMed Scopus (106) Google Scholar). Consistent with this we find that one of the protonated carboxyl groups has a vibrational frequency characteristic of a nonhydrogen-bonded carbonyl group (1758 cm−1) (8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar,10Troullier A. Gerwert K. Dupont Y. Biophys. J. 1996; 71: 2970-2983Abstract Full Text PDF PubMed Scopus (48) Google Scholar). The frequency of the second protonated carboxyl group (1710 cm−1) indicates that it is hydrogen-bonded (8Georg H. Barth A. Kreutz W. Siebert F. Mäntele W. Biochim. Biophys. Acta. 1994; 1188: 139-150Crossref PubMed Scopus (29) Google Scholar, 10Troullier A. Gerwert K. Dupont Y. Biophys. J. 1996; 71: 2970-2983Abstract Full Text PDF PubMed Scopus (48) Google Scholar). This, however, does not necessarily mean that it is in a hydrophilic environment, because the hydrogen bond donor may be a protein residue in an otherwise hydrophobic protein environment. Ca2+ uptake is paralleled by lumenal alkalinization, which has been attributed to proton binding to E2-P (33Yamaguchi M. Kanazawa T. J. Biol. Chem. 1985; 260: 4896-4900Abstract Full Text PDF PubMed Google Scholar, 34Steiner M. Bauer H. Krassnigg F. Schill W.-B. Adam H. Clin. Chem. Acta. 1988; 177: 107-114Crossref PubMed Scopus (5) Google Scholar, 35Polvani C. Sachs G. Blostein R. J. Biol. Chem. 1989; 264: 17854-17859Abstract Full Text PDF PubMed Google Scholar, 36Yu X. Carrol S. Rigaud J.-L. Inesi G. Biophys. J. 1993; 64: 1232-1242Abstract Full Text PDF PubMed Scopus (144) Google Scholar) (for a different view see Ref. 37Madeira V.M.C. Arch. Biochem. Biophys. 1979; 193: 22-27Crossref PubMed Scopus (14) Google Scholar). Because this has been observed at 4 °C, pH 6.0–6.4 (33Yamaguchi M. Kanazawa T. J. Biol. Chem. 1985; 260: 4896-4900Abstract Full Text PDF PubMed Google Scholar, 38Yamaguchi M. Kanazawa T. J. Biol. Chem. 1984; 259: 9526-9531Abstract Full Text PDF PubMed Google Scholar) and at 17–25 °C, pH 7.2 (23Levy D. Seigneuret M. Bluzat A. Rigaud J.-L. J. Biol. Chem. 1990; 265: 19524-19534Abstract Full Text PDF PubMed Google Scholar, 36Yu X. Carrol S. Rigaud J.-L. Inesi G. Biophys. J. 1993; 64: 1232-1242Abstract Full Text PDF PubMed Scopus (144) Google Scholar), conditions not far removed from the 1 °C, pH 7.0 used to record the Ca2 E 1-P →E 2-P spectra, we suggest that the protons responsible for the protonation signals of E 2-P in these spectra (Fig. 2) are those that cause lumenal alkalinization and therefore originate from the lumen. Protonation of protein residues of E 2-P has been found to increase the stability of the phosphoenzyme (39Inesi G. Lewis D. Murphy A.J. J. Biol. Chem. 1984; 259: 996-1003Abstract Full Text PDF PubMed Google Scholar) and to ensure the low affinity for Ca2+ (40de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4253-4261Crossref Scopus (181) Google Scholar, 41de Meis L. Inesi G. J. Biol. Chem. 1982; 257: 1289-1294Abstract Full Text PDF PubMed Google Scholar). It may well be that the protonated carboxyl groups that we detect in the conversion spectra mediate both effects. The C=O bands of the two (or more) protonated carboxyl groups of E and E 2-P are observed at the same characteristic wavenumbers in the difference spectra, clearly indicating that each group experiences a hydrogen bonding strength that is the same in E and E 2-P. This coincidence strongly suggests that the same amino acid residues become protonated in the two enzyme states. For E, they have been associated with the high affinity binding sites (see above). For E 2-P, we therefore propose that residues that form the high affinity sites of Ca2 E 1 become protonated when Ca2+ is released from the phosphoenzyme Ca2 E 1-P →E 2-P. This requires that lumenal protons have access to these residues during this reaction via a proton conducting network or a channel. In the latter case Ca2+ would probably also have access to these residues, which then would be involved in high as well as low affinity binding of Ca2+. The above line of argument concludes from the identical positions of the two bands in both Ca2+ release reactions that the same two carboxyl groups are involved in proton binding to E and E 2-P and locates them in the high affinity Ca2+ binding sites of Ca2 E 1. They are thought to be those Ca2+ ligands that are involved in the H+/Ca2+ exchange. Pursuing this, one might look for possible candidates that meet the criterion of participating in both, Ca2+ and H+ binding. Site-directed mutagenesis studies have identified several carboxyl groups that are thought to be Ca2+ ligands of the high affinity binding site: Glu309, Glu771, Asp800, and perhaps Glu908 (4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar). Of these, only Glu309 and Glu771 seem to be crucial for phosphoenzyme hydrolysis E 2-P → E, and this has been explained with their possible role in proton binding to E 2-P and proton countertransport (4Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar). Because these residues seem to bind protons in the E 2-P state and Ca2+ in Ca2 E 1and Ca2 E 1-P, they are likely candidates for the carboxyl groups that give rise to the protonation signals in the Ca2 E 1 →E and Ca2 E 1-P →E 2-P spectra. As a working hypothesis, we therefore tentatively assign the two protonation signals in both Ca2+ release spectra to Glu309 and Glu771. A definite assignment would require studies with site-directed mutants, which are presently not available in the quantities needed for infrared experiments. The simplest model consistent with our data would assume only one pair of Ca2+ binding sites on the ATPase, serving both Ca2+ translocation and H+ countertransport. However, more complicated models with two pairs of binding sites cannot be ruled out (reviewed in Refs. 2Mintz E. Guillain F. Biosci. Rep. 1995; 15: 377-385Crossref PubMed Scopus (7) Google Scholar and 13Martonosi A.N. Biochim. Biophys. Acta. 1996; 1275: 111-117Crossref PubMed Scopus (34) Google Scholar). Our results then require that residues of the high affinity binding sites have access to lumenal protons at some stage of the Ca2+ release reaction Ca2 E 1-P →E 2-P." @default.
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