Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Characterization of the Ca2+-binding sites of annexin II tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca(2+)-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca(2+)-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca(2+), the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca(2+)-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca(2+)-dependent binding to F-actin, the binding of the TCM AIIt was Ca(2+)-independent. These results suggest that the type II or type III Ca(2+)-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca(2+), the type II and type III Ca(2+)-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca(2+) binding to these sites, or the inactivation of these Ca(2+)-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca(2+) for binding to heparin, we also propose that novel Ca(2+)-binding sites regulate this binding event.     

Interaction of alkaline metal ions with Ca(2+)-binding sites of Ca(2+)-ATPase of sarcoplasmic reticulum: 23Na-NMR studies.

The analysis of the 23Na-NMR signal shape variations in the presence of vesicles of light sarcoplasmic reticulum (SR) shows the existence of sodium sites on the membranes with Kd values of about 10 mM. Other monovalent cations displace Na+ from SR fragments in a competitive manner according to the row K+ greater than Rb+ greater than Cs+ greater than Li+. Calcium ions also reduce Na+ binding, the Na+ desorption curve being of a two-stage nature, which, as suggested, indicates the existence of two types of Ca(2+)-sensitive Na+ binding sites (I and II). Sites of type I and II are modified by Ca2+ in submicromolar and millimolar concentrations, respectively. Analysis of sodium (calcium) desorption produced by calcium (sodium) allowed us to postulate the competition of these two cations for sites I and identity of these sites to high-affinity Ca(2+)-binding ones on the Ca(2+)-ATPase. Sites I weakly interact with Mg2+ (KappMg approximately 30 mM). Reciprocal effects of sodium and calcium on binding of each other to sites II cannot be described by a simple competition model, which indicates nonhomogeneity of these sites. A portion of sites I (approximately 70%) interacts with Mg2+ (KappMg = 3-4 mM). The pKa value of sites II is nearly 6.0. The number of sites II is three times greater than that of sites I. In addition, sites with intermediate affinity for Ca2+ were found with Kd values of 2-5 microM. These sites were revealed due to the reducing of the sites II affinity for Na+ upon Ca2+ binding to SR membranes. It can thus be concluded that in nonenergized SR there are binding sites for monovalent cations of at least three types: (1) sites I (which also bind Ca2+ at low concentrations), (2) magnesium-sensitive sites II and (3) magnesium-insensitive sites II.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

pH-Dependent structural changes at Ca(2+)-binding sites of coagulation factor IX-binding protein

Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

Ca(2+)-dependent annexin self-association on membrane surfaces

Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1:1 or 1:3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1:0, 1:1, or 1:3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca(2+)-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levels where the protein may bind two membranes as a monomer.     

Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding sites

Ca2+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Ca(2+)-binding sites with a mean dissociation constant (KD) of 0.79 mM were titrated in the absence, and 23 sites with a KD of 0.88 mM in the presence of 3 mM Mg2+. No cooperativity was observed. For Mg2+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg2+/protein with a KD of 2mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca(2+)- and Mg(2+)-binding sites. After correction for proton release, the enthalpy change for Ca2+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg2+) was therefore virtually the sole driving force for Ca2+ binding. Mg2+ binding is slightly more exothermic (-12.6 kJ/protein), but as for Ca2+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg(2+)-saturated calsequestrin was clearly different from that in the Ca(2+)-saturated protein, but that the (Ca2+ + Mg2+)-saturated protein was not distinct from the Ca(2+)-saturated protein. Thus, in addition to the thermodynamic characterization of the Ca2+/calsequestrin interaction, our data indicate that Ca2+ and Mg2+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca(2+)-Mg2+ exchange in vivo.     

Annexins--a new family of Ca(2+)-binding proteins

Annexins, the Ca(2+)- and phospholipid-binding proteins, are able to induce Ca(2+)-dependent aggregation of biomembranes. All the representatives of this family contain four or eight tandem repeats, 60-80 amino acids each. All these repeats include a highly conservative 17-member amino acid consensus sequence (an endonexin fold). The central domain comprises all these repeats and contains, in addition, the site(s) with a binding affinity for Ca2+ and phospholipids. Annexins are devoid of the classical "EF-hand" Ca(2+)-binding domain and can therefore be assigned to a new family of Ca(2+)-binding proteins.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.

Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied. In each mutant, a conserved glutamic acid residue at position 12 in all of the Ca(2+)-binding loops has been mutated in one site. In one series the residue is changed to glutamine; in the second series the change is to lysine. The Ca(2+)-binding properties of these mutants and the wild-type protein under pseudo-physiological conditions are presented. In addition, Ca(2+)-induced changes to the environment of the single tyrosine residue (Tyr-138) have been studied for some of the mutants. Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of these eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Nonuniform distribution of Ca(2+) uptake sites within human neutrophils

The rate at which Ca(2+) returns towards the basal concentration is controlled by the action of Ca(2+) pumps, both on the plasma membrane and on organelles within the cytosol. The distribution of Ca(2+) uptake sites within the cytosol was investigated using rapid confocal imaging (55 ms/frame) of fluo3-loaded human neutrophils. In some zones within the cell, the uptake of Ca(2+) from the cytosol followed a single exponential time course, whereas in others, there was accelerated kinetics after about 3 s. Using the full array of data, to produce a cell-map of Ca(2+) uptake rates a clear nonuniformity of Ca(2+) uptake sites throughout the neutrophil cytosol was observed. The location of the Ca(2+) uptake sites did not correlate with the granules or the main body of the nucleus, but Ca(2+) uptake was highest near the vestigial Golgi/ER, the edges of the nuclear lobes and at the leading cell edge. The possibility exists that the nonuniform distribution of Ca(2+) uptake sites plays a role in restricting Ca(2+) signals with the neutrophil cytosol.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

Near-infrared Yb(3+) vibronic sideband spectroscopy: application to Ca(2+)-binding proteins.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.     

Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.