The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase

The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.     

The charge transfer band in horseradish peroxidase correlates with heme in-plane distortions induced by calcium removal

Horseradish peroxidase C (HRPC) is a class III peroxidase whose structure is stabilized by the presence of two endogenous calcium atoms. Calcium removal has been shown to decrease the enzymatic activity of the enzyme. The spin state of the iron, a mixture of high spin (HS) and mixed quantum spin state (QS) consisting of intermediate spin (IS) 3/2 + (HS) 5/2, is also significantly affected by calcium removal, going from a predominant QS component to a predominant HS component upon removal of one calcium. Removal of both calcium ions, however, results in the appearance of a significant LS contribution, easily monitored in the charge transfer (CT) band region by low-T absorption. Normal structural decomposition (NSD) calculations of the in-plane (ip) modes of the heme extracted from HRPC native and Ca(2+)-depleted models show that removal of the proximal calcium is associated with perturbed E(u) and increased A(1g) ip distortions of the heme. The effect of complete or distal calcium removal on the heme also results in increased A(1g) ip distortions, but in significantly decreased E(u) distortions. The overall effect is to decrease the nonplanarity of the heme: the total ip distortion of the native HRPC heme is 0.200 and 0.134 A for the Ca(2+)-depleted species. Our NSD results corroborate the role proposed for the protein matrix, namely to fine-tune the active site by inducing subtle changes in heme planarity and spin state of the iron.     

Role of calcium in metalloenzymes: effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and M?ssbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and M?ssbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.     

Resonance Raman spectroscopy study of change of iron spin state in horseradish peroxidase C induced by removal of calcium

Resonance Raman spectroscopy is used to probe the effect of calcium depletion on the heme group of horseradish peroxidase C at pH 8. Polarized Raman spectra are recorded with an argon ion laser at eight different wavelengths to provide a sound database for a reliable spectral decomposition. Upon calcium depletion, the spectrum is indicative of a predominantly pentacoordinated high spin state of the heme iron coexisting with small fractions of hexacoordinated high and low spin states. The dominant quantum mixed spin state of native ferric horseradish peroxidase, which is characteristic for class III peroxidases, is not detectable in the spectrum of the enzyme with partial distal Ca(2+) depletion. The quenching of the quantum mixed spin state and the predominance of the pentacoordinated high spin state are likely to arise from distortions induced by distal calcium depletion, which translates into a weaker Fe-N(epsilon)(His) bond and a more tilted imidazole. A correlation is proposed between the lower enzyme activity and the elimination of the pentacoordinated quantum mixed state upon Ca(2+) depletion.     

The structure of horseradish peroxidase C characterized as a molten globule state after Ca(2+) depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.     

Chelator-induced disappearance of carboxylate stretching vibrational modes in S2/S1 FTIR spectrum in oxygen-evolving complex of photosystem II.

Fourier transform infrared (FTIR) spectroscopy has been applied toward studies of photosynthetic oxygen evolution, especially on the effects of Ca(2+) depletion and chelating agents using S(2)/S(1) FTIR difference spectrum in the mid-IR region. Ca(2+) depletion showed little influences on the symmetric (1365/1404 cm(-1)) and the asymmetric (1587/1562 cm(-1)) stretching bands of a carboxylate, which are typical of the S(2)/S(1) vibrational features induced by the oxidation of the Mn-cluster; however, minor changes were observed in the amide regions. Addition of a chelating agent (EDTA or EGTA) to the Ca(2+)-depleted membranes resulted in the disappearance of the carboxylate bands concurrent with large modifications of the amide bands with an apparent K(d) value of approximately 0.49 mM (for EDTA). The carboxylate bands and the greater part of the amide bands were restored by the replenishment of CaCl(2), and the chelators did not affect the spectrum in the nondepleted control membranes, indicating that the effects of the chelator are reversible and manifest only in the cases in which the Ca(2+) site is unoccupied by Ca(2+). Ca(2+)-depleted membranes showed the normal S(2)Q(A)(-) thermoluminescence band, and further addition of EDTA did not show any effects on the peak temperature and peak intensity. Moreover, the Ca(2+)-depleted membranes in the presence of EDTA exhibited the S(2) multiline EPR signal with nearly the normal hyperfine splittings. These results demonstrated that the Mn-cluster is oxidized to the S(2) state with normal redox and magnetic properties in the presence of the chelator despite the loss of the carboxylate bands in the FTIR spectra. The results are interpreted as indicating that the chelator interacts with the Mn-cluster as a replacement of the native carboxylate ligand. This prevents the structural changes of the Mn-cluster and protein backbone which are induced upon the oxidation of the Mn-cluster up to the S(2) state, but preserve the redox and magnetic properties of the S(2) state Mn-cluster. The roles of Ca(2+) in the photosynthetic oxygen evolution are also discussed.     

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca2+ depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S(1)-to-S(2) transition was obtained with Ca(2+)-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca(2+) to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S(2)/S(1) FTIR spectrum upon Ca(2+) depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca(2+) depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K(+) at the Ca(2+) site. In the present study, the preparation of the Ca(2+)-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K(+). The obtained S(2)/S(1) spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm(-1) and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K(+). This result indicates that the drastic FTIR changes are a pure effect of Ca(2+) depletion, and provides solid evidence for the general view that Ca(2+) is strongly coupled with the Mn cluster.     

Mechanism of versatile peroxidase inactivation by Ca(2+) depletion

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.     

Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase

A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.     

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca2+ removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.     

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.     

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.     

Capacitative calcium entry in the nervous system

Capacitative calcium entry is a process whereby the depletion of Ca(2+) from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca(2+) channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca(2+) store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca(2+) stores when their Ca(2+) levels become depleted, or a more direct protein-protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca(2+) influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.     

Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways

Human polymorphonuclear neutrophil (PMN) responses to G protein-coupled chemoattractants are highly dependent upon store-operated Ca(2+) entry (SOCE). Recent research suggests that SOCE currents can be mediated by a variety of related channel proteins of the transient receptor potential superfamily. SOCE has been regarded as a specific response to depletion of cell calcium stores. We hypothesized that net SOCE might reflect the contributions of more than one calcium entry pathway. SOCE was studied in normal human PMN using Ca(2+) and Sr(2+) ions. We found that PMN SOCE depends on at least two divalent cation influx pathways. One of these was nonspecific and Sr(2+) permeable; the other was Ca(2+) specific. The two pathways show different degrees of dependence on store depletion by thapsigargin and ionomycin, and differential sensitivity to inhibition by 2-aminoethyoxydiphenyl borane and gadolinium. The inflammatory G protein-coupled chemoattractants fMLP, platelet-activating factor, and IL-8 elicit unique patterns of Sr(2+) and Ca(2+) influx channel activation, and SOCE responses to these agonists displayed differing degrees of linkage to prior Ca(2+) store depletion. The mechanisms of PMN SOCE responses to G protein-coupled chemoattractants are physiologically diverse. They appear to reflect Ca(2+) transport through a variety of channels that are independently regulated to varying degrees by store depletion and by G protein-coupled receptor activation.     

The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca(2+) in proximity to the two hemes [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. This Ca(2+) did not readily dissociate; however, after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca(2+)-dependent changes in heme spin state and conformation. Addition of H(2)O(2) to the Ca(2+)-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca(2+) to the Ca(2+)-depleted MauG, full TTQ biosynthesis activity and reactivity toward H(2)O(2) were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca(2+) binding to Ca(2+)-depleted MauG indicated a two-step mechanism. Ca(2+) initially reversibly binds to Ca(2+)-depleted MauG (K(d) = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10(-3) s(-1)) but highly favorable (K(eq) = 4.2) conformational change, yielding an equilibrium dissociation constant K(d,eq) value of 5.3 μM. The circular dichroism spectra of native and Ca(2+)-depleted MauG were essentially the same, consistent with Ca(2+)-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Calcium-dependent heme structure in the reduced forms of the bacterial cytochrome c peroxidase from Paracoccus pantotrophus

This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca(2+), the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca(2+), the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca(2+) binding are indicated by spectral differences in the structure-sensitive marker lines, the out-of-plane low-frequency macrocyclic modes, and the vibrations associated with the heme substituents of that heme. The Ca(2+)-dependent appearance of a strong gamma 15 saddling-symmetry mode for the mixed-valence form is consistent with a strong saddling deformation in the active peroxidatic heme, a feature seen in the Raman spectra of other peroxidases. For the fully reduced form in the presence of Ca(2+), the resonance Raman spectra show that the peroxidatic heme remains high-spin.     

The role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of unstimulated rat lymphocytes

The mechanism responsible for the increase in cytosolic free Ca2+ concentration ([Ca2+]i) during mitogenic stimulation of lymphocytes has been widely investigated. By contrast, little is known about the processes underlying Ca2+i homeostasis in resting (unstimulated) cells. It has been suggested that [Ca2+]i is an important determinant of the rate of Ca2+ influx following mitogenic activation. Using rat thymic lymphocytes, we investigated whether the resting influx pathway is similarly controlled by [Ca2+]i. Otherwise untreated cells were Ca(2+)-depleted by loading with Ca2+ chelators while suspended in Ca(2+)-free solution. Ca2+ depletion induced an 8-fold increase in the rate of unidirectional Ca2+ uptake. The depletion-activated flux was voltage-sensitive and was blocked by La3+ and by compound SK&F 96365, a receptor-operated Ca2+ channel blocker. Upon reintroduction to Ca(2+)-containing solution, the increased influx brought about a rapid recovery of [Ca2+]i. Detailed analysis of the magnitude of the 45Ca2+ flux during this recovery indicated that [Ca2+]i is not the primary determinant of the plasmalemmal Ca2+ permeability. Instead, depletion of an internal thapsigargin-sensitive store correlates with and appears to be responsible for the increased permeability of the plasma membrane. Accordingly, the Ca2+ fluxes induced by intracellular Ca2+ depletion and by thapsigargin were pharmacologically indistinguishable. Mitogenic lectins also released Ca2+ from a thapsigargin-sensitive store and activated a plasmalemmal Ca2+ permeability displaying identical pharmacology. The data support the existence of a coupling process whereby the degree of filling of an internal Ca2+ store dictates the Ca2+ permeability of the plasma membrane. This coupling mechanism is important not only in mediating the effects of mitogens and other agonists, as suggested before, but seemingly also in the control of resting Ca2+i homeostasis in unstimulated cells.