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.

Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide. They include peroxidases of plant, fungal, and prokaryotic origins that can be divided into three classes based on sequence alignment (1). Additional support for such a classification was gained as the crystal structures of representative enzymes of each class became available. Class I contains bacterial peroxidases and peroxidases from plant mitochondria and chloroplasts, for example most ascorbate peroxidases and cytochrome c peroxidase. Class II contains extracellular fungal peroxidases such as Coprinus cinereus peroxidase (a peroxidase essentially identical to Arthromyces ramosus peroxidase) and lignin-degrading peroxidases. Class III contains secretory plant peroxidases, typified by the classical horseradish peroxidase isoenzyme C (HRPC). 1 The peroxidases of classes II and III share a number of structural elements considered to be of importance for maintaining protein stability and activity. These include calciumbinding sites proximal and distal to the heme and four disulfide bridges (1)(2)(3)(4)(5); the latter was in different locations in class II with respect to class III peroxidases. These features are absent in class I peroxidases. Class III peroxidases also have a loop insertion in the sequence, composed of the DЈ, FЈ, and FЉ helices, not found in the other classes. The relationship between calcium binding and enzyme inactivation has been treated primarily by studies on lignin peroxidase (LIP) (6,7), manganese peroxidase (MNP) (8,9), and HRPC (10,11). Thermal (6) and alkaline (7) inactivation of LIP has been correlated with loss of calcium ions and the formation of an inactive bis-histidyl hexacoordinate low-spin (6-c LS) heme state. In apparent contrast with alkaline inactivation, which results in the loss of both calcium ions, thermal inactivation causes the loss of only the more weakly bound distal calcium ion. Likewise, thermal inactivation of MNP has been correlated with loss of calcium and the formation of an inactive 6-c LS state proposed to result from the dissociation of the distal calcium only (8,9). The x-ray crystal structure of HRPC (4) confirmed previous analyses of calcium content indicating 2 mol of calcium per mol of enzyme. A schematic representation of the locations of the two calcium ions and heme pocket residues is shown in Fig. 1. ApoHRPC has been found not to refold properly in the absence of calcium ions (12), and removal of the bound calcium ions from native HRPC results in a considerable decrease in activity and thermal stability (10,11,13,14). These observations demonstrate the essential role played by the structural calcium ions in HRPC in maintaining a heme pocket architecture conducive to high activity. The present work extends previous spectroscopic studies of Ca 2ϩ -depleted HRPC to obtain further insight into the consequences on the heme environment of removing calcium from the enzyme. On the basis of the present results, the relative importance of the proximal and distal calcium ions in determining the heme pocket structural properties is discussed.

EXPERIMENTAL PROCEDURES
HRPC Ca 2ϩ Ion Depletion and Reconstitution-In a manner similar to the procedure reported by Haschke and Friedhoff (10), the Ca 2ϩ -* This work was supported by the Italian CNR, the Ministero Universitá e Ricerca Scientifica e Tecnologica and Cofin. MURST 97 CFSIB (to G. S.), and European Union Grant BIO4-97-2031 (to G. S). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Reconstitution of the Ca 2ϩ -depleted protein (ϳ30 M) was achieved by incubation for 18 h at 4°C with 30 mM CaCl 2 and a heme solution, prepared fresh before use, of final concentration 50% that of the protein.
After incubation the protein was passed through a Sephadex G-25 column equilibrated and eluted with 50 mM Tris/HCl at pH 7.8. In the preparation of both Ca 2ϩ -depleted and reconstituted HRPC, identical results were obtained using a 23 ϫ 1-cm column of Sephacryl S100 high resolution gel (Amersham Pharmacia Biotech). However, only in the determination of the Ca 2ϩ ion content of the Ca 2ϩ -depleted protein by reconstitution with 45 Ca 2ϩ ions, where a greater resolution is required, was the Sephacryl S100 column used enabling a clearer separation of the protein fraction of the elute from that of free 45 Ca 2ϩ ions (see below).
Determination of Calcium Content-The calcium content of Ca 2ϩdepleted HRPC was determined by reconstitution of the protein in the presence of radioactive 45 Ca 2ϩ ions. The Ca 2ϩ -depleted protein was reconstituted as described above except for the addition of 45 CaCl 2 (30 mM, 223,502 cpm/mol) (Amersham Pharmacia Biotech) to 250 l of protein solution. After the 18-h incubation period the enzyme was loaded onto a Sephacryl S100 column, equilibrated and eluted with 50 mM Tris/HCl, pH 7.8. The protein concentration of eluted fractions (0.8 ml) was determined from the electronic absorption spectrum and the level of radioactivity by liquid scintillation counting.
As a control, the reconstitution experiment in the presence of 45 CaCl 2 was carried out as described above using native rather than Ca 2ϩdepleted HRPC. This enables the possible presence of adventitiously bound or exchangeable Ca 2ϩ ions to be ascertained.
Sample Preparation-Plant HRPC was obtained from Biozyme Laboratories Ltd. as salt-free lyophilized powder (code HRP4B). The preparation has RZ (Reinheitszahl) or purity index values of 3.1 and was used without further purification.
Samples of ferrous enzymes for electronic absorption and resonance Raman spectroscopy were prepared by addition of 2 l of dithionite (20 mg ml Ϫ1 ) to 50 l of deoxygenated peroxidase solution. The CO complexes were prepared by first flushing the ferric protein solution with nitrogen, then flushing with CO (Rivoira, Italy), and reducing the protein as described above. Benzohydroxamic acid (BHA) complexes were prepared by adding aliquots of 0.2 M benzohydroxamic acid (Sigma) in 50 mM Tris/HCl, pH 7.8, to the enzyme samples to a final concentration of 5 mM. The dissociation constant, K d , for BHA binding to Ca 2ϩ -depleted HRPC in a 5 mM EDTA, 50 mM Tris/HCl solution at pH 7.8, 25°C was determined following the procedure of Smith and co-workers (15,16). Imidazole complexes were prepared by adding solid imidazole to the ferric protein solution, to a final concentration of ϳ3 M.
Enzyme concentrations were determined spectrophotometrically using extinction coefficients of 112 and 107 mM Ϫ1 cm Ϫ1 at the Soret maxima for native and Ca 2ϩ -depleted HRPC, respectively (Table I). Extinction coefficients were determined in triplicate by the pyridine hemochrome method (17).
Determination of Enzyme Activity-Peroxidase activity was measured in 60 mM phosphate at pH 7.0, using the substrate ABTS (12). Initial reaction rates were measured by following the increase in absorbance at 405 nm resulting from the formation of the ABTS cation radical product. A final concentration of 0.3 mM ABTS and a saturating concentration of 1 mM H 2 O 2 were used for the assays, which were initiated by addition of enzyme to a final concentration in the range of 0.05-0.5 nM. For each enzyme (native, Ca 2ϩ -depleted, and reconstituted HRPC), the value of V max was determined for three different concentrations, and the values were plotted to determine the resultant slope by regression analysis. The percentage of activities reported in Table I has been obtained by such a measurement of the slope of V max against enzyme concentration for each enzyme, taking as reference the corresponding slope of native HRPC which is assigned a value of 100%. Each value was determined three or more times to obtain the standard deviation.
Electronic Absorption and Resonance Raman Spectroscopy-Electronic absorption spectra were measured with a Cary 5 spectrophotometer using a cuvette of 1-or 10-mm path length. RR spectra were obtained at room temperature with excitation from the 406.7, 530.9, and 568.2 nm lines of a Kr ϩ laser (Coherent, Innova 90/K), 496.5 and 514.5 nm lines of an Ar ϩ ion laser (Coherent, Innova 90/5), and from the 441.6 nm line of a HeCd laser (Liconix). The back-scattered light from a slowly rotating NMR tube was collected and focused into a computercontrolled double monochromator (Jobin-Yvon HG2S) equipped with a cooled photomultiplier (RCA C31034A) and photon counting electronics. To minimize local heating of the protein by the laser beam, the sample was cooled by a gentle flow of N 2 gas passed through liquid N 2 . RR spectra were calibrated to an accuracy of 1 cm Ϫ1 for intense isolated bands, with indene as the standard for the high frequency region and with indene and CCl 4 for the low frequency region. Polarized spectra were obtained by inserting a polaroid analyzer between the sample and the entrance slit of the monochromator. The depolarization ratios of the bands at 314 and 460 cm Ϫ1 of CCl 4 were measured to check the reliability of the polarization measurements. The values obtained, 0.73 and 0.00, compared well with the theoretical values of 0.75 and 0.00, respectively. Peak intensities were determined using a curve-fitting program to simulate experimental spectra with Lorentzian line shapes.

RESULTS
Specific Activity and Calcium Content-The specific activity at pH 7.0 of Ca 2ϩ -depleted HRPC is reported in Table I. The loss of 50% activity compared with native protein noted for the Ca 2ϩ -depleted form is reversed upon reconstitution by incubation with heme and excess CaCl 2 (see "Experimental Procedures"). Reconstitution leads to a rise in the specific activity, achieving 85% that of native HRPC.
When Ca 2ϩ -depleted HRPC was reconstituted with heme and 45 Ca-labeled CaCl 2 radioactivity equivalent to 1.18 g atom of 45 Ca 2ϩ ions per mol of enzyme was found to co-chromato-  graph on gel filtration ( Fig. 2A). This implies that 1 mol of calcium per mol of enzyme is present in the Ca 2ϩ -depleted protein instead of the 2 mol of calcium per mole of enzyme present in native HRPC (4). To determine whether adventitiously bound or exchangeable Ca 2ϩ ions were present in our protein preparations, native HRPC was incubated with heme and 45 CaCl 2 without prior treatment to remove the Ca 2ϩ . Fig.  2B shows that a very small level of radioactivity (equivalent to 0.07 g atom of 45 Ca 2ϩ ions per mol of enzyme) co-chromatographs with the native protein. Although it is not possible to ascertain whether this is the result of adventitiously bound or exchangeable 45 Ca 2ϩ ions, the effective value of radioactivity that co-chromatographs with the reconstituted protein is, consequently, reduced from 1.18 to 1.11 g atom of 45 Ca 2ϩ .
Electronic Absorption and Resonance Raman of Ferric Enzymes-The electronic absorption spectra of Ca 2ϩ -depleted HRPC and native enzyme at pH 7.8 are shown in Fig. 3A. The blue shift of the absorption maxima of the Ca 2ϩ -depleted form compared with those of the native can arise either as a consequence of a different degree of vinyl conjugation with the porphyrin macrocycle or from the presence of a quantum mechanically mixed spin (QS) state of the heme iron (18), which results from the quantum mechanical mixing of high-and intermediate-spin states (S ϭ 5/2 and 3/2, respectively). The extinction coefficient of the Soret maximum of Ca 2ϩ -depleted HRPC is unchanged with respect to that of the native (Table I).
A comparison between the high frequency RR spectra for Soret excitation (406.7 nm) of Ca 2ϩ -depleted and native HRPC at pH 7.8 is presented in Fig. 3B. The spectrum of the native has been reported previously and found to be a mixture of 5-coordinate (5-c) and 6-c heme forms, both of which display anomalously high core size marker band frequencies (19). On the basis of this observation and other data available for HRPC, it has been proposed that resting HRPC at room tem-perature cannot be adequately described by pure HS heme states and that the two heme forms should be considered as QS states having a very low level of IS state admixed with the HS state (20,21). The most striking difference between the spectra of native and Ca 2ϩ -depleted HRPC is the higher frequencies of the core size marker bands of the Ca 2ϩ -depleted form. This immediately indicates the presence of a QS heme state, which is more fully expressed compared with that of the native protein having a greater proportion of IS in the quantum mechanical admixture, since no bands due to an LS heme were detected in the corresponding absorption spectrum (22)(23)(24). All subsequent reference to the QS state of the Ca 2ϩ -depleted enzyme will be in regard to this fully expressed QS state and not that of the native protein in which only a low level of IS is apparently present. It is noteworthy that reconstitution of the Ca 2ϩ -depleted protein gave rise to electronic absorption and RR spectra which were identical to those of native HRPC (data not shown). This clearly demonstrates that the Ca 2ϩ depletion process is reversible, in agreement with the activity measurements noted above, and hence that the observed variations in enzyme activity and spectral features are a direct consequence of conformational changes induced by calcium removal.
RR excitation at wavelengths in the visible region (Fig. 4) and the use of polarized light has enabled the band frequencies of all the core size marker bands to be determined. A comparison of these frequencies with those noted for model heme compounds (25), native HRPC (26), and a number of other class III peroxidases, which display a QS state (22)(23)(24), has resulted in a detailed assignment of the RR bands of Ca 2ϩ -depleted HRPC (Table II). RR spectra recorded in polarized light (data not shown) reveal that while the band at 1638 cm Ϫ1 (Fig. 4) is depolarized the bands at 1622 and 1631 cm Ϫ1 have intermediate polarization (0.4 and 0.5, respectively). The difference in polarization ratios between polarized modes (0.125) and depolarized modes (0.75) suggests that each of the two latter bands results from the overlap of a polarized and a depolarized mode. Therefore, the band at 1638 cm Ϫ1 is assigned to a 10 mode, whereas the bands at 1622 and 1631 cm Ϫ1 are considered to be each a superposition of a vinyl (polarized) and 10 (depolarized) mode. The band at 1576 cm Ϫ1 is inversely polarized and is, thus, assigned to a 19 mode. The bands at 1548 and 1553 cm Ϫ1 are depolarized and can be assigned to 11 modes. The vinyl bands are evident at 1622 and 1631 cm Ϫ1 for all the visible excitation wavelengths shown in Fig. 4. This effect has been observed previously for other heme proteins (18). It is noted that the frequencies of the vinyl stretching modes at 1622 and 1631 cm Ϫ1 are not influenced by Ca 2ϩ depletion, indicating that the orientation of the vinyl groups is unchanged.
The 3 band at 1503 cm Ϫ1 together with the 2 mode at 1574 cm Ϫ1 (only the mean frequency can be given, due to overlap with the 2 modes of other heme species), 19  It is worthwhile noting that the low frequency RR spectrum of Ca 2ϩ -depleted HRPC, obtained with excitation at 406.7 nm, is essentially unchanged with respect to that of native HRPC (data not shown). This spectral region is characterized by outof-plane heme modes and the bending modes of the peripheral substituents, vinyl and propionate groups (18), indicating that removal of calcium does not have a significant effect on heme deformation. Furthermore, the close similarity of the electronic absorption spectra of native and Ca 2ϩ -depleted HRPC at pH 10.0 (data not shown) indicates that the behavior of the Ca 2ϩdepleted protein at alkaline pH remains invariant compared with the native. At alkaline pH native HRPC binds a hydroxyl group giving rise to a 6-c LS heme species (28,29), which has a pK a for the alkaline transition of 11.1 (30). Hence, the present data demonstrate that the pK a of the alkaline transition is effectively unchanged in the Ca 2ϩ -depleted protein.
Carbon Monoxide and BHA Binding to Ca 2ϩ -depleted HRPC-A study of ligand binding at the sixth coordination site of the heme iron can be an effective means of identifying differences between the distal heme cavities of native and Ca 2ϩdepleted HRPC. The electronic absorption and RR spectra of the Fe(II)-CO complex of Ca 2ϩ -depleted HRPC at pH 7.0 (data not shown) show close similarities with the corresponding spectra of the native protein. This indicates that, as in the native protein, two conformers are formed in the CO complex of Ca 2ϩdepleted HRPC corresponding to hydrogen bonding between CO and either the distal Arg or distal His residue (31).
The BHA molecule is a useful probe of the distal heme cavity as it enters into a number of hydrogen bond interactions with Arg 38 , His 42 , Pro 139 (32), and a distal water molecule coordinated to the heme iron, resulting in a 6-c QS complex (33,34). The electronic absorption spectra of the native and Ca 2ϩ -depleted HRPC in the presence of saturating amounts of BHA are very similar (data not shown). The only difference of note is a slight blue shift of the spectrum of the Ca 2ϩ -depleted BHA complex compared with that of the native, particularly with respect to the CT1 band (4 nm). The high frequency RR spectra of the Ca 2ϩ -depleted and native BHA complexes are also very similar (data not shown); the minor differences evident can be explained in terms of a slightly different relative proportion of  a The population of the spin species is too small to be able to assign with precision its vinyl mode frequencies.
b Only the mean frequency can be determined due to overlap of the bands.
the two hexacoordinate states, 6-c HS and 6-c QS, which characterize the native BHA complex (19,34). In fact, subtraction of the spectrum of the native complex from that of the Ca 2ϩdepleted form (data not shown) indicates an increased proportion of the 6-c HS state ( 3 at 1486 cm Ϫ1 , 2 at 1562 cm Ϫ1 , 37 at 1579 cm Ϫ1 , and 10 at 1615 cm Ϫ1 ) in the Ca 2ϩ -depleted complex. An increased proportion of the 6-c HS species with respect to 6-c QS in the Ca 2ϩ -depleted BHA complex compared with the native complex is also supported by the blue shift of the CT1 band. It has been reported previously (26) that the 6-c HS form of HRPC has a CT1 band significantly blue-shifted compared with the 6-c QS form. Furthermore, the low frequency RR spectra of the native and Ca 2ϩ -depleted BHA complexes were found to be identical (data not shown). The finding of only small spectroscopic differences between the native and Ca 2ϩ -depleted BHA complexes is reflected in the relatively small difference between the K d values for the complexes (Table I).
Electronic Absorption and RR Spectra of Ferrous Enzymes-The electronic absorption spectra of the reduced Ca 2ϩ -depleted HRPC at pH 7.8 and 9.6 are shown in Fig. 5, together with those of native ferrous HRPC (pH 6.7) and its imidazole (Im) complex (pH 10.1). The spectrum of Ca 2ϩ -depleted HRPC at pH 7.8 has marked similarities with that of the HRPC⅐Im complex and is considerably different from that of the native which, between pH 4 and 10, is characterized by a 5-c HS heme species (35). Consequently, Ca 2ϩ -depleted HRPC at pH 7.8 is mainly characterized by a bis-histidyl LS heme with a small amount of HS, as indicated by a shoulder at 586 nm. The transition to the LS species in Ca 2ϩ -depleted HRPC is even more complete at pH 9.6. At pH values higher than 9.6 the protein began to denature.
The high frequency RR spectrum of Fe(II) Ca 2ϩ -depleted HRPC at pH 7.8 is reported in Fig. 6, together with those of native ferrous HRPC and its imidazole complex, for 441.6 nm excitation, where the HS form is selectively resonance-enhanced. The spectrum of the native protein recorded at 441.6 nm is essentially identical to that reported previously for excitation at 457.9 nm (36) and is characteristic of a 5-c HS heme. The same experiment was also carried out with 413.1 nm excitation, where the LS form is selectively resonance enhanced (data not shown). For both excitation wavelengths (441.6 and 413.1 nm) the spectrum of Ca 2ϩ -depleted HRPC at pH 7.8 (identical to that at pH 9.6) appears similar to that of the HRP⅐Im complex and, hence, contains a significant proportion of a bis-histidyl 6-c LS heme (25,37,38). Nevertheless, in accord with the absorption spectra, the presence of two 3 bands at 1471 and 1492 cm Ϫ1 , corresponding to HS and LS species, respectively, together with the relative intensity of the bands at 1556 and 1580 cm Ϫ1 (corresponding essentially to 2 bands of HS and LS species, respectively) indicate that in Ca 2ϩ -depleted HRPC an additional minor HS component is also present.
In the low frequency RR spectra of the native protein at pH 6.7 for 441.6 nm excitation (Fig. 7A), the intense band at 244 cm Ϫ1 has been assigned previously to the (Fe-Im) stretching mode between the heme iron atom and the Im of the proximal ligand (35), which is active only in the 5c HS Fe(II) state (39). The bands at 312, 350, 373, and 406 cm Ϫ1 are assigned to ␥ 6 , 8 , and the bending modes of the propionate and vinyl groups, respectively. It is immediately evident that the whole of the low frequency RR spectrum undergoes significant changes upon Ca 2ϩ depletion, assuming a form distinctly reminiscent of the Fe(II) HRPC⅐Im complex and, hence, a bis-histidyl LS heme. The only striking difference is the presence of a new band at 217 cm Ϫ1 , not present in the HRPC⅐Im complex. This band is assigned to a Fe-Im stretching mode, which replaces the Fe-Im band observed at 244 cm Ϫ1 in native HRPC. The 27-cm Ϫ1 shift to lower frequencies indicates that Ca 2ϩ depletion determines a considerable weakening of the Fe-Im bond. Confirmation that the band at 217 cm Ϫ1 results from a Fe-Im stretching mode can be derived from the near-absence of the band for excitation at 413.1 nm. This wavelength is out of resonance for the HS heme species (Soret maximum at 437 nm); hence, the Fe-Im stretching mode, which is strongly coupled to the Soret resonance (40), is considerably weakened. This effect is also evident from the reduced intensity of the 244 cm Ϫ1 (Fe-Im) mode of the native protein upon 413.1 nm excitation and has been observed in a recent study of Fe(II) catalase-peroxidase (41).

DISCUSSION
The two calcium ions and four disulfide bridges present in class II and III peroxidases are structural elements that have attracted much interest. The determination of their precise roles in maintaining the heme pocket structure, with consequent important implications for enzyme activity, offers significant scope for resolving questions of peroxidase structure- function properties and identifying potential biotechnological applications. The essential nature of these features is, in fact, clearly revealed by Ca 2ϩ depletion of HRPC. Both the present and previous (10, 11) work on Ca 2ϩ -depleted HRPC demonstrate that removal of calcium leads to profound changes in enzyme activity and electronic structure of the heme iron. In all cases the specific activity is reduced to ϳ50% that of native after calcium removal despite that, in the present study, only one Ca 2ϩ ion is lost upon Ca 2ϩ depletion, whereas both Ca 2ϩ ions were reported to be lost in the previous work. Nevertheless, it is clear that Ca 2ϩ ions are necessary for optimum catalysis but are not essential for low levels of activity. Similar behavior was found for another class III peroxidase upon loss of both Ca 2ϩ ions, peanut peroxidase (42), whereas the class II peroxidases MNP and LIP were rendered inactive following loss of either one or two Ca 2ϩ ions after a less drastic thermal or alkaline pH treatment, respectively (6 -8). Such contrasting behavior between proteins of the two peroxidase classes, in which the Ca 2ϩ ions appear indispensable for activity in class II peroxidases, may be due to the different locations of the disulfide bridges in the two classes. In the specific cases of HRPC and LIP, it has been noted that the cysteine present in distal helix B of HRPC can form a disulfide bridge and limit flexibility of the helix. Formation of such a disulfide bridge is not possible in LIP, which thus favors binding of the distal His residue in LIP to the heme iron after calcium release forming an inactive LS species (7). Furthermore, the close proximity of this disulfide bridge (Cys 44 -Cys 49 ) to the distal calcium site may have a stabilizing influence on the local structure, absent in LIP, hindering loss of the distal Ca 2ϩ ion in HRPC. The foregoing considerations find support in a recent study of MNP (43). The insertion of an additional disulfide bond close to the distal Ca 2ϩ ion-binding site resulted in an apparent increase in the stability of the heme environment and active state of the protein. The particular significance of the Ca 2ϩ ions and disulfide bridges to protein stability has been underlined by a comparative study of HRPC and the class I peroxidase cytochrome c peroxidase in the presence of denaturants (44). The considerably lower kinetic stability found for cytochrome c peroxidase was attributed to the absence of these two stabilizing structural elements.
The details of the local structure in the proximity of the distal and proximal Ca 2ϩ ion-binding sites are now available from the x-ray structure determination of native HRPC (Fig. 1) (4). Both Ca 2ϩ ion sites are seven-coordinate with ligands composed of side chain oxygens and carbonyl groups and one water molecule in the case of the distal site. The distal and proximal Ca 2ϩ ions are structurally coupled to the active site (His 42 and His 170 , respectively) through an intermediate residue adjacent in sequence (Asp 43 and Thr 171 , respectively). Each of these intermediate residues provides two bonds to the corresponding Ca 2ϩ ion; therefore, the loss of either Ca 2ϩ ion has considerable potential to perturb the active site structural and catalytic properties. Nevertheless, there have been some indications from previous work that one of the two Ca 2ϩ ions may play a more essential role in maintaining the heme pocket structural characteristics necessary for high catalytic activities (11,45). The observation of NMR spectral changes upon titration of Ca 2ϩ ions to Ca 2ϩ -depleted HRPC, where both Ca 2ϩ ions were reported lost, indicated that one Ca 2ϩ ion was determinant for the protein structure (11,45). A similar Ca 2ϩ ion titration NMR study of the Ca 2ϩ -depleted (with loss of both Ca 2ϩ ions) class III peanut peroxidase, was inconclusive concerning the relative contribution of each Ca 2ϩ ion site to the structural properties and activity of the enzyme (46). Reconstitution of Ca 2ϩ -depleted HRPC with 45 Ca demonstrates that in the present study only one Ca 2ϩ ion is lost upon Ca 2ϩ depletion. However, as will become particularly evident from the following discussion, the structural properties of both the distal and proximal sides of the heme are modified by Ca 2ϩ depletion of HRPC. To shed further light on the question of which Ca 2ϩ ion is lost from HRPC upon Ca 2ϩ depletion, the evidence that favors either the distal or proximal Ca 2ϩ ion is now discussed on the basis of the body of currently available data for HRPC.
The present study clearly reveals that, contrary to the native protein, a bis-histidyl LS heme is formed in reduced Ca 2ϩdepleted HRPC. This indicates, as suggested by a previous study (11), that loss of calcium has induced a conformational change in the heme pocket. This leads to alterations of the distal side heme structure enabling the imidazole group of the distal histidine to bind to the sixth coordination position of the reduced heme iron. It is noteworthy that such effects are not noted in the ferric Ca 2ϩ -depleted form. In fact, the behavior of the ferric enzyme at alkaline pH appears to follow closely that of native HRPC, indicating that hydroxyl binding to the heme iron is favored over coordination of the distal His and, hence, that the ferric distal pocket is not extensively perturbed by Ca 2ϩ depletion. The formation of a bis-histidyl LS heme following Ca 2ϩ depletion has been noted for LIP (with loss of either only the distal Ca 2ϩ ion or both Ca 2ϩ ions) (6, 7) and MNP (upon loss of the distal Ca 2ϩ ion) (8,9); however, in these cases the LS species was observed in both the ferric and ferrous states. The presence of a bis-histidyl LS heme in Fe(II) Ca 2ϩdepleted HRPC apparently favors the case of loss of the distal Ca 2ϩ ion, as proposed for MNP (8). Conversely, the similarities of the Fe(III) Ca 2ϩ -depleted properties at alkaline pH to those of the native protein suggest a relatively minor disturbance of the distal cavity occurs upon Ca 2ϩ depletion. If one recalls that LS heme is formed in MNP (47) and Fe(II) HRPC (27) following mutation of the proximal Phe residue, which is stacked approximately parallel to the proximal histidine ligand, it is evident that proximal effects can be transmitted to the distal cavity inducing some flexibility in the position of the distal histidine. It is therefore suggested that the contrasting observations noted for the ferric and ferrous states can be reconciled if the proximal Ca 2ϩ ion is lost from HRPC upon Ca 2ϩ depletion. The example of the proximal Phe 221 3 Met HRPC mutant is particularly appropriate as it mimics in many ways the behavior of the Ca 2ϩ -depleted enzyme. In common with Ca 2ϩ -depleted HRPC, the ferric mutant protein at neutral pH is characterized by a dominant QS heme state (see below) and the ferrous state by the presence of a significant proportion of LS heme. Nevertheless, the spectral characteristics of the ferrous CO and ferric BHA adducts closely resemble those of the native. Analogous behavior is observed for Ca 2ϩ -depleted HRPC in the presence of CO and BHA, suggesting that modification of the distal cavity by Ca 2ϩ depletion is not particularly extensive and/or can be easily reversed. Furthermore, it provides evidence that strongly favors loss of the proximal Ca 2ϩ ion as the origin of these distal effects.
A particularly relevant study in the context of the present discussion is that of the Glu 64 HRPC mutants (48). The Glu 64 residue is linked to the distal Ca 2ϩ ion through a hydrogen bond with a water molecule. Changes in the position of the distal His and Arg residues in the mutants, leading to a substantial depression of the catalytic activity, was attributed to dissociation of the distal Ca 2ϩ ion. Interestingly, despite loss of the distal Ca 2ϩ ion, the geometry of the heme in the Glu 64 mutants was found to be very similar to the native protein, and there was only minor perturbation around the proximal His. Hence, the structural properties of the Glu 64 mutants have little in common with those displayed upon Ca 2ϩ depletion, where completely contrasting behavior is observed, the distal cavity suffering only minor changes whereas the proximal cavity is considerably perturbed (see below). Consequently, this supports loss of the proximal Ca 2ϩ ion in Ca 2ϩ -depleted HRPC. Moreover, it suggests that the distal Ca 2ϩ ion is of limited importance and that the proximal Ca 2ϩ ion is responsible for retaining the heme geometry and coordination structure of the proximal ligand.
The marked downshift (27 cm Ϫ1 ) of the Fe-Im stretching frequency observed in reduced Ca 2ϩ -depleted HRPC compared with the native indicates a significant weakening of the bond between the iron atom and the proximal histidine residue. This reflects a marked change in the strength of the hydrogen bond between the conserved proximal His and Asp residues characteristic of peroxidases, which imparts a pronounced imidazolate character to the proximal His and results in higher (Fe-Im) frequencies than found for other heme proteins (39). Furthermore, it demonstrates that substantial structural changes of the heme environment in the vicinity of the proximal histidine are induced by Ca 2ϩ depletion. In fact, the Fe-Im stretching frequency occurs at 220 cm Ϫ1 in myoglobin, where the proximal His is hydrogen-bonded with a neutral peptide carbonyl group (49), and at 196 cm Ϫ1 in the Fe 2ϩ protoporphyrin IX histidine complex, where the proximal His is not involved in hydrogen bonding interactions (50). In this context, it should be recalled that the residue adjacent to the proximal His (Thr 171 ) provides two coordination bonds to the proximal Ca 2ϩ ion ( Fig. 1) (4); hence one might reasonably expect that loss of this Ca 2ϩ ion would cause a change in the disposition of the histidine leading to a weakening of the His-Asp hydrogen bond and, therefore, of the heme iron-His 170 interaction.
Native Fe(III) HRPC is characterized at room temperature by a QS state having a very low level of intermediate-spin admixed with the high-spin state (19 -21). In ferric Ca 2ϩ -depleted HRPC at pH 7.8, however, the pentacoordinate QS state becomes more fully expressed compared with the native protein, having a greater relative proportion of IS compared with HS in the quantum mechanical admixture, as demonstrated by the higher frequencies of the core size marker bands of the Ca 2ϩ -depleted form. It constitutes, together with a 5-c HS form, one of the two major heme states of Ca 2ϩ -depleted HRPC; 6-c HS and 6-c LS states are also present at very low levels. The appearance of the QS state is particularly interesting. This heme species is uncommon in biological systems. Known examples include the neutral pH form of cytochrome cЈ (51,52) and class III peroxidases (18, 23). Unfortunately, the biological significance and structural properties of the heme that give rise to the QS state remain obscure (24). Hence, the presence of this unusual heme state cannot be exploited as a means to uncover subtle modifications to the heme environment associated with Ca 2ϩ depletion. In this regard, however, a recent report (27) has revealed that mutation of the proximal Phe 221 residue of HRPC also produces a QS heme state, whereas the many distal mutants of HRPC that have been characterized lack such a state (18). Therefore, the appearance of a QS heme state in Ca 2ϩ -depleted HRPC in concomitance with a significant weakening of the Fe-Im bond suggests that disturbance of the proximal, rather than distal, heme domain may be more important in the generation of this heme state. It is noted that, in contrast to the Ca 2ϩ -depleted protein, in the case of the Phe 221 mutant a strengthening of the Fe-Im bond was observed. This was attributed to the loss of theinteraction between the approximately parallel stacked aromatic rings of the His 170 and Phe 221 residues, reduction in the steric constraints imposed on the proximal His residue and, consequently, a modification of the His disposition.
In summary, evaluation of the experimental data available for HRPC, both from this study and previous work, firmly favors the proximal Ca 2ϩ ion as that which is lost upon Ca 2ϩ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.