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The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

Monique Laberge^*, Qing Huang, Reinhard Schweitzer-Stenner and Judit Fidy^*

^* Institute of Biophysics and Radiation Biology, Semmelweis University, Puskin u. 9, Budapest H-1088, Hungary; and Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico PR00931, USA

Correspondence: Address reprint requests to Monique Laberge, Semmelweis University, Dept. of Biophysics and Radiation Biology, P.O. Box 263, Budapest, H-1444, Hungary. Tel.: 36-1-267-6261; Fax: 36-1-266-6656; E-mail: laberge{at}puskin.sote.hu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS COMPUTATIONAL METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca²⁺-bound and Ca²⁺-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B_1g- and B_2g-type distortions in the Ca²⁺-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca²⁺-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion.

	INTRODUCTION

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Horseradish peroxidase is a secretory plant peroxidase that catalyzes the oxidation of small aromatic substrates, such as plant hormones and lignin precursors, by hydrogen peroxide (Dunford, 1991; Smith and Veitch, 1998). Isoenzyme C (HRPC) is the most abundant isoenzyme found in horseradish roots. In the resting state, the HRPC prosthetic group is a typical class III peroxidase pentacoordinate ferric heme b with the iron described as a quantum mixed state (QMS) consisting of a low contribution from an intermediate spin (IS) species admixed with a predominant high spin (HS) state (Maltempo and Moss, 1976; La Mar et al., 1980; de Ropp et al., 1997; Howes et al., 2001; Smulevich, 1998). Class III peroxidases are also characterized by the presence of two calcium binding sites, respectively proximal and distal to the heme. The x-ray crystal structure of HRPC (Gajhede et al., 1997) shows that the proximal Ca²⁺ ion is located at 13.52 Å from the heme iron and the distal at 15.93 Å (Fig. 1). The role of calcium has been the object of several studies showing that Ca²⁺ binding is essential for catalysis and maintaining the structural features of the enzyme required for its catalytic activity (Haschke and Friedhoff, 1978; Morishima et al., 1986; Shiro et al., 1986; Smith et al., 1990; Chattopadhyay and Mazumdar, 2000). Ca²⁺ has also been shown to be required for the stability of the enzyme: in the presence of calcium, the free-energy change during unfolding is 16.7 kJ/mol for the native enzyme compared to 9.2 kJ/mol in the absence of Ca²⁺ (Pappa and Cass, 1993).

View larger version (59K): [in this window] [in a new window]   FIGURE 1  The seven-coordinate calcium binding sites in HRPC. The proximal site (purple) is partially solvent-exposed (dotted surface) in a more flexible region of the protein while the distal site (blue) is buried in the protein core. Image from the energy-minimized structure generated using the 1ATJ.PDB coordinates (Gajhede et al., 1997). Solvent-accessible surface (115.92 Å) was calculated using the Connolly algorithm (Connolly, 1983) with a 1.4-Å probe radius.

In this work, we use Polarized Resonance Raman Dispersion Spectroscopy (PRRDS) to investigate the heme distortions resulting from partial Ca²⁺ removal. We also use molecular dynamics simulations (MDS) to generate equilibrated structural models of HRPC and of the Ca²⁺-depleted species from the available x-ray structure (Gajhede et al., 1997). Further, we extract the hemes from the averaged MDS trajectory structures and subject them to normal coordinate structural decomposition (NSD). The NSD method has been used extensively to describe and analyze the out-of-plane distortions of porphyrins in heme proteins (Shelnutt, 2000). It classifies the porphyrin distortions in terms of equivalent displacements along the lowest frequency normal coordinate of the porphyrin and provides a computational procedure allowing to determine the out-of-plane and in-plane displacements along all the normal coordinates of the porphyrin (Jentzen et al., 1997). NSD results have been published characterizing the out-of-plane distortions of peroxidases belonging to different classes (Jentzen et al., 1998; Howes et al., 1999) and it was recently applied to study the calcium-dependent conformation of a heme in a diheme peroxidase (Pauleta et al., 2001), thus providing a database for comparison.

	MATERIALS AND METHODS

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Sample preparation
Horseradish peroxidase isoenzyme C (code HRP4B) was purchased from Biozyme Laboratories Ltd. (San Diego, CA). This preparation has a Reinheitszahl (RZ) of 3.4 and contains 90% isoenzyme C and it was used without further purification. Calcium was partially removed following the procedure of Haschke and Friedhoff (1978). To achieve depletion of one mole of calcium, the enzyme was dissolved in 100 mM Tris/HCl pH 8 and incubated for 4 h in 6 M guanidine hydrochloride in 10 mM EDTA at room temperature, followed by 12 h dialysis against 5 mM EDTA, pH 7 and 4 h dialysis against water. As reported elsewhere (Huang et al., 2003), total reflection x-ray fluorescence spectroscopy was used to analyze the calcium contents of this preparation which yields 0.43 mol of Ca²⁺/mole of enzyme. PRRDS measurements were performed on 1 mM native and Ca²⁺-depleted HRPC dissolved in 50 mM Tris/HCl buffer at pH 8.0.

Polarized resonance Raman spectroscopy
All Raman spectra were measured in 135° backscattering geometry using a tunable Argon ion laser (Lexel 95). Its excitation radiation lines cover the resonant region of the Q-band of HRP. The laser beam, polarized perpendicular to the scattering plane, was filtered by a set of interference filters and focused onto a sample in a quartz cell mounted in a macro-chamber at room temperature. The laser power was adjusted to values between 10 mW to 50 mW depending of the excitation wavelength used. The scattered light was collimated and collected by an imaging lens system into an entrance slit of 100 um width of a triple-grating spectrometer (Jobin-Ivon). Polarization analyzer and scrambler were inserted between collimator and entrance slit of the spectrometer in order to measure the two components polarized perpendicular (I_y) and parallel (I_x) to the polarization of the incident laser beam. The scattered light was then dispersed by a Jobin Ivon T65000 triple monochromator, which is equipped with 1800 groove/mm gratings. The photons were counted with a liquid nitrogen cooled CCD camera (CCD3000 from Jobin-Ivon) with 1024 x 512 pixel array chip. The data were digitized and stored on a Dell computer with Pentium III processor for further analysis. The spectral resolution of the spectrometer ranged from 3.8 cm^-1 (457.9 nm) to 2.8 cm^-1 (514.5 nm). Calibrated with 934 cm^-1 of ClO₄^-, the recorded Raman spectra have an accuracy of 1 cm^-1.

All spectra were analyzed by the program MULTIFIT (Jentzen et al., 1996). Each Raman band was fitted with a Voigtian profile, which results from the convolution of its Lorentzian line profile and the Gaussian line profile of spectrometer slit function. The spectra were decomposed consistently by using identical parameters such as half-width, frequency position, and band profile for all eight excitation wavelengths. Thus, the observed spectra were subjected to a global fit. Many attempts with different guess values for the spectral parameters were carried out and the best fitting parameters and the respective statistical errors were found with the smallest ²-values. The intensities of the polarized bands were derived from their band areas. The depolarization ratios of the spectral lines were calculated as

where I_x and I_y were measured parallel and perpendicular to the polarization of the exciting laser beam. The accuracy of the depolarization ratio was verified with the 216 cm^-1 line of CCl₄. The measured depolarization ratio was found to be identical with its expectation value of 0.75 ± 0.02 at all excitation wavelengths. The depolarization ratio of the 934 cm^-1 line of the internal standard ClO₄^- was always close to 0, as theoretically expected. This shows that the contamination by the collimator optics is negligibly small for polarized and depolarized lines.

	COMPUTATIONAL METHODS

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Generating the models and energy minimization
All simulations were performed using the academic CHARMM version 27 (Brooks et al., 1983; MacKerell et al., 1998) on a Silicon Graphics R-10000 O2 workstation linked to the NIIF supercomputing environment. The x-ray coordinates of HRPC were obtained from the RCSB protein database (Bernstein et al., 1977); namely PDB1ATJ.ENT (Gajhede et al., 1997). The structure was corrected for x-ray disordered and modified regions. Specifically, Met1, introduced for gene expression, was removed and Asn307 and Ser308, disordered and not included in the coordinates, were added in extended mode using the Biopolymer module of the InsightII software package (Accelerys, San Diego, CA). The stereochemical quality of the model was verified using the PROCHECK program (Laskowski et al., 1993) and three calcium-depleted models were generated by removing the proximal calcium site, the distal site and finally, both calciums.

Parameterization
Charges for the ferric heme and the Ca²⁺-coordination spheres were calculated at the Hartree-Fock level using a 6-311G basis set and electrostatic potential fitting as previously described (Schay et al., 2001) and incorporated into CHARMM. van der Waals parameters were from Cates et al. (2002).

Energy minimization
All crystallographic water molecules were retained (151, less one molecule which is a ligand to Ca352) since they have been shown to have an important structural role by participating in the H-bond network, especially the waters found in the heme crevice (Gajhede et al., 1997). Explicit hydrogens were added using the HBUILD module and the amino acid residues were protonated so as to be consistent with neutral pH. The propionic acid side chains were also considered ionized as discussed elsewhere (Schay et al., 2001) and the disulfide bridges were explicitly modeled, namely: Cys11-Cys91, Cys44-Cys49, Cys97-Cys301, and Cys177-Cys209. The structures were solvated in a 36-Å sphere of 5000 explicit TIP3 waters (Jorgensen et al., 1983) using a spherical shape quartic boundary potential within the Miscellaneous Mean Field Potential approximation as implemented in CHARMM. All models were subjected to the same energy minimization protocol. First, the hydrogens of the solvent waters were relaxed while imposing fixed constraints on all other atoms with 30 steps of steepest descent minimization. Then the solvent was relaxed keeping the protein constrained. Finally, harmonic constraints were used to progressively relax the R-groups, then the backbone and the heme group from 240 to 0 kcal mol^-1 using conjugate gradient minimization until the derivatives reached 0.9 mol^-1 Å^-1. An Adopted Basis Newton-Raphson final minimization of all models completed the minimization protocol with final derivatives of 0.06 mol^-1 Å^-1.

Molecular dynamics
Molecular dynamics were performed using the SHAKE algorithm so as to remove the highest frequencies from the system and allow use of a 0.001 ps timestep for integration. The van der Waals cutoff distance was 14 Å using a smooth switching function at a distance of 10.0 Å and a shifting function was used for the electrostatic interactions at a cutoff of 13.0 Å. A dielectric constant of 1 was used. The structures were brought to 300 K in 10 ps using a stepwise heating stage. The heating stage was followed by an equilibration stage until energy stabilized. After a 50 ps-equilibration stage, 200-ps trajectories were acquired for analysis. The average structure was calculated for all 4 trajectories and subjected to progressive energy minimization as described above until the derivatives reached 0.3 mol^-1 Å^-1.

Normal coordinate structural decomposition
The hemes of the four models were extracted for NSD, performed using version 2.0 of the NSD program (Jentzen et al., 1997) with the hemes oriented as shown in Fig. 2. The computational procedure is based on group theory in that the atomic distortions of the 24 porphyrin atoms from ideal D_4h symmetry can be described in terms of 3N - 6 = 66 normal coordinates. The program projects out the out-of-plane and in-plane distortions along all the 66 normal coordinates of the porphyrin. For heme proteins however, it has been shown that the total distortion from planarity can statistically be adequately described by using only the six lowest out-of-plane frequency modes, namely B_2u, B_1u, E_g(x), E_g(y), A_1u, and A_2u because these modes contribute the most to heme nonplanarity (Jentzen et al., 1997; Jentzen et al., 1998).

View larger version (18K): [in this window] [in a new window]   FIGURE 2  Orientation of the heme for the NSD calculations.

	RESULTS AND DISCUSSION

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View larger version (40K): [in this window] [in a new window]   FIGURE 3  Polarized Raman spectra of native Ca2+-depleted HRPC (pH 8) excited at 476.5 and 457.9 nm (a) and at 514.5 and 496.5 nm (b), which cover both B-preresonance and Qv-resonance excitation.

View larger version (22K): [in this window] [in a new window]   FIGURE 4  Comparison of DPRs for native and Ca2+-depleted HRPC: 4 (top), 21 (middle), and 11 (bottom).

Fig. 4 c compares the DPRs of the B_1g type mode ₁₁ between native and Ca²⁺-depleted HRPC. The DPRs of ₁₁ in the Ca²⁺-depleted species increase monotonically with excitation wavelength, and cross both the expectation line of 0.75 under D_4h and the DPR curve of ₁₁ in native HRPC. This shows that this mode is subject to A_2g-type and A_1g-type vibronic perturbations resulting from B_2g and B_1g in-plane distortions of the macrocycle, respectively. The dispersion is more pronounced for Ca²⁺-depleted HRPC, indicating that both types of distortions increase with respect to native HRPC. In fact, the contribution of A_1g-perturbation/B_1g-distortion which gives rise to the DPR decrease at higher excitation wave numbers must be considered as particularly strong, since the excitation is still far away from the B-band resonance position at which such an effect would be expected to be maximal. If all these observations involve the proximal histidine, it follows that the type of perturbations induced by Ca²⁺ removal could cause both a decrease of the His:NE2-Fe distance or a smaller azimuthal angle of the imidazole ring with respect to the N-Fe-N line, as theoretically argued elsewhere (Schweitzer-Stenner, 1989).

Molecular dynamics
All structures were subjected to initial 50-ps equilibration MDS runs which yielded fully equilibrated structures after 20 ps. These runs were followed by the acquisition of 200-ps production trajectories. All runs were quite stable as shown by their constant potential energies and temperature. The energy minimization protocol was also successful in reaching comparable energy minima for the four models. The stability of the fluctuation of the total energy was also examined by calculating the ratio between the average rms fluctuation of the total energy and the average energy. For all models, this ratio did not exceed 0.1, thus showing that energy was conserved during the simulations and that the models were well equilibrated.

Table 2 presents some useful quantities calculated from the simulations. The radius of gyration is the radius of a sphere containing an equivalent volume to that of the molecule. With removal of one calcium, the R_gyr values do not change with respect to that of the native structure. However, complete Ca²⁺ removal leads to a slight expansion of the protein matrix, evidenced by the increase in the radius of gyration. The backbone and R-group RMSD values are lowest for the native structure (0.987 Å and 1.211 Å). They reflect the dynamics of the protein matrix. The values calculated for the Ca²⁺-depleted models (1.044, 1.163, and 1.140 Å for the backbone and 1.451, 1.543, and 1.526 Å for the R-groups) are higher and reflect an increased flexibility of the secondary structure as a result of calcium removal. Also relevant are the backbone RMSD values comparing only the effect of calcium removal. They fall in the same range (1.213, 1.220, and 1.210 Å) and attest to a role played by calcium in modifying the dynamics of HRPC. Fig. 5 shows the backbone C-traces of the native (top) and Ca²⁺-free structures. Removal of calcium does not affect the secondary structure fold to a significant extent besides relaxing it to a looser form. This is probably due to the presence of the four disulfide bridges, Cys11-Cys91, Cys44-Cys49, Cys97-Cys301, and Cys177-Cys209, that have been shown to stabilize helices A, B, C, D, F1, and F2 (Chattopadhyay and Mazumdar, 2000). The two regions showing the most backbone reorganization are on the proximal side of the heme, namely short insertion helix F' consisting of Met181, Asp182, Arg183, and Leu 184 and part of helix H, namely Asp247 and Ser246. Fig. 6 compares the R-group displacements in the vicinity of the heme for both HRPC and the Ca²⁺-free model. They are quite significant especially in the reorganization of both distal and proximal Phe aromatic clusters. The distance of distal His42 to the heme iron shortens upon calcium removal from 6 to 5.5 Å and the heme pocket water closest to the iron in the native structure (3.20 Å) moves further away from the iron in the calcium-depleted species toward Arg38. All of the R-group displacements necessarily must reorganize the nonbonded interaction network at the catalytic site. Detailed ¹H-NMR studies performed on HRPC have provided a detailed assignment of the hyperfine proton resonances of the heme macrocycle and of the amino acid resonances in the heme pocket (La Mar et al., 1980; Thanabal et al., 1987, 1988). A comparison with calcium-depleted HRPC would be required to experimentally verify if calcium can indeed cause residue reorientation with respect to the heme, as shown in the case of cationic peanut peroxidase (Barber et al., 1995).

View larger version (47K): [in this window] [in a new window]   FIGURE 5  Stereo view of the backbone traces of the HRPC (top) and Ca2+-depleted (bottom) average structure after 200 ps of molecular dynamics.

View larger version (22K): [in this window] [in a new window]   FIGURE 6  The effect of calcium removal in the vicinity of the heme of HRPC (left) and Ca2+-free HRPC (right). The aromatic cluster consisting of Phe41, 143, 152, and 179 on the distal side and Phe172, 221, 229, and 277 on the proximal side are rendered purple. Pro141 and His42 are yellow, and wat90 is a red sphere.

View larger version (25K): [in this window] [in a new window]   FIGURE 7  View of the his170 imidazole orientation with respect to the Npyrr-Fe-Npyrr line in the hemes extracted from the HRPC models. Clockwise from top left: HRPC + 2Ca2+; Ca2+-depleted HRPC; HRPC + distal Ca2+; and HRPC + distal Ca2+.

View larger version (25K): [in this window] [in a new window]   FIGURE 8  Out-of-plane displacements in Å of the minimal basis set for the hemes in the average structures extracted from the MDS trajectories. Deformations along the six lowest frequency normal coordinates of the following D4h symmetry types: B2u (saddling), B1u (ruffling), A2u (doming), Eg(x) (x-waving), Eg(y) (y-waving), and A1u (propellering).

The NSD results are clearly different for the hemes of the Ca²⁺-depleted models. Calcium depletion predominantly affects the saddling and ruffling deformations. Saddling is drastically reduced in the Ca²⁺-free model, with a deformation of -0.173 Å compared to -0.841 Å for the native model, and -0.398 Å and -0.337 Å in the structures with either proximal or distal Ca²⁺ present. Ruffling, which is not significant in the native model (-0.128), increases to moderate values upon Ca²⁺ removal (-0.597, -0.537, and -0.449 Å). It has been proposed that ruffling has a stronger effect on the high frequency lines than an equivalent saddling deformation (Franco et al., 2000). In a recent 600-ps MDS study of the myoglobin-CO heme deformations, ruffling was shown to exhibit the largest transient deformations (exceeding 0.5 Å), which were already well characterized on the timescale of our trajectories (Kiefl et al., 2002). The authors proposed that they were probably part of ns-oscillations in heme deformations. Clearly, longer timescale trajectories are required to fully average these ruffling excursions, especially if they are correlated to tertiary structure rearrangements of the protein matrix. Our NSD results also show that the doming deformation increases and changes sign when compared to the native model. But since the His:NE2-Fe bond length does not change upon calcium removal, this doming cannot correspond to the classical displacement observed in myoglobin or hemoglobin as they cycle between oxy and deoxy forms with the concomitant significant change of axial ligand-metal bond length. It should also be noted that the doming deformation in the NSD context does not involve the iron, but only the 24 atoms of the porphyrin ring. We therefore propose that increased doming probably results from smaller distances between the imidazole carbons and the C_a and N_pyrr atoms of the porphyrin which is also consistent with our observed increase of both B_2g and B_1g-type distortions. These could be induced by a rearrangement of the weak nonbonded interactions—such as van der Waals and hydrogen bonding (Laberge, 1998)—resulting from the sidechain reorganization occurring in the vicinity of the heme as a result of calcium removal (compare to Fig. 5). Moreover, the HRPC heme is not covalently linked to the protein matrix and is thus conceivably more sensitive to respond to altered nonbonded interactions in its vicinity.

	CONCLUSIONS

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In resting HRPC, calcium depletion results in reorganization of the residues of the heme pocket. These rearrangements modulate the conformation of the heme prosthetic group. Specifically, they increase B_2g and B_1g in-plane distortions that contribute to a change in the spin state of the iron and also increase the overall planarity of the heme. This supports the concept of nonplanar distortions providing a mechanism for the modulation of function by the protein (Shelnutt et al., 1998) and confirm the structural role of Ca²⁺ in maintaining the heme of HRPC in a geometry required for efficient catalysis.

	ACKNOWLEDGEMENTS

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We gratefully acknowledge the Hungarian Government's National Information Infrastructure Development Program for providing computing time on the IIF parallel supercomputing environment, and J.A. Shelnutt for kindly providing the NSD program.

This research was supported by Hungarian OTKA grant T-032117 (J.F.), a Senior NATO Science Fellowship (M.L.), and by grants from the National Institutes of Health (COBRE-program, P20 RR16439-01) and the Petroleum Research Funds (PRF#38544-AC4) (R.S.S.).

Submitted on September 26, 2002; accepted for publication December 11, 2002.

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