Structure of Barley Grain Peroxidase Refined at 1.9-Å Resolution
A PLANT PEROXIDASE REVERSIBLY INACTIVATED AT NEUTRAL pH*

Anette HenriksenDagger §, Karen G. Welinder, and Michael GajhedeDagger

From the Dagger  Department of Physical Chemistry, Chemical Institute, University of Copenhagen, Universitetsparken 5, DK-2100 København Ø, Denmark and the  Department of Protein Chemistry, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 København K, Denmark

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 Å. The refined model includes amino acid residues 1-309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

All living organisms utilize peroxidases in a variety of biosynthetic or degradable processes and in defense against pathogens or oxidative pressure. The majority of known peroxidases belong to the plant peroxidase superfamily, which is characterized by a central heme group sandwiched between a distal and a proximal protein domain. The plant peroxidase superfamily is subdivided into three classes based on structural divergence (1). Class I constitutes intracellular peroxidases of prokaryotic origin, and class I peroxidases are found in yeast (cytochrome c peroxidase; CCP),1 plants (pea cytosolic ascorbate peroxidase), and bacteria. Class II and III peroxidases are found in fungi and plants, respectively, and are largely extracellular. Both class II and class III peroxidases contain two conserved calcium ions, one in each of the distal and proximal domains. Class II includes the lignin-degrading, manganese-dependent (MnP), and Coprinus peroxidases (synonymous with Arthromyces ramosus peroxidase). Class III includes the classic horseradish peroxidase (HRP C); peanut peroxidase (PNP), for which the first crystal structure of a class III peroxidase was solved (9); and the major peroxidase from barley grain (BP 1).

In common with other heme-containing peroxidases, BP 1 catalyzes the following multistep reaction.
<UP>Peroxidase</UP> (<UP>Fe</UP><SUP>3<UP>+</UP></SUP>)+<UP>H</UP><SUB>2</SUB><UP>O</UP><SUB>2</SUB> → <UP>Compound I</UP> (<UP>Fe</UP><SUP>4<UP>+</UP></SUP>)<SUP>+</SUP>·+<UP>H</UP><SUB>2</SUB><UP>O</UP>
<UP>Compound I</UP> (<UP>Fe</UP><SUP>4<UP>+</UP></SUP>)<SUP>+</SUP>·+<UP>AH</UP> → <UP>Compound II</UP> (<UP>Fe</UP><SUP>4<UP>+</UP></SUP>)+<UP>A<SUP>⋅</SUP></UP>
<UP>Compound II</UP> (<UP>Fe</UP><SUP>4<UP>+</UP></SUP>)+<UP>AH</UP> → <UP>Peroxidase</UP> (<UP>Fe</UP><SUP>3<UP>+</UP></SUP>)+<UP>A<SUP>⋅</SUP></UP>+<UP>H<SUB>2</SUB>O</UP>
<UP>R<SC>eaction</SC></UP> 1
Still, with its unique pH and calcium dependent activity profile (2, 3), BP 1 is the most interesting and atypical of the class III peroxidases. In the presence of calcium and following a pH-induced conformational change with a pKa < 3.7, BP 1 has a rate constant for compound I formation comparable with the rate constant for HRP C. The similarities between BP 1 and HRP C include N-terminal signal peptides, C-terminal propeptides suggesting a vacuolar targeting, isoelectric points near 9, four disulfide bridges, and an amino acid sequence identity of 42% including all active site residues (4). However, in contrast to HRP C, BP 1 can only form compound I at pH < 5 (3).

A number of spectroscopic studies have demonstrated structural versatility within the superfamily of plant peroxidases. At alkaline pH, CCP collapses to a bis-His-coordinated form (5). A similar observation has been made for thermally inactivated MnP, shown to have lost only the distal calcium ion (6). The Coprinus peroxidase mutant D245N showed reversible structural changes in response to pH changes in the form of unusual spin and coordination variants (7, 8). These findings emphasize the importance of obtaining crystallographic evidence to characterize the nature of structural rearrangements occurring in peroxidases. Here we present the crystal structure of barley grain peroxidase, BP 1, in the inactive form, which is reversibly activated at pH < 5. The crystallographic data show that a distal segment comprising residues 64-84 has been significantly rearranged as compared with the crystal structures of active HRP C2 and PNP (9). This rearrangement leads to a marked displacement of the side chain of catalytic distal histidine and provides an explanation for the loss of activity for this plant peroxidase at neutral pH. The rearrangement has the potential of being influenced by pH and the distal cation and its ligands.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Crystallization and Data Collection-- Purification and characterization of BP 1, obtained in a glycosylated form, BP 1a, and nonglycosylated form, BP 1b, was reported previously (10). Crystallization was achieved at pH 8.5 (BP 1a) and pH 7.5 (BP 1b) (11). A pH 5.5 form was obtained by soaking BP 1b crystals crystallized at pH 7.5 in 15% polyethylene glycol 6000, 10 mM KI, 0.1 M MES buffer, pH 5.5, for 1 week. Data were collected from single BP 1a and BP 1b crystals using a Rigaku R-axis IIC image plate system with a Rigaku RU200 rotating anode as x-ray source. The anode was operated at 50 kV and 180 mA and equipped with a graphite monochromator and a 0.5-mm collimator. The BP 1b crystals at pH 7.5 belong to the space group P21212 with a = 71.9 Å, b = 105.1 Å, and c = 41.0 Å and one molecule in the asymmetric unit. Fifty-six frames of 3.0° oscillations were collected. BP 1a crystals belong to the space group P212121 with a = 62.9 Å, b = 66.3 Å, and c = 71.0 Å also with one molecule in the asymmetric unit. Seventy frames of 1.5° oscillations were collected. Integration of intensities was carried out with the program Denzo (12), and further data processing was performed with programs from the CCP4 program package (13). Heavy atom derivative data for BP 1a were collected similarly to the collection of native BP 1a data. Data collection statistics are listed in Table I. An additional data set was collected for BP 1b crystals soaked at pH 5.5. The pH 5.5 BP 1b crystals were highly nonisomorphous, with cell dimensions a = 66.9 Å, b = 104.3 Å, and c = 43.2 Å, corresponding to changes of 7, 1, and 5% relative to the pH 7.5 crystal, but they still belonged to space group P21212.

Molecular Replacement-- Molecular replacement was carried with the program AMoRe (14). The molecular replacement search used a polyalanine model based on peanut peroxidase residues 1-294 and the heme group (9). The amino acid sequence identity between peanut peroxidase and BP 1 is 44%. For BP 1a, the successful replacement appeared second in the rotation function peak list and first in the translation function peak list with a correlation coefficient of 40.7 and an R-factor of 51.6. The second highest peak had a correlation coefficient of 29.5. After rigid body refinement, the values improved to 45.9 and 49.0, respectively. For BP 1b at pH 7.5, the corresponding appearance of the successful replacement was first in both the rotation function and the translation function peak list, with a correlation coefficient of 44.0 and an R-factor of 50.9. In this search, the second highest peak had a correlation coefficient of 28.5. After rigid body refinement, the correlation coefficient and R-factor improved to 48.5 and 48.8, respectively. Data from 20 to 5 Å were used for molecular replacement both for BP 1a at pH 8.5 and BP 1b at pH 7.5.

Heavy Atom Phases-- Data from three successful heavy atom derivatives of BP 1a were collected. Hg(CH3COO)2 was used in all cases and added directly to the mother liquor to a final concentration of 0.1-0.2 mM. The crystals were soaked with derivative solutions for 1-2 weeks. An unambiguous analysis of difference Patterson maps was not achieved, whereas difference Fourier maps, using phases from the molecular replacement solution, gave clear mercury signals in all derivative data. Heavy atom parameters were refined using "mlphare" from the CCP4 program package (13) without including anomalous data. B-Factors could not be refined for any of the heavy atom sites and were fixed at 30 Å2. Three Hg(CH3COO)2 sites were common for all soaks but were occupied to different extents. In the third data set, an additional Hg(CH3COO)2 site was found. The results of the refinement of heavy atom parameters and statistics for derivative data are listed in Table I. Interpreting electron density maps calculated only from multiple isomorphous replacement phases was not possible, and therefore, heavy atom derived phases and model phases for BP 1a to 3.5 Å were combined with "sfall" and "sigmaa" of the CCP4 program package. Phase refinement and extension were performed with "dm" from CCP4 using histogram matching, solvent leveling, and Sayre's equation. No successful heavy atom soaks were achieved for BP 1b.

                              
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Table I
Data collection statistics

Model Building and Refinement-- Model building was initiated in the space group of BP 1a using "O" (15), since unbiased multiple isomorphous replacement phase information was available for this enzyme. The first round of model building, which did not include the main chain and side chain atoms of 56 amino acid residues in areas of poor electron density, was followed by slow cool simulated annealing minimization using the procedure of X-PLOR (16, 17). Electron density maps were calculated in both space groups, and model building proceeded in the space group of BP 1b at pH 7.5 as areas of unresolved electron density in the BP 1a electron density map were well resolved in the BP 1b map. For all X-PLOR minimizations, the Engh and Huber parameters (18) were used, and except for the last round of minimization, a subset of 10% of randomly selected reflections was used as a test set for free R-factor calculation (17). The weight for x-ray refinement was kept at approximately 0.3 times the recommended value from CHECK to keep a strict geometry. Individual B-factors were refined but restrained to be correlated with the B-factors of neighboring atoms. For BP 1b the initial model including 253 amino acid residues and heme gave an R-factor/free R-factor of 0.486 for 8.0-2.2-Å data. The conventional R-factor was 0.248, and the free R-factor was 0.307 after model rebuilding and X-PLOR refinement including all BP 1 residues: 1-309, two calcium ions, and heme. Further model rebuilding, refinement, the inclusion of low resolution data, the inclusion of bulk solvent correction, the replacement of distal calcium with sodium, and the addition of 146 solvent molecules gave an R-factor of 19.3 and a free R-factor of 23.0 using 38-1.9-Å data. The addition of water molecules was based on Fo - Fc electron density, hydrogen bonding possibilities, and resulting 2Fo - Fc electron density. No additional solvent molecules were included in the model for the final cycle of refinement including the test data set. The structure of BP 1b, pH 7.5, was compared with BP 1a, pH 8.5, and BP 1b, pH 5.5, after molecular replacement, rigid body refinement, model corrections using "O" and one round of X-PLOR slow cool simulated annealing using the refined BP 1b, pH 7.5, structure as the molecular replacement model.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Model Quality-- The final model of barley peroxidase BP 1b at pH 7.5 includes 2361 nonhydrogen protein atoms, 43 heme group atoms, one calcium ion, one sodium ion, and 146 solvent atoms in the asymmetric unit. The solvent-exposed side chain Trp15 was not included, since its presence was not justified by the electron density map. The conventional Rcryst for the resolution range 38-1.90 Å with no sigma (F) cutoff applied was 19.2% with an Rfree of 23.0% before the last refinement cycle. r.m.s. deviations from ideal geometry and the refinement statistics are listed in Table II. 90.4% of the model's phi -psi angles fall in the most favored regions of the Ramachandran plot as calculated with the program PROCHECK (19) excluding Gly and Pro residues; 8.8% fall in additionally allowed regions; and Ser292 falls in a disallowed region. The Pro195-Arg196 bond was refined as a cis-peptide bond to fit the electron density.

                              
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Table II
Summary of crystallographic refinement

Description of the Molecule-- The central heme group of BP 1 is sandwiched between a distal N-terminal domain and a proximal C-terminal domain (Fig. 1), and the overall structure closely resembles the structures of HRP C2 and PNP (9). The r.m.s. difference between the BP 1 and HRP C Calpha atoms is 1.3 Å, including 276 residues based on a 3.8-Å distance cutoff. Residues 65-83 from the BC loop are among the excluded residues. A total of 12 alpha -helices are present in BP 1, of which 10 are conserved among all known heme peroxidases of the plant peroxidase superfamily. The F' alpha -helix found in HRP C and PNP is replaced by a 310-helix in BP 1. An overview of the secondary structure elements is given in Table III. Besides the secondary structures described for HRP C and PNP, two antiparallel two-stranded beta -sheets are found in BP 1. The strands beta 2-(183-185), beta 3-(221-223) flank the large F-G insertion, which is characteristic of plant peroxidases and which contains helices F' and F". The two-residue strands beta 1-(132-133) and beta 4-(290-291) connect the extended structure between helices D and D' of the N-terminal domain and the C-terminal segment extending after helix J. Two short 310-helices (59-61 and 76-78) are found between helices B and C in BP 1. The major difference between the structure of inactive BP 1 at pH 5.5, 7.5, and 8.5 and those of active HRP C and PNP is found in the last part of the region between helix B and C (residues 64-84) (Fig. 1). Minor structural differences between BP 1 and HRP C are seen in the solvent-exposed loops, 99-102, 193-197, 215-219, and 254-256, and in the direction of helix D'-(139-145). The unique features of BP 1 include an N-terminal extension of seven residues as compared with HRP C and the majority of plant peroxidases (21). These seven residues are well ordered with several intramolecular hydrogen bonds and hydrophobic contacts. No significant structural differences were observed between the protein structures of BP 1b at pH 7.5, BP 1a at pH 8.5, and BP 1b at pH 5.5 (r.m.s. differences of 1.2 and 0.9 Å, respectively, including all protein atoms).


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Fig. 1.   The structure of barley grain peroxidase compared with that of HRP C. Top, stereographic view of barley grain peroxidase, BP 1, viewed along the substrate access channel. The N and C termini (N-term and C-term) are labeled, as well as the 64-84 region and the B, C, F', and F" helices. Disulfide bonds appear as stick models. The protein regions with the largest r.m.s. deviations from the HRP C structure are magenta. Bottom, stereographic view of HRP C. Overall, the structures of BP 1 and HRP C closely resemble one another. However, residues 64-84 of BP 1, comprising the last part of the loop between helices B and C are very different. This loop contains Asn77 and Glu71 of BP 1, the residues equivalent to Asn70 and Glu64 in HRP C and PNP, which provide the hydrogen-bonded link from distal histidine to distal calcium. In BP 1, this network is severely disrupted.

                              
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Table III
Segments of secondary structure in BP 1 
Secondary structure elements are shown as defined by Kabsch and Sander (20) and calculated by the program PROCHECK (19).

Active Site Structure-- The proximal heme side of BP 1 closely resembles that of HRP C. The proximal ligand of the heme iron, His179, is hydrogen-bonded to the invariant proximal aspartate, Asp250, as shown in Fig. 2. In contrast, the distal crevice in BP 1 (Fig. 2) is slightly compressed, since the distances between heme iron and the Calpha atoms of catalytic histidine and arginine are approximately 1 Å shorter than found in HRP C, PNP, CCP, and A. ramosus peroxidase. The loop between helix B and C is displaced significantly relative to HRP C, allowing for a rotation of catalytic distal His by 96° around the Calpha -Cbeta bond, and 109° around the Cbeta -Cgamma bond to a position where the His49 Ndelta 1-Fe distance is 8.2 Å compared with HRP C, where the corresponding His42 Nepsilon 2-Fe distance is 5.9 Å. As a consequence of the movement of distal His, the structural water 626 between distal His and distal Arg is removed away from the heme iron (water-iron distance, 5.7 Å), and Phe48 is rotated to a position with a greater extent of pi -orbital stacking with heme than seen in HRP C. Catalytic distal Arg45 participates in hydrogen bonds with the heme propionates through water molecules 620 and 707 and with backbone atoms Asn77 nitrogen and Thr79 oxygen (Fig. 2) from the extended structure between helix B and helix C. The heme is embedded in a hydrophobic environment. It is noteworthy that the heme propionates are hydrogen-bonded to residues near and in the F', F" insertion unique to class III peroxidases (Leu183 nitrogen, His185 nitrogen, and Ser188 Ogamma ) to Arg81 Nepsilon , Arg81 Neta 2, and through a water molecule to catalytic distal arginine.


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Fig. 2.   The active site of BP 1. Top, stereographic view of the active site of BP 1. A unique hydrogen bond from distal histidine to Thr67 Ogamma from the loop between helix B and C is seen. Two structural water molecules are found in the active site of BP 1. The water molecule between catalytic histidine and arginine is displaced, however, compared with other higher plant peroxidases. Middle, superimposition of the active site of BP 1 (light) and HRP C (dark). The distal histidine of BP 1, His49 is displaced as compared with HRP C to a position 8.25 Å from the heme iron, where it can no longer catalyze compound I formation. Phe48 has moved toward the heme iron, and in doing so the accessibility of the heme iron is diminished. Bottom, Fo - Fc omit electron density map of Phe48 and His49 superimposed on the native BP 1 structure. The contours are 7sigma , with sigma  being the r.m.s. density computed over an entire asymmetric unit.

Cation Binding Sites-- Two sites for cation binding found in the fungal peroxidases lignin peroxidase (22, 23), MnP (24), and Coprinus peroxidase (25, 26) and in plant peroxidases HRP C and PNP are also found in BP 1. The proximal cation binding site is very similar to the corresponding sites in HRP C and PNP and has seven amino acid oxygen ligands with a mean coordination distance of 2.4 Å. Two of the coordinating side chains are aspartates. The cation has been refined as a calcium ion based on coordination number, coordination distance, and structural similarity with HRP C and PNP. The proximal cation is connected to the proximal histidine through several interaction pathways. An extended hydrogen bonding network connects Asp225 Odelta 1-Asp239 Odelta 2-water 614-Leu224 oxygen-water 617-Asp250 Odelta 2 to proximal histidine. No variation in the mean coordination distance for the proximal cation (2.4 Å) is seen when comparing the BP 1 structures at pH 5.5, pH 7.5, and pH 8.5. The distal cation in BP 1 is only six-coordinate, all six ligands being donated by amino acid oxygens, while the distal Ca2+ in both HRP C and PNP is seven-coordinate. Furthermore, the binding between Asp57 Odelta 1 and the cation in BP 1 is weakened as compared with HRP C and PNP, since Asp57 Odelta 2 is only 3.7 Å from Arg290 Nepsilon (corresponding to HRP C Leu290) and participates in an extended hydrogen bonding network through water molecules, Asp57 Odelta 2-water 652-Ser135 Ogamma , Asp57 Odelta 2-water 680-Arg288 oxygen, and Asp57 Odelta 2-water 680-Arg290 Nepsilon . Refining the distal cation as a calcium ion gave a temperature factor for the calcium of 32.5 Å2, which is more than twice the temperature factor for the proximal calcium in BP 1. In addition, the B-factors of the distal cation ligands are in the range 12.8-17.3 Å2 and do not indicate great thermal mobility at the distal site. In the structure of BP 1b at pH 5.5, which had been soaked for 1 week in 10 mM K+, the mean coordination distance for the distal cation is found to be 2.6 Å, while the distance is 2.5 Å in the structures of BP 1b at pH 7.5 and at pH 8.5, which had been exposed to 10 mM Na+. Because of the presence of only six ligands, the weaker negative charge density matching the distal cation in BP 1, the variance in mean coordination distance, and the high temperature factor, the electron density in the distal cation site of BP 1b at pH 7.5 was also refined assuming either a water molecule or a sodium ion. Refining the cation site as a water molecule with an occupancy of 1.0 gave a B-factor of 8.9 Å2, whereas a sodium ion gave a B-factor of 17.1 Å2, similar to the B-factor of the surrounding ligands. Additionally, the sodium model gave a small drop in Rfree of 0.1%, while the water model did not affect the Rfree value. Sodium has an average metal-oxygen distance of 2.45 Å (27), and this is also the average metal-ligand distance found at the distal cation binding site. Protein data base studies have also revealed that sodium has a smaller average number of ligands than calcium (27). Finally, purified BP 1 was stored in 3 M ammonium sulfate containing 2 mM CaCl2 but was dialyzed exhaustively against 10 mM sodium acetate buffer prior to crystallization without including calcium in the buffer. Consequently, the electron density in the distal cation site has been assigned to a sodium ion in the BP 1b structure at pH 7.5, although a partly occupied calcium site could give a similar value of the temperature factor.

Carbohydrate Structure-- A single round of X-PLOR slow cool simulated annealing of the BP 1b, pH 7.5, structure against BP 1a data caused a distinct Fo - Fc density in connection to Asn300. The glycan sequence was previously determined as Manalpha 1-6(Xylbeta 1-2)Manbeta 1-4GlcNAcbeta 1-4(Fucalpha 1-3)GlcNAc (4), and the Fo - Fc density confirms the 1-3 and 1-4 branch points of the first GlcNAc.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Proximal Domain

Substrate Binding Site-- In HRP C, PNP, and BP 1, two beta -strands flank the helical insertion F', F", which so far is unique to class III peroxidases. The two beta -strands are conserved in CCP (28), where they are part of a larger sheet structure that also comprises beta -strands occupying roughly the same three-dimensional space as the F', F" helices. The remaining beta -strands of this sheet in CCP arise from an insertion between helices G and H. Two short beta -strands are also found in the fungal MnP (24), Coprinus peroxidase (25, 26), and lignin (22, 23) peroxidases. In these structures, the F', F" space is filled with a coil structure connecting helices G and H. Ascorbate peroxidase (29) has neither the class III plant peroxidase insertion between helices F and G, nor the insertion between helix G and helix H characteristic of CCP or the fungal peroxidases. The two short beta -strands restrain the structural flexibility, and an invariant disulfide bond at this site in class III peroxidases (Cys186-Cys213) additionally stabilizes the beta -sheet, allowing for a higher degree of structural diversity in plant peroxidases in the unique insertion between the two strands. Alignment of the F', F" substructure of BP 1, HRP C, and PNP shows divergences in the structure of the F' helix, in the loop between F' and F", and in the structure succeeding helix F". A general sequence alignment of class III heme peroxidases shows that this region is one of the most variable among plant peroxidases (30). In particular, the loop connecting F' and F" varies in length and amino acid composition. The ends of the segment between beta 2 and beta 3 appear to be functionally important, since the side chains interact directly with a heme propionate (Ser188 Ogamma forms a hydrogen bond (2.6 Å) to heme propionate 7) and also form one side of the substrate access channel. In HRP C, the structurally equivalent residue of Ser188 is Phe179, which interacts directly with aromatic substrates (31). However, the major part of the class III insert is far from the heme and not believed significantly to influence substrate binding.

Calcium Site-- Two structural calcium binding sites have been proposed for the class III heme peroxidases (30), based on primary sequence homology with class II peroxidases, that have been positively identified as calcium-containing proteins by x-ray crystallographic studies (22, 26) and the calcium content of HRP C (32). Endogenous calcium has been shown to be important for the activity of the classical class III peroxidase, HRP C (33). The class III peroxidases HRP C, PNP, and BP 1 lack a ligand in the proximal cation site compared with the proximal calcium binding site of the fungal class II peroxidases. Whereas Asp225 in BP 1 is a monodentate ligand, the corresponding aspartate in A. ramosus peroxidase, lignin peroxidase, and MnP is a bidentate ligand. The insertion of a single residue (Val226 in BP 1) changes the backbone conformation of class III peroxidases and causes a rotation of the aspartate.

Heme Environment-- The proximal sides of the heme crevice of the three class III peroxidase structures, HRP C, PNP, and BP 1, closely resemble one another. Asp250, imparting an imidazolate character to proximal histidine, His179, is in barley peroxidase hydrogen-bonded to Tyr236 Oeta (2.7 Å) a residue replaced by a hydrophobic residue in CCP, lignin peroxidase, A. ramosus peroxidase, and MnP but conserved in class III peroxidases and in pea cytosolic ascorbate peroxidase. Asp250 is additionally hydrogen-bonded to Ser249 Ogamma , Ser249 oxygen, and Ser179 Ogamma through water molecules, a hydrogen bonding network also found in HRP C.

Distal Domain

Active Site-- Kinetic analysis of BP 1 suggests that BP 1 has a distal histidine in the common arrangement with heme after pH-induced activation (3). The activation has a pKa < 3.7, and the resultant peroxidase exhibits slow compound I formation with unusual kinetic properties. Further activation of BP 1 can be achieved upon calcium addition, compatible with the presence of a calcium site with a KD of 4 mM. The result of calcium ion activation is a peroxidase with kinetic properties very similar to the kinetic properties of HRP C. The region of largest structural difference between BP 1 and HRP C or PNP is residues 64-84 in the loop connecting helices B and C (Fig. 1). The displacement of this region in BP 1 relative to HRP C is independent of pH in the range 5.5-8.5, and its orientation has a major impact on the localization of the amino acid side chains in the distal heme crevice. Earlier studies (34-36) have shown the distal histidine to be a key residue in compound I formation. However, with a distance of 8.25 Å between heme iron and catalytic distal histidine in BP 1, the distal His can no longer catalyze the reaction between peroxidase and peroxide. Additionally, access to heme iron is sterically hindered by the position of Phe48 (Fig. 2). The result is an inactive peroxidase. Distal phenylalanine (Phe48) and histidine (His49) are situated near the end of helix B (residues 38-52) and followed immediately by the 10-residue cation binding loop (residues 50-59) (Fig. 3). This loop is further restrained by an invariant disulfide bond, Cys51-Cys56 (Fig. 1). The segment containing these three features is highly conserved in class III peroxidases, both in amino acid sequence and protein fold (Fig. 1). This essential segment is largely covered by and interacting with residues 64-84, the segment profoundly differentiating BP 1 and HRP C (Fig. 1). The molecular mechanism of BP 1 activation by protonation, and additionally by the binding of calcium ion, is therefore closely linked to the differences in interactions between these two segments, observed in the inactive BP 1 structure and in the active HRP C structure. The reversible change between inactive and active BP 1 seems also facilitated by an increased flexibility in the segment 64-84 in BP 1, which contains three additional glycine residues, Gly64, Gly67, and Gly70, as compared with HRP C. 


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Fig. 3.   Distal cation site in BP 1 and PNP. Top, stereographic view of the distal cation site in BP 1. The side chain (Asp50, Asp57, Ser59) and backbone (Asp50, Val53, Gly55) ligands are homologous to those of HRP C and PNP. However, an additional water ligand is seen in HRP C and PNP, and the charge density on Asp57 is reduced by hydrogen bonding networks as described in the discussion of the distal cation site. An unusual hydrogen bonding network (Asp143 Odelta 1-Gln54 Nepsilon , Gln54 Oepsilon -Ser59 Ogamma makes Ser59 unavailable as hydrogen bond donor to Glu71 Oepsilon , a conserved interaction in other plant peroxidases. The cation in BP 1 is identified as a sodium ion. Bottom, stereographic view of the distal cation site in PNP.

pH-dependent Activation-- The structures of active HRP C and PNP suggest the following hydrogen bonding network connecting the orientation of catalytic distal histidine and the distal cation site to be typical of an active class III peroxidase (HRP C residue numbers with BP 1 numbers in parentheses). Glu64(71) Oepsilon 1 accepts a hydrogen bond from Ser52(59) Ogamma , a distal calcium ligand. Glu64 Oepsilon 2 in turn accepts a hydrogen bond from a water molecule, which is also a calcium ligand, and additionally accepts a hydrogen bond from another water molecule hydrogen-bonded to Asn70(77) Ndelta 2. Additionally, Asn70(77) Ndelta 2 donates a hydrogen bond to Glu64(71) oxygen, while Asn70(77) Odelta 1 accepts a hydrogen bond from His42(49) Ndelta 1. We also expect the pattern to exist in the calcium-bound form of active BP 1, but in BP 1 at pH 5.5, 7.5, and 8.5 this hydrogen-bonded network is entirely disrupted. His49 (Fig. 2) is hydrogen-bonded to a water molecule in the heme crevice, as seen in most peroxidase structures but additionally has a nonstandard hydrogen bond to Thr67 Ogamma 1. Glu71 and Asn77 are caught up in unusual hydrogen bonding patterns and are unavailable for the normal hydrogen bonding partners. Another unusual hydrogen bonding pattern is seen in BP 1 in connection with the distal cation site (Fig. 3). Ser59 Ogamma , a calcium ligand, serves in BP 1 as a hydrogen bond donor to Gln54 Oepsilon 1, which is restricted in its orientation by a hydrogen bond from Asp143 Odelta 1 to Gln54 Nepsilon 2. Asp143 (corresponding to Leu133 in HRP C and Ser133 in PNP) is part of the D' helix noted to have a slightly different orientation in BP 1 as compared with HRP C and PNP. With Ser59 and a water cation ligand unavailable as hydrogen bond donors, the typical interactions of Glu71 are lacking. Protonation of Asp143 will presumably influence the orientation of Gln54 and make Ser59 Ogamma available for the typical hydrogen bond donation to Glu71 (Fig. 3). We predict that protonation of Asp143 is the event triggering pH-dependent activation of BP 1.

An extended hydrogen bonding network in the distal heme crevice of BP 1 involving active site residue Arg45, structural water, Thr79 oxygen, and Asn77 nitrogen ties catalytic arginine up in an orientation close to what is found in HRP C (Fig. 2). However, since both Thr79 and Asn77 are parts of the BC loop and could be expected to move upon a pH decrease, the crystallographic data cannot support speculations about the orientation of distal arginine in the pH-activated form of BP 1.

Cation Site-- The distal cation site in BP 1 has a short intramolecular distance between the cation ligand Asp57 Odelta 2 and Arg290 Nepsilon . In HRP C and PNP, a leucine is the residue structurally equivalent to Arg290. BP 1 additionally lacks the structural water molecule that is the seventh distal cation ligand in HRP C. Consequently, the ligand field in the distal cation site in BP 1 is weaker than what is found in e.g. HRP C. Under the crystallization conditions used, the result is a distal sodium site. No spatial distortions between the distal cation ligands of BP 1 and HRP C or PNP are found (Fig. 3). The calcium activation observed in kinetic studies could be calcium binding to the distal cation site, which from the crystallographic data must be expected to have a lower KD than the similar site in HRP C. The presence of calcium in the distal site will stabilize the typical hydrogen bonding network, thereby reducing the mobility of the catalytic distal histidine. To maintain a fully intact distal domain in BP 1, calcium might be necessary to act as a kind of zipper for the distal hydrogen bonding network. A supporting observation for this hypothesis is that distal calcium is necessary to avoid a collapse of the distal heme crevice in MnP (6). The possibility of an alternative site of calcium binding with KD = 4 mM leading to a BP 1 with typical peroxidase kinetics is discussed in Ref. 3.

In conclusion, barley peroxidase BP 1 is an inactive peroxidase in the pH range 5.5-8.5 due to the reorientation of distal histidine. The movement of the distal histidine is possible, since the extended structure between helix B and helix C has different intramolecular interactions in BP 1 compared with the structures of the other class III peroxidases HRP C and PNP. This arrangement has the potential of being influenced by pH and the distal cation and its ligands. The present study demonstrates unambiguously how the distal domain of a plant peroxidase can adopt a stable conformation different from the active form and shows the importance of the distal cation to structural integrity.

    ACKNOWLEDGEMENTS

We are thankful to Drs. D. J. Schuller and T. L. Poulos for kindly distributing the peanut peroxidase coordinates prior to publication and to Dr. Christine B. Rasmussen for purification of barley peroxidase.

    FOOTNOTES

* This work was supported by Danish Natural Science Research Council Grant 11-9136 and by the Danish National Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors for barley peroxidase BP 1 (codes 1bgp and r1bgpsf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§ To whom correspondence should be addressed. Tel.: 45 35320273; Fax: 45 35320299; E-mail: anette{at}jerne.ki.ku.dk.

1 The abbreviations used are: CCP, Yeast cytochrome c peroxidase; BP, barley peroxidase; HRP C, horseradish peroxidase C; PNP, peanut peroxidase; MnP, manganese peroxidase; MES, 2-(N-morpholino)ethanesulfonic acid; r.m.s., root mean square; r.m.s.d., r.m.s. deviation; Rcryst, Sigma ||Fo- |Fc||/Sigma |Fo|.

2 Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T. and Poulos, T. L. (1997) Nat. Struct. Biol. 4, 1032-1038.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Welinder, K. G. (1992) Curr. Opin. Struct. Biol. 2, 388-393[CrossRef]
  2. Rasmussen, C. B., Bakovic, M., Welinder, K. G., Dunford, H. B. (1993) FEBS Lett. 321, 102-105[CrossRef][Medline] [Order article via Infotrieve]
  3. Rasmussen, C. B., Hiner, A. N. P., and Welinder, K. G. (1998) J. Biol. Chem. 273, 2232-2240[Abstract/Free Full Text]
  4. Johansson, A., Welinder, K. G., Harthill, J. E., Rasmussen, S. K. (1992) Plant Mol. Biol. 18, 1151-1161[Medline] [Order article via Infotrieve]
  5. Smulevich, G., Miller, M. A., Kraut, J., and Spiro, T. G. (1991) Biochemistry 30, 9546-9558[Medline] [Order article via Infotrieve]
  6. Sutherland, G. R. J., Zapanta, L. S., Tien, M., Aust, S. D. (1997) Biochemistry 36, 3654-3662[CrossRef][Medline] [Order article via Infotrieve]
  7. Smulevich, G., Neri, F., Marzocchi, M. P., Welinder, K. G. (1996) Biochemistry 35, 10576-10585[CrossRef][Medline] [Order article via Infotrieve]
  8. Veitch, N. C., Gao, Y., and Welinder, K. G. (1996) Biochemistry 35, 14370-14380[CrossRef][Medline] [Order article via Infotrieve]
  9. Schuller, D. J., Ban, N., van Huystee, R. B., McPherson, A., Poulos, T. L. (1996) Structure 4, 311-321[Medline] [Order article via Infotrieve]
  10. Rasmussen, C. B., Henriksen, A., Abelskov, A. K., Jensen, R. B., Rasmussen, S. K., Hejgaard, J., Welinder, K. G. (1997) Physiol. Plant. 100, 102-110[CrossRef]
  11. Henriksen, A., Petersen, J. F. W., Svensson, A., Hejgaard, J., Welinder, K. G., Gajhede, M. (1992) J. Mol. Biol. 228, 690-692[Medline] [Order article via Infotrieve]
  12. Otwinowski, Z. (1993) Proceedings of the CCP 4 Study Weekend: Data Collection and Processing, pp. 56-62, SERC Daresbury Laboratory, Daresbury, United Kingdom
  13. Collaborative Computer Project, Number 4. (1994) Acta Crystallogr. Sec. D 50, 760-763 [CrossRef][Medline] [Order article via Infotrieve]
  14. Navaza, J. (1994) Acta. Crystallogr. Sec. A 50, 157-163[CrossRef]
  15. Jones, T. A., Zou, J.-Y., Cowan, S. W., Kjeldgaard, M. (1991) Acta Crystallogr. Sec. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
  16. Brünger, A. T., Kuriyan, J., and Karplus, M. (1987) Science 235, 458-460
  17. Brünger, A. T. (1992) X-PLOR version 3.1: A System for X-ray Crystallography and NMR, Yale University Press, New Haven, CT
  18. Engh, R. A., and Huber, R. (1991) Acta Crystallogr. Sec. A 47, 392-400[CrossRef]
  19. Laskowski, R. A., MacArthur, M. W., Moss, S. D., Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291 [CrossRef]
  20. Kabsch, W., and Sander, C. (1983) Biopolymers 22, 2577-2637[Medline] [Order article via Infotrieve]
  21. Welinder, K. G. (1992) in Plant Peroxidases 1980-1990 (Penel, C., Gaspar, T., and Greppin, H., eds), pp. 1-24, University of Geneva, Switzerland
  22. Poulos, T. L., Edwards, S. L., Wariishi, H., and Gold, M. H. (1993) J. Biol. Chem. 268, 4429-4440[Abstract/Free Full Text]
  23. Piontek, K., Glumoff, T., and Winterhalter, K. (1993) FEBS Lett. 315, 119-124[CrossRef][Medline] [Order article via Infotrieve]
  24. Sundaramoorthy, M., Kishi, K., Gold, M. H., Poulos, T. L. (1994) J. Biol. Chem. 269, 32759-32767[Abstract/Free Full Text]
  25. Petersen, J. F. W., Kadziola, A., and Larsen, S. (1994) FEBS Lett. 339, 291-296[CrossRef][Medline] [Order article via Infotrieve]
  26. Kunishima, N., Fukuyama, K., Matsubara, H., Hatanaka, H., Shibano, Y., and Amachi, T. (1994) J. Mol. Biol. 235, 331-344[Medline] [Order article via Infotrieve]
  27. Glusker, J. P. (1991) Adv. Protein Chem. 42, 1-76[Medline] [Order article via Infotrieve]
  28. Poulos, T. L., Freer, S. T., Alden, R. A., Edwards, S. L., Skogland, U., Takio, K., Eriksson, B., Xuong, N., Yonetani, T., Kraut, J. (1980) J. Biol. Chem. 255, 575-580[Free Full Text]
  29. Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341[Medline] [Order article via Infotrieve]
  30. Welinder, K. G., and Gajhede, M. (1993) in Plant Peroxidases: Biochemistry and Physiology (Welinder, K. G., Rasmussen, S. K., Penel, C., and Greppin, H., eds), pp. 35-42, University of Geneva, Switzerland
  31. Veitch, N. C., Williams, R. J. P., Bone, N. M., Burke, J. F., Smith, A. T. (1995) Eur. J. Biochem. 233, 650-658[Abstract]
  32. Haschke, R. H., and Friedhoff, J. M. (1978) Biochem. Biophys. Res. Commun. 80, 1039-1042[Medline] [Order article via Infotrieve]
  33. Morishima, I., Kurono, M., and Shiro, Y. (1986) J. Biol. Chem. 261, 9391-9399[Abstract/Free Full Text]
  34. Erman, J. E., Vitello, L. B., Miller, M. A., Shaw, A., Brown, K. A., Kraut, J. (1993) Biochemistry 32, 9798-9806[Medline] [Order article via Infotrieve]
  35. Miller, M. A., Shaw, A., and Kraut, J. (1994) Nat. Struct. Biol. 1, 524-531[Medline] [Order article via Infotrieve]
  36. Fülöp, V., Phizackerley, R. P., Soltis, S. M., Clifton, I. J., Wakatsuki, S., Erman, J., Hajdu, J., Edwards, S. L. (1994) Structure 2, 201-208[Medline] [Order article via Infotrieve]


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