From the 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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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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.
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EXPERIMENTAL PROCEDURES |
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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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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.
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RESULTS |
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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 (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
-
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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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
C 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
-helices are present in BP 1, of
which 10 are conserved among all known heme peroxidases of the plant peroxidase superfamily. The F
-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
-sheets
are found in BP 1. The strands
2-(183-185),
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
1-(132-133) and
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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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 C 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
C
-C
bond, and 109° around the
C
-C
bond to a position where the
His49 N
1-Fe distance is 8.2 Å compared with
HRP C, where the corresponding His42 N
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
-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 O
)
to Arg81 N
, Arg81
N
2, and through a water molecule to catalytic distal
arginine.
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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 O1-Asp239
O
2-water 614-Leu224 oxygen-water
617-Asp250 O
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 O
1
and the cation in BP 1 is weakened as compared with HRP C and PNP,
since Asp57 O
2 is only 3.7 Å from
Arg290 N
(corresponding to HRP C
Leu290) and participates in an extended hydrogen bonding
network through water molecules, Asp57
O
2-water 652-Ser135 O
,
Asp57 O
2-water 680-Arg288
oxygen, and Asp57 O
2-water
680-Arg290 N
. 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
Man
1-6(Xyl
1-2)Man
1-4GlcNAc
1-4(Fuc
1-3)GlcNAc (4),
and the Fo
Fc density confirms
the 1-3 and 1-4 branch points of the first GlcNAc.
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DISCUSSION |
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Proximal Domain
Substrate Binding Site--
In HRP C, PNP, and BP 1, two
-strands flank the helical insertion F
, F", which so far is unique
to class III peroxidases. The two
-strands are conserved in
CCP (28), where they are part of a larger sheet structure that also
comprises
-strands occupying roughly the same three-dimensional
space as the F
, F" helices. The remaining
-strands of this sheet in
CCP arise from an insertion between helices G and H. Two short
-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
-strands restrain the
structural flexibility, and an invariant disulfide bond at this site in
class III peroxidases (Cys186-Cys213)
additionally stabilizes the
-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
2 and
3 appear
to be functionally important, since the side chains interact directly
with a heme propionate (Ser188 O
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
O (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 O
, Ser249 oxygen, and
Ser179 O
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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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)
O1 accepts a hydrogen bond from Ser52(59)
O
, a distal calcium ligand. Glu64
O
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) N
2. Additionally,
Asn70(77) N
2 donates a hydrogen bond to
Glu64(71) oxygen, while Asn70(77)
O
1 accepts a hydrogen bond from His42(49)
N
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 O
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
O
, a calcium ligand, serves in BP 1 as a hydrogen bond
donor to Gln54 O
1, which is restricted in
its orientation by a hydrogen bond from Asp143
O
1 to Gln54 N
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
O
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.
Cation Site--
The distal cation site in BP 1 has a short
intramolecular distance between the cation ligand Asp57
O2 and Arg290 N
. 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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, ||Fo|
|Fc||/
|Fo|.
2 Gajhede, M., Schuller, D. J., Henriksen, A., Smith, A. T. and Poulos, T. L. (1997) Nat. Struct. Biol. 4, 1032-1038.
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REFERENCES |
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