(Received for publication, March 6, 1997, and in revised form, March 24, 1997)
From the Department of Molecular Biology & Biochemistry and Physiology & Biophysics, University of California,
Irvine, California 92697-3900 and § Department of
Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate
Institute of Science & Technology,
Portland, Oregon 97291-1000
Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.
White-rot basidiomycete fungi are capable of degrading the plant cell wall polymer, lignin (1-4), and a wide variety of aromatic pollutants (5-9). The best-studied lignin-degrading fungus, Phanerochaete chrysosporium, secretes two types of extracellular heme peroxidases, lignin peroxidase (LiP)1 and manganese peroxidase (MnP), which, along with an H2O2-generating system, are the major extracellular components of its lignin-degrading system (1, 2, 4, 10-12). Both LiP and MnP depolymerize lignin in vitro (11-13). Moreover, MnP is produced by all white-rot fungi known to degrade lignin extensively (14-16).
P. chrysosporium MnP has been characterized by a variety of biochemical and biophysical methods (4, 17-24). In addition, the sequences of cDNA and genomic clones encoding several P. chrysosporium MnP isozymes (mnp1, mnp2, and mnp3) have been determined (4, 25-30). Biophysical studies and DNA sequences suggest that the heme environment and catalytic cycle of MnP are similar to those of other heme peroxidases, such as horseradish peroxidase and LiP (31, 32). However, MnP is unique in its ability to catalyze the one-electron oxidation of MnII to MnIII (18, 20, 23) in a multi-step reaction cycle (see Reactions 1-3).
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(Reaction 1) |
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(Reaction 2) |
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(Reaction 3) |
Recently, the crystal structures of both LiP and MnP have been reported (36-39). Both enzymes have the same tertiary fold and share topology with other heme peroxidases (39). These structures also confirm that the heme environments of LiP and MnP are similar to those of cytochrome c peroxidase, plant, and fungal peroxidases (38, 39). However, MnP has a unique cation binding site consisting of Glu-35, Glu-39, Asp-179, and one of the heme propionates, and this site has been proposed as the MnII binding site (39, 40). The recent characterization of MnP site-directed mutants at Asp-179, Glu-35, and Glu-39 (41, 42) suggests that these residues form the manganese binding site. In the present study, we have crystallized MnP in the presence of various amounts of MnII to further probe the MnII binding site of this protein. In addition, we have solved and refined the crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) of amino acid ligands at the MnII binding site.
Wild-type MnP isozyme 1 was purified
from the extracellular medium of acetate-buffered, agitated cultures of
P. chrysosporium strain OGC101, a derivative of strain
BKM-F-1767, as described (17, 21). The enzyme concentration was
determined at 406 nm using an extinction coefficient of 129 mM1 cm
1 (17). In an attempt to
remove the enzyme-bound MnII ion, MnP was applied to a
Chelex 100 (Bio-Rad) column (1.0 × 20 cm), equilibrated with 100 mM sodium phosphate buffer (pH 6.5) at room temperature,
and eluted with the same buffer. The protein was desalted by
ultrafiltration. The MnII and CaII content of
the Chelex-treated MnP (MnP*) was determined by atomic absorption
spectroscopy.
Site-directed mutagenesis was carried out by overlap extension (43) using the polymerase chain reaction as described (41, 42). Transformation of P. chrysosporium mutants was carried out as described (42, 44). Production and purification of variant proteins were as described previously (41, 42).
CrystallizationCrystals of MnP*, the D179N single mutant MnP and the E35Q,D179N double mutant MnP, were grown by the hanging drop vapor diffusion method as described (45). Approximately 5 µl of the protein solution (9-19 mg/ml) were mixed with an equal volume of 30% polyethylene glycol 8000, 0.2 M ammonium sulfate, and 0.1 M sodium cacodylate buffer (pH 6.5) and equilibrated against 1 ml of the same buffer for 1-2 days. The protein solution drops were microseeded by first touching the crystals of native MnP crystals with a very thin metal wire and then touching the protein solution drops. For macroseeding, the small seed crystals grown by touch seeding were washed successively in solutions containing 35, 32.5, and 30% polyethylene glycol 8000. A washed seed crystal was added to a freshly pre-equilibrated protein solution drop, after which diffraction quality crystals grew to the required size in a few weeks.
Co-crystallization of MnP* in the presence of MnII or EDTA was carried out by mixing the appropriate reagent with the protein solution drops. MnP* and MnII co-crystals [MnP*(Mn)] were grown with 2 mM MnSO4 in the protein solution. EDTA (2 mM) was included in the drops to grow crystals of MnP*(EDTA).
During crystallization at ambient temperature, it was observed that either the protein denatured or the crystals bleached within 2 weeks, except in the case of MnP*(Mn); therefore, MnP*, MnP*(EDTA), E35Q,D179N, and D179N crystals were grown at 7-8 °C.
Data CollectionThe crystals were very stable at room
temperature during data collection and diffracted to high resolution so
that complete data sets could be collected with one crystal in each
case. All data sets were collected on a Siemens area detector system
using a rotating anode x-ray source equipped with focusing optics. The data were indexed in the C2 space group with the same unit cell as the
native crystal (a = 163.24 Å, b = 45.97 Å, c = 53.57 Å and = 97.16°)
and processed using the XENGEN software package (46). The details of
the data collections are provided in Table I.
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The native MnP crystal structure reported
earlier (39) was used as the starting model for refinement in all
cases. With MnP*, MnP*(EDTA), and MnP*(Mn), a 50-cycle positional
refinement with B factors set at 15 Å2 was
carried out, followed by 10 cycles of group B factors and 20 cycles of individual B factors for all non-hydrogen atoms
using X-PLOR (47) (Table II). Objective estimates of the
relative occupancies of the MnII site were obtained by
refining the models using the common reflections observed in all the
data sets in the 8.0-2.3-Å resolution range with a F > 2(F) cutoff. Fo
Fc omit electron density maps were generated by
removing MnII from the models, followed by 20 rounds of
positional refinement. When MnII was included in the
refinement, the B factor for MnII was set equal
to the overall temperature factor obtained from the Wilson plot for the
same set of reflections, and only the occupancy of MnII was
refined in the last round of refinement. The refinements converged
typically in 6-7 cycles. Alternatively, the occupancy for
MnII was set to unity, and the B factor was
refined for all the data sets. Various measurements of the relative
occupancies of the MnII site, i.e. Fo
Fc electron density, B factor, and
occupancy, are listed in Table III for all data
sets.
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In the refinement of the mutant structures, the MnII and
its ligands, with the exception of the heme, were omitted from the refinement, and electron density maps were calculated. The changes around the MnII were discernible in the difference Fourier
maps. The side chains and solvent structure around the mutated sites
were rebuilt guided by the 2Fo Fc and Fo
Fc maps using the TOM FRODO graphics software (48),
followed by refinements of the model iteratively until the maps were
fitted satisfactorily and R factors converged. At the final
stage, omit maps were calculated excluding the remodeled side chains
and solvent molecules from 25 cycles of positional refinement. The
details of refinement are provided in Table II.
The crystal structure of the native MnP was reported earlier, and
the proposed MnII binding site was based on this structure
(39). The obligatory substrate for the enzyme, MnII, binds
to a heme propionate and is coordinated to five other ligands in an
octahedral geometry (Fig. 1). Three of the
MnII ligands are acidic amino acid side chains, Glu-35,
Glu-39, and Asp-179, and the remaining two are oxygen atoms of water
molecules. The site is at the surface of the protein and is accessible
to the solvent.
Crystal Structure of MnP* in the Presence of EDTA or Excess MnII
When grown at room temperature, the crystals of
the Chelex-treated protein, MnP*, bleached before growing to a size
suitable for diffraction. However, the crystals were stable at 7 °C
and grew to full size, although at a slower rate. The data set for MnP*
extended to a slightly lower resolution compared with the average
resolution of the native MnP crystals previously obtained (Table I)
(39). The difference Fourier map calculated with the MnII
excluded from the structure showed a reduced, but significant, electron
density peak at the MnII site indicating that
MnII was not removed completely (Table III). Crystals grown
in the presence of EDTA [MnP*(EDTA)] were similar to MnP* crystals
but exhibited a much lower peak in the Fo Fc electron density map, which was close to the
average peak height of water (Fig. 2A and
Table III). However, there was no change in the orientation of the
MnII ligands, suggesting that the MnP*(EDTA) crystal still
might have a cation bound, either MnII with much lower
occupancy or possibly another cation such as sodium.
MnP* crystals grown in the presence of excess MnII [MnP*(Mn)] showed approximately the same peak height at the MnII binding site as observed in the initial MnP structure determination (Fig. 2B and Table III) (39), indicating that the proposed MnII site in the enzyme was at least partially occupied by MnII ion throughout the purification process. Importantly, the addition of excess MnII to MnP* did not lead to a new electron density peak, strongly suggesting that there is no other major MnII binding site in MnP.
To gain some insight into relative occupancies and disorder, we used two different refinement approaches, considering only the common reflections in the 8.0- to 2.3-Å resolution range for all of the crystals. First, the occupancies were held constant at 1.0 for all non-hydrogen atoms, including the MnII ion, and the crystallographic temperature or B factors were refined. The B factor of the MnII ion refined to a value of 33.5 Å2 compared with 8.4 and 10.0 Å2 for the two calcium sites in the native data set. On the other hand, the B factor for the MnII site for MnP*(EDTA) data increased to 58.0 Å2. In the second set of refinements, the B factor of the MnII site was fixed at the value determined from Wilson statistics, and the occupancy of the MnII site was refined. The occupancy fell well below 1.0 in all cases, the lowest being 0.37 for MnP*(EDTA). These results suggest that the MnP*(Mn) and native MnP crystals were more fully occupied with MnII, whereas crystals grown in the absence of MnII or in the presence of EDTA were only partially occupied with MnII or possibly occupied with another cation such as sodium.
Structure of the Single MutantThe Fo Fc maps for the D179N mutant data set, calculated
using the native MnP coordinates, including and excluding the
MnII and its ligands, suggested the absence of a cation in
the MnII site (Fig. 3A). The maps
are very noisy in this region, indicating large changes in the
structure as a result of the mutation. The mutated residue, Asn-179,
undergoes very little conformational change from its native position,
except a small rotation of
30° about the
C
---C
bond. However, the other two
MnII ligands, Glu-35 and Glu-39, undergo large changes
(Fig. 4A). Both Glu-35 and Glu-39 turn away
from the MnII site and, consequently, the solvent structure
in this region rearranges. The Glu-35 side chain rotates almost 110°
about the C
---C
bond and becomes
solvent-exposed. This leaves a void that fills with two solvent
molecules (Wat-653 and Wat-441). Wat-653 is about 1.5 Å from the
MnII site and still interacts with the side chains of
Asn-179 and Glu-39 and with the propionate. Wat-520, bridging the two
propionates and a ligand to MnII in the native structure,
moves about 2.0 Å. In its new position, this water forms a hydrogen
bond interaction with the side chain amide of Asn-179.
Structure of the Double Mutant
Similar to the single mutant,
the difference Fourier map calculated using the E35Q,D179N double
mutant data set and the coordinates of native MnP, including and
excluding MnII and its non-heme ligands, do not show any
significant positive density that can be interpreted as a cation in the
vicinity of the MnII site. On the other hand, the
difference map calculated using the complete set of native MnP
coordinates showed a large negative peak in the MnII site
(Fig. 3B), indicating the absence of a cation or only
partial occupancy of this site. The refined structure of the mutant
around the MnII site is shown in Fig. 4B. One of
the mutated residues, Gln-35, is disordered and appears to be in
multiple conformations. Gln-35 was modeled in two conformations, one
pointing outward and the other pointing inward as in the native
conformation, with 50% occupancy of each. In one of the two
conformations, Gln-35 retains a hydrogen bond with the side chain of
Arg-177. In the conformation pointing outward, the void is occupied by
a water molecule (Wat-653) which is about 1.5 Å from the
MnII site. This is similar to the single mutant structure
(Fig. 4, A and B). The other mutated residue,
Asn-179, undergoes little conformational change from the native
position of Asp-179. There is a 30° rotation about the
C
---C
bond, which does not alter its
position or local interactions significantly. The side chain carbonyl
oxygen of Asn-179 retains the hydrogen bond with the backbone amide of
Ala-187 and the side chain amino group hydrogen bonds with the
invariant solvent molecule (Wat-459), analogous to the native
structure. In the double mutant protein, Glu-39 undergoes a dramatic
conformational change in the absence of a cation in the proposed
MnII site, swinging out and away from the MnII
site. One of the side chain carboxylate oxygens of Glu-39 in the mutant
forms a weak hydrogen bond (3.2-3.3 Å) with both conformations of the
Gln-35 side chain. In the absence of the cation in the double mutant,
one of the solvent ligands, Wat-520, moves by 2.0 Å out of the plane
formed by the heme propionates, while still bridging the heme
propionates as was observed in the single mutant. Another feature of
the mutant structure is the large movement of the distal Arg-42 toward
the peroxide binding pocket, so as to form a hydrogen bond with the
distal Wat-556. This Arg is invariant in non-mammalian heme peroxidases
and has been implicated as an important residue in cleavage of the
H2O2 O---O bond during the formation of
compound I (49). Such movement of the distal Arg has been observed in
the crystal structure of compound I of CcP (50).
MnP is a unique heme peroxidase that oxidizes MnII to
MnIII (18, 21, 23). The enzyme-generated MnIII,
complexed with an organic acid such as oxalate, oxidizes either the
terminal phenolic substrate (18, 35) or a mediator (13, 22). Our
earlier crystallographic study (39), as well as homology modeling of
MnP (40), predicts a MnII binding site close to the surface
of the protein, consisting of three acidic amino acid residues,
Asp-179, Glu-35, and Glu-39 and one of the heme propionates.
Site-directed mutagenesis studies on the amino acid ligands in the
manganese binding site demonstrate that this is the productive binding
site (41, 42). In contrast, earlier work by Harris et al.
(51) and Banci et al. (24) suggested a MnII
binding site close to the -meso edge of the heme.
The crystals of MnP* and of MnP*(EDTA) exhibit reduced electron density at the proposed MnII binding site, indicating reduced MnII occupancy. However, our results indicate that the MnII is not completely removed from the MnP* or MnP*(EDTA) crystals, although atomic absorption spectroscopic analyses indicate that these proteins contain less than 0.2% MnII ion (data not shown). Since MnP has a higher affinity for MnII at pH 6.5 (the pH of the cacodylate buffer used for crystallization) than at the physiological pH of 4.5 (data not shown), a trace amount of contaminating MnII in the buffer may bind to MnP during crystallization. MnP* crystals grown in the presence of excess MnII exhibit sharply increased electron density at the proposed binding site, suggesting that the electron density at this site is, indeed, due to MnII (Table III). Furthermore, crystals grown in the presence of excess MnII exhibit no additional large positive peaks in the electron density map, indicating that there is no other strong MnII binding site in MnP.
Characterization of site-directed mutations at the MnII binding site of MnP, including the D179N, E35Q, and E39Q single mutations and the D179N,E35Q double mutations, strongly suggests that this is the productive MnII binding site of MnP (41, 42). Kinetic analyses of the single mutants, E35Q, E39Q, and D179N, yielded Km values for the substrate MnII that were ~50-fold greater than the corresponding Km value for the wild-type enzyme. Similarly, the kcat values for MnII oxidation were ~300-fold lower than that for the wild-type MnP. The E35Q,D179N double mutant had a Km value for MnII that was ~120-fold greater and a kcat value that was ~1000-fold less than those for the wild-type MnP. Transient-state kinetic analysis for the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first-order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were 200- and ~4000-fold less, respectively, than that for the wild-type enzyme. In contrast, the Km values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. Thus, these mutants affect both binding and electron transfer from MnII to compound II but do not affect the formation of compound I (41, 42).
The present study provides a structural basis for understanding the functional consequences of mutating the MnII ligands. The structures of Chelex-treated MnP (MnP*) and MnP* crystals grown in the presence of EDTA exhibit greatly diminished electron density at the proposed MnII site. The electron density returns upon co-crystallizing MnP* in excess MnII, with no other peaks of electron density appearing elsewhere in the protein. This indicates that the previously observed (39) electron density at this site is due to manganese rather than another cation. These results also suggest that MnP contains only one major MnII binding site. The positions of Asp-179, Glu-35, and Glu-39 do not change significantly in MnP*, probably because MnII remains bound at very low occupancy with the site being shared by another cation or water. Alternatively, manganese is completely replaced by another smaller buffer ion at this position. In contrast, we observe a large change in the position of Glu-35 and Glu-39 in the mutant MnPs. These changes in structure are consistent with the increasing Km and KD values for the mutants. Most likely, in the absence of a cation, these anionic ligands rotate to lower the strong negative charge at this site. It is also interesting that the steady-state kcat values and first-order rate constants for compound II reduction are significantly lower in the mutants. The lower electron transfer rate, as reflected in the first-order rate constant, is probably the result of weaker binding of MnII. Weaker binding of MnII by the mutant protein might decrease the amount of electrostatic stabilization of the MnII ion by the negatively charged carboxylates that, in turn, would result in a higher redox potential for the MnII in the binding site compared with that for the wild-type enzyme. This higher redox potential would negatively affect the electron transfer rate. There is some support for this idea since mutagenesis results with other peroxidases show that decreasing the electronegative character of the proximal His heme ligand results in an increase in heme redox potential (53).
The mutations also alter the electrostatic environment at the binding
site. In the wild-type protein, the MnII is surrounded by
four carboxylates, one of which pairs with Arg-177, yielding a net
charge of 3. In the single mutant one of these negative charges is
removed, and in the double mutant two negative charges are removed. The
excess negative charge in the wild-type protein may promote oxidation
of MnII to MnIII. The loss of this
electrostatic energetic incentive in the mutants may also partially
explain the decrease in the electron transfer rate.
Previous work shows that the MnIII produced by the enzyme
is released as a MnIII-chelator complex. The latter forms a
stable diffusible oxidant (23). The wild-type and mutant structures may
help to elucidate this part of the catalytic cycle. Unlike other
peroxidases, the heme propionate side chains of MnP are
solvent-exposed, allowing access for MnII binding (Fig.
5A). The metal ligand distances of the
MnII ligands increase in the following order: OD1 of heme 6 propionate (2.34 Å), OD1 of Asp-179 (2.57 Å), OE1 of Glu-35 (2.69 Å), and OE1 of Glu-39 (2.82 Å). The B factor or temperature factor
for these ligands increases in the same order. These subtle differences suggest that, although still required for MnII binding,
Glu-35 and Glu-39 are weaker ligands than the heme propionate or
Asp-179. Comparison of the native and mutant MnP structures also
suggests that the Glu-35 and Glu-39 side chains assume different conformations depending upon whether or not MnII is bound.
When the MnII is bound, the ligands are oriented toward the
metal. In the absence of manganese, the side chains of Glu-35 and
Glu-39 swing away to disperse the negative charge, resulting in the
formation of an open cavity. This suggests that these two ligands may
act as a gate for MnII/III, binding the incoming
MnII in their closed conformations and releasing the
oxidized MnIII in their open conformations. Fig. 5,
B and C, shows that the propionates in the mutant
structures are more solvent-exposed when Glu/Gln-35 and Glu-39 are in
their open conformations. Such a gate could facilitate productive
catalysis, particularly since MnIII must bind to a
dicarboxylic acid to serve as a diffusible oxidant. Glu-35 and Glu-39
may facilitate the release of the MnIII to an incoming
dicarboxylic acid.
It also is possible that the open nature of the MnII binding site in the manganese-free protein might facilitate the binding of a MnII-oxalate complex in the manganese-saturated MnP. When the amino acid ligands form a closed site, the MnII-chelator complex may not be able to enter. Although free MnII can bind to the enzyme, as demonstrated here and in our previous work (23, 39), a range of kinetic experiments suggest that a MnII-chelator complex is the best substrate for the enzyme (17, 33, 34). If the MnII-chelator complex is the real MnP substrate, the two water molecules in the MnP crystal structure (39) would be replaced by the chelator. To date, we have not been able to obtain a co-crystal of MnP and a MnII-chelator complex.
Despite the presence of this unique MnII binding site, the
overall structure of MnP is very similar to all other non-mammalian heme peroxidases for which structures are available. Apparently the
localized structural alterations near the surface of the protein required to form the MnII site do not induce significant
changes in the core peroxidase structure. For example, the structure of
P. chrysosporium LiP is very similar to that of MnP but
lacks the MnII site. LiP has only one of the three acidic
residues, Glu-39 (Glu-40 in LiP)2
(Fig. 6). In place of Glu-35 and Asp-179, LiP contains
alanine (Ala-36) and asparagine (Asn-182), respectively (39). Although it is possible to accommodate an aspartic acid in place of asparagine (Asn-182) in the LiP structure, the space occupied by the side chain of
Glu-35 in MnP is filled by the backbone structure of the C terminus in
LiP. MnP has a longer C terminus, which deviates considerably in its
course from that of LiP. In addition, Arg-177 pushes the polypeptide
chain out and away from the main body of the protein to form the
MnII site in MnP. The corresponding residue in LiP is an
alanine (Ala-180). Finally, MnP has an extra disulfide that helps to
force the polypeptide chain away from the body of the protein. These
differences result in the formation of space for Glu-35 near the cation
binding site. These comparisons suggest that constructing a productive
MnII binding site in LiP by protein engineering may require
more than a few simple amino acid substitutions, although it should be
possible by a combination of additional genetic, kinetic, and
structural studies to more precisely elucidate the electron transfer
pathway in the MnP enzyme system.
The atomic coordinates and structure of the MnP1 crystal structure (code 1MNP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.