Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase*

(Received for publication, March 6, 1997, and in revised form, March 24, 1997)

Munirathinam Sundaramoorthy Dagger , Katsuyuki Kishi §, Michael H. Gold § and Thomas L. Poulos Dagger

From the Dagger  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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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.


INTRODUCTION

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).
<UP>MnP</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP><SUB>2</SUB> → <UP>MnP compound I</UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP> (Reaction 1)
<UP> MnP compound I</UP>+<UP>Mn<SUP>II</SUP></UP> → <UP>MnP compound II</UP>+<UP>Mn<SUP>III</SUP></UP> (Reaction 2)
   <UP>MnP compound II</UP>+<UP>Mn<SUP>II</SUP></UP> → <UP>MnP</UP>+<UP>Mn<SUP>III</SUP></UP>+<UP>H</UP><SUB>2</SUB><UP>O</UP> (Reaction 3)
The enzyme-generated MnIII is complexed with a dicarboxylic acid such as oxalate, which is also secreted by the fungus (23, 33, 34). The MnIII-organic acid complex, in turn, oxidizes phenolic substrates, including lignin model compounds (35), lignin (11), chlorinated phenols (9), and mediators (13, 22).

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.


MATERIALS AND METHODS

Enzyme Preparation

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 mM-1 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 and Purification

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).

Crystallization

Crystals 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 Collection

The 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 beta  = 97.16°) and processed using the XENGEN software package (46). The details of the data collections are provided in Table I.

Table I. Crystallographic data collection summary


Crystal MnP* MnP*(Mn) MnP*(EDTA) E35Q-D179N D179N

Data observed 67,431 98,031 73,221 91,040 96,757
Number of reflections 18,298 31,331 20,888 27,424 27,441
Rsym (%)a 13.93 9.12 11.04 10.61 10.35
Highest resolution 2.3 1.9 2.2 2.0 2.0
I/sigma (I) at highest resolution 2.3 1.9 2.3 1.0 1.4
Completeness 100 96 99 98 98

a Rsym = Sigma |Ii - < Ii> |/Sigma Ii, where Ii is the intensity of the ith observation and < Ii> is the mean intensity.

Refinement

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 > 2sigma (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.

Table II. Crystallographic refinement summary


Crystal MnP* MnP*(Mn) MnP*(EDTA) E35Q-D179N D179N

Resolution range (Å) 8.0-2.3 8.0-2.0 8.0-2.2 8.0-2.0 8.0-2.0
Reflections measured 16,384 25,562 18,753 25,117 25,321
Reflections used, F > 2 sigma (F) 14,511 23,907 16,495 22,248 23,362
R factora 0.182 0.200 0.182 0.187 0.213
RMS deviation ofb
  Bond lengths (Å) 0.009 0.008 0.009 0.008 0.009
  Bond angles (°) 1.431 1.388 1.420 1.359 1.398

a R = Sigma ||Fo- |Fc||/Sigma |Fo|.
b The RMS deviations of bond parameters represent the root mean square deviations from expected values. Engh and Huber parameters (52) were used in the refinement.

Table III. Comparison of MnII binding in different crystals


Crystal MnP* (Mn) Native MnP MnP* MnP*(EDTA)

Resolution range (Å) 8.0-2.3
Number of common reflections 13,508
Peak height in Fo - Fc
  Omit map (sigma ) 14.6 12.6 10.7 7.6
B factor of MnII2)
  Occupancy = 1.0 31.47 33.53 43.60 57.95
  Overall B factor (Å2)a 8.48 9.60 9.57 9.83
  Occupancy when B factor fixed at overall B 0.63 0.57 0.49 0.37

a The overall B factors were calculated from the Wilson plot using the common reflections observed in the individual data sets.

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.


RESULTS

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.


Fig. 1. Stereo view of the manganese binding site in the wild-type MnP structure with manganese and its ligands (heme propionate, Glu-35, Glu-39, Asp-179, and the two solvent oxygens). The MnII is represented as a solid sphere and the solvent oxygens as open circles.
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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.


Fig. 2. The Fo - Fc omit maps for the MnII site in the structures of MnP*(EDTA) (A) and MnP*(Mn) (B). Both maps are contoured at 5 sigma . The lowest density at the metal site is about 7 sigma , observed in A, and the highest is 14 sigma  in B.
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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 Mutant

The 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 approx 30° about the Cbeta ---Cgamma 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 Cbeta ---Cgamma 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.


Fig. 3. Difference Fourier maps (|Fo(mutant) - Fc(native)| expiphi (native)) of the D179N single mutant (A) and the E35Q,D179N double mutant (B). The positive density (3 sigma ) is contoured in thick lines, and negative density (3 sigma ) is in thin dashed lines. The thick bonds represent the refined structures of the mutants, and the native structure is superimposed in thin bonds.
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Fig. 4. Stereo representations of refined structures and interactions around the MnII binding site in the D179N single mutant (A) and in the E35Q,D179N double mutant (B). In E35Q,D179N, Gln-35 is modeled in two conformations, and Wat-653 is present only in the open conformation of Gln-35. In D179N Wat-653 is fully occupied, and the extra space left by the movement of Glu-35 is occupied by Wat-441. Wat-653 forms hydrogen bond interaction with the heme propionate, Glu-39, Asn-179, and a solvent (Wat-441 in the single mutant and Wat-650 in the double mutant).
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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 approx 30° rotation about the Cbeta ---Cgamma 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).


DISCUSSION

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 delta -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.


Fig. 5. Edge-on view of van der Waals surface representations of native MnP (A), the D179N single mutant, down the MnII binding site (B), and the E35Q,D179N double mutant (C). The color coding is as follows: heme, red; side chain ligands, green; MnII, yellow; and mutated side chains, purple.
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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.


Fig. 6. Stereo view of the MnII binding site of MnP (shaded bonds) superimposed on LiP (open bonds). The LiP residues are shown in parentheses. The differences in length and courses of the C-terminal chains in both structures are shown. The extra disulfide bond in the longer C terminus of MnP also is shown.
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FOOTNOTES

*   This research is supported by National Science Foundation Grants MCB-9405128 (to T. L. P.) and MCB-9405978 (to M. H. G.) and by U.S. Department of Energy, Division of Energy Biosciences Grant DE-FG06-93ER20093 (to M. H. G.).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 of the MnP1 crystal structure (code 1MNP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.


   To whom correspondence should be addressed: Dept. of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900. Tel.: 714-824-7020; Fax: 714-824-3280; E-mail: poulos{at}uci.edu.
1   The abbreviations used are: LiP, lignin peroxidase; MnP, manganese peroxidase; polyethylene glycol MnP*, Chelex-treated MnP; MnP*(EDTA), MnP* crystallized in the presence of EDTA; MnP*(Mn), MnP* crystallized in the presence of manganese; Wat, water molecule.
2   LiP residue numbers are shown in parentheses throughout.

REFERENCES

  1. Buswell, J. A., and Odier, E. (1987) CRC Crit. Rev. Biotechnol. 6, 1-60
  2. Kirk, T. K., and Farrell, R. L. (1987) Annu. Rev. Microbiol. 41, 465-505 [CrossRef][Medline] [Order article via Infotrieve]
  3. Gold, M. H., Wariishi, H., and Valli, K. (1989) ACS Symp. Ser. 389, 127-140
  4. Gold, M. H., and Alic, M. (1993) Microbiol. Rev. 57, 605-622 [Abstract]
  5. Bumpus, J. A., and Aust, S. D. (1987) BioEssays 6, 166-170
  6. Hammel, K. E. (1989) Enzyme Microb. Technol. 11, 776-777
  7. Valli, K., Brock, B. J., Joshi, D. K., and Gold, M. H. (1992) Appl. Environ. Microbiol. 58, 221-228 [Abstract]
  8. Valli, K., Wariishi, H., and Gold, M. H. (1992) J. Bacteriol. 174, 2131-2137 [Abstract]
  9. Joshi, D. K., and Gold, M. H. (1993) Appl. Environ. Microbiol. 59, 1779-1785 [Abstract]
  10. Kuwahara, M., Glenn, J. K., Morgan, M. A., and Gold, M. H. (1984) FEBS Lett. 169, 247-250 [CrossRef]
  11. Wariishi, H., Valli, K., and Gold, M. H. (1991) Biochem. Biophys. Res. Commun. 176, 269-275 [Medline] [Order article via Infotrieve]
  12. Hammel, K. E., Jensen, K. A., Jr., Mozuch, M. D., Landucci, L. L., Tien, M., and Pease, E. A. (1993) J. Biol. Chem. 268, 12274-12281 [Abstract/Free Full Text]
  13. Bao, W., Fukushima, Y., Jensen, K. A., Moen, M. A., Jr., and Hammel, K. E. (1994) FEBS Lett. 354, 297-300 [CrossRef][Medline] [Order article via Infotrieve]
  14. Perie, F. H., and Gold, M. H. (1991) Appl. Environ. Microbiol. 57, 2240-2245 [Medline] [Order article via Infotrieve]
  15. Orth, A. B., Royse, D. J., and Tien, M. (1993) Appl. Environ. Microbiol. 59, 4017-4023 [Abstract]
  16. Hatakka, A. (1994) FEMS Microbiol. Rev. 13, 125-135 [CrossRef]
  17. Glenn, J. K., and Gold, M. H. (1985) Arch. Biochem. Biophys. 242, 329-341 [Medline] [Order article via Infotrieve]
  18. Glenn, J. K., Akileswaran, L., and Gold, M. H. (1986) Arch. Biochem. Biophys. 251, 688-696 [Medline] [Order article via Infotrieve]
  19. Mino, Y., Wariishi, H., Blackburn, N. J., Loehr, T. M., and Gold, M. H. (1988) J. Biol. Chem. 263, 7029-7036 [Abstract/Free Full Text]
  20. Wariishi, H., Akileswaran, L., and Gold, M. H. (1988) Biochemistry 27, 5365-5370 [Medline] [Order article via Infotrieve]
  21. Wariishi, H., Dunford, H. B., MacDonald, I. D., and Gold, M. H. (1989) J. Biol. Chem. 264, 3335-3340 [Abstract/Free Full Text]
  22. Wariishi, H., Valli, K., Renganathan, V., and Gold, M. H. (1989) J. Biol. Chem. 264, 14185-14191 [Abstract/Free Full Text]
  23. Wariishi, H., Valli, K., and Gold, M. H. (1992) J. Biol. Chem. 267, 23688-23695 [Abstract/Free Full Text]
  24. Banci, L., Bertini, I., Bini, T., Tien, M., and Turano, P. (1993) Biochemistry 32, 5825-5831 [Medline] [Order article via Infotrieve]
  25. Pribnow, D., Mayfield, M. B., Nipper, V. J., Brown, J. A., and Gold, M. H. (1989) J. Biol. Chem. 264, 5036-5040 [Abstract/Free Full Text]
  26. Pease, E. A., Andrawis, A., and Tien, M. (1989) J. Biol. Chem. 264, 13531-13535 [Abstract/Free Full Text]
  27. Godfrey, B. J., Mayfield, M. B., Brown, J. A., and Gold, M. H. (1990) Gene (Amst.) 93, 119-124 [CrossRef][Medline] [Order article via Infotrieve]
  28. Mayfield, M. B., Godfrey, B. J., and Gold, M. H. (1994) Gene (Amst.) 142, 231-235 [CrossRef][Medline] [Order article via Infotrieve]
  29. Alic, M., Akileswaran, L., and Gold, M. H. (1997) Biochim. Biophys. Acta 1338, 1-7 [Medline] [Order article via Infotrieve]
  30. Orth, A. B., Rzhetskaya, M., Cullen, D., and Tien, M. (1994) Gene (Amst.) 148, 161-165 [CrossRef][Medline] [Order article via Infotrieve]
  31. Dunford, H. B., and Stillman, J. S. (1976) Coord. Chem. Rev. 19, 187-251 [CrossRef]
  32. Renganathan, V., and Gold, M. H. (1986) Biochemistry 25, 1626-1631
  33. Kuan, I-C., Johnson, K. A., and Tien, M. (1993) J. Biol. Chem. 268, 20064-20070 [Abstract/Free Full Text]
  34. Kishi, K., Wariishi, H., Marquez, L., Dunford, H. B., and Gold, M. H. (1994) Biochemistry 33, 8694-8701 [Medline] [Order article via Infotrieve]
  35. Tuor, U., Wariishi, H., Schoemaker, H. E., and Gold, M. H. (1992) Biochemistry 31, 4986-4995 [Medline] [Order article via Infotrieve]
  36. Edwards, S. L., Raag, R., Wariishi, H., Gold, M. H., and Poulos, T. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 750-754 [Abstract]
  37. Piontek, K., Glumoff, T., and Winterhalter, K. (1993) FEBS Lett. 315, 119-124 [CrossRef][Medline] [Order article via Infotrieve]
  38. Poulos, T. L., Edwards, S. L., Wariishi, H., and Gold, M. H. (1993) J. Biol. Chem. 268, 4429-4440 [Abstract/Free Full Text]
  39. Sundaramoorthy, M., Kishi, K., Gold, M. H., and Poulos, T. L. (1994) J. Biol. Chem. 269, 32759-32767 [Abstract/Free Full Text]
  40. Johnson, F., Loew, G. H., and Du, P. (1993) in Plant Peroxidases: Biochemistry and Physiology (Welinder, K. G., Rasmussen, S. K., Penel, C., and Greppin, H., eds), pp. 31-34, University of Geneva, Geneva, Switzerland
  41. Kusters-van Someren, M., Kishi, K., Lundell, T., and Gold, M. H. (1995) Biochemistry 34, 10620-10627 [Medline] [Order article via Infotrieve]
  42. Kishi, K., Kusters-van Someren, M., Mayfield, M. B., Sun, J., Loehr, T. M., and Gold, M. H. (1996) Biochemistry 35, 8986-8994 [CrossRef][Medline] [Order article via Infotrieve]
  43. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59 [CrossRef][Medline] [Order article via Infotrieve]
  44. Alic, M., Clark, E. K., Kornegay, J. R., and Gold, M. H. (1990) Curr. Genet. 17, 305-311
  45. Sundaramoorthy, M., Kishi, K., Gold, M. H., and Poulos, T. L. (1994) J. Mol. Biol. 238, 845-848 [CrossRef][Medline] [Order article via Infotrieve]
  46. Howard, A. J., Gilliland, G. L., Finzel, B. F., Poulos, T. L., Olendorh, D. H., and Salemme, F. R. (1987) J. Appl. Crystallogr. 20, 824-844 [CrossRef]
  47. Brünger, A. T. (1992) X-PLOR Manual, Version 3.1, Yale University, New Haven, CT
  48. Jones, T. A. (1985) Methods Enzymol. 115, 157-171 [Medline] [Order article via Infotrieve]
  49. Poulos, T. L., and Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205 [Free Full Text]
  50. Edwards, S. L., Nguyen, H. X., Hamlin, R. C., and Kraut, J. (1987) Biochemistry 26, 1503-1511 [Medline] [Order article via Infotrieve]
  51. Harris, R. Z., Wariishi, H., Gold, M. H., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 8751-8758 [Abstract/Free Full Text]
  52. Engh, R. A., and Huber, R. (1991) Acta Crystallogr. A 47, 110 [CrossRef][Medline] [Order article via Infotrieve]
  53. Goodin, D. B., and McRee, D. E. (1993) Biochemistry 32, 3313-3324 [Medline] [Order article via Infotrieve]

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