Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase*,

Xiaotang WangDagger §, Hiroyasu TachikawaDagger , Xianwen Yi, Kelath M. Manoj, and Lowell P. Hager||

From the Dagger  Department of Chemistry, Jackson State University, Jackson, Mississippi 39217 and the  Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

Received for publication, September 16, 2002, and in revised form, December 9, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The heme active site structure of chloroperoxidase (CPO), a glycoprotein that displays versatile catalytic activities isolated from the marine mold Caldariomyces fumago, has been characterized by two-dimensional NMR spectroscopic studies. All hyperfine shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues including the heme iron ligand (Cys29) attributable to the unique catalytic properties of CPO have been firmly assigned through (a) measurement of nuclear Overhauser effect connectivities, (b) prediction of the Curie intercepts from both one- and two-dimensional variable temperature studies, (c) comparison with assignments made for cyanide derivatives of several well characterized heme proteins such as cytochrome c peroxidase, horseradish peroxidase, and manganese peroxidase, and (d) examination of the crystal structural parameters of CPO. The location of protein modification that differentiates the signatures of the two isozymes of CPO has been postulated. The function of the distal histidine (His105) in modulating the catalytic activities of CPO is proposed based on the unique arrangement of this residue within the heme cavity. Contrary to the crystal state, the high affinity Mn(II) binding site in CPO (in solution) is not accessible to externally added Mn(II). The results presented here provide a reasonable explanation for the discrepancies in the literature between spectroscopists and crystallographers concerning the manganese binding site in this unique protein. Our study indicates that results from NMR investigations of the protein in solution can complement the results revealed by x-ray diffraction studies of the crystal form and thus provide a complete and better understanding of the actual structure of the protein.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Chloroperoxidase is a monomeric glycoprotein (42 kDa) secreted by the mold Caldariomyces fumago (1). Like most members of the peroxidase superfamily, CPO1 contains an iron protoporphyrin IX moiety (heme b; Fig. 1) as its prosthetic group and shares major common reaction intermediates with other heme peroxidases (1). However, extensive biochemical and biophysical studies carried out on this enzyme have revealed dramatic structural and catalytic differences between CPO and traditional heme peroxidases. For example, the axial ligand to the heme iron in CPO is a cysteine (Cys29) sulfur atom rather than a histidine nitrogen atom commonly found in most heme peroxidases (2-5). Furthermore, CPO employs a glutamic acid (Glu183) as the distal acid-base catalyst, whereas most other heme peroxidases use a histidine to fulfill the same function (6).


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Fig. 1.   Schematic presentation of different conformations of iron protoporphyrin IX in peroxidases (CPO and HRP (A) and CcP and MnP (B)).

The unique active site structure of CPO dictates a broad spectrum of catalytic activities such as oxidation of organic substrates (peroxidase activity) (7), dismutation of hydrogen peroxide (catalase activity) (8, 9), and monooxygenation of many organic molecules (monooxygenase activity) (10-13). Furthermore, CPO has a unique ability to utilize halide (except fluoride) ions to halogenate a wide variety of organic acceptor molecules in the presence of hydrogen peroxide or other organic hydroperoxides (14-16). Most importantly, CPO is adept in catalyzing the stereoselective epoxidation of alkenes (10, 17, 18), hydroxylation of alkynes (19, 20), and oxidation of organic sulfides (21-23).

The versatile catalytic activities of CPO have attracted much interest in understanding the structural properties of the enzyme. Especially, the increasing current interest in chiral synthesis has made CPO an attractive candidate for making important chiral synthons that are of both industrial and medicinal significance. Therefore, detailed structural insight into this structurally unique and catalytically diverse heme enzyme would help to further understand the structure-activity relationship of heme proteins in general and the structural basis for the broad range of activities displayed by CPO in particular.

Many chemical and spectroscopic techniques are now available for structural investigations of heme proteins. Among them, NMR spectroscopy and x-ray crystallography represent the most powerful methods for high resolution structural characterization of paramagnetic metalloenzymes. The two methods are complementary in most cases, and it is difficult to determine which one is better. Despite the great success with cytochrome c peroxidase in the early 1980s (24, 25), x-ray crystallography of heme peroxidases has suffered from difficulties in obtaining suitable diffracting protein crystals. Nonetheless, the solid-state structure of CcP has served as a convenient and independent structural basis for evaluating results derived from NMR studies of the protein in solution. Consequently, CcP has served as a prototype model for NMR spectroscopists interested in hyperfine resonance assignment and structural refinement of the protein in solution, a state that is more closely related to the physiological conditions under which the enzyme functions (26-34).

Compared with the extensive and in depth NMR studies on CcP (28-36) and horseradish peroxidase (34, 37-53), relatively few NMR studies of CPO have been reported (54-58). Furthermore, the most powerful NMR approach, the two-dimensional NMR technique that has led to the unambiguous assignment of major hyperfine-shifted signals in a number of heme peroxidases (59), has not been applied to the investigation of CPO. As a result, no extensive and definitive resonance assignments are available for this structurally unique yet functionally diverse enzyme.

Here we report the first application of the two-dimensional NMR method to the elucidation of the active site structure of CPO in solution. The observation of both COSY and NOESY connectivities among paramagnetically shifted signals as well as NOESY connectivities between hyperfine shifted resonances and signals within the crowded diamagnetic envelope, in combination with the Curie intercepts obtained from variable temperature experiments coupled with previous one-dimensional NOE studies performed on this enzyme (54) have allowed us to assign most of the signals from the heme group, which in turn allows the assessment of the conformations of the heme side chains. Of particular importance, the two-dimensional studies have allowed firm assignment of the heme iron ligand, Cys29 spin system that is critical to the unique catalytic properties of CPO. Surprisingly, the addition of excess Mn(II) to CPO resulted in no detectable effects on the NMR spectral properties of the protein. This is in sharp contrast with the results observed for CPO isolated from cultures grown in the presence of manganese (60) and from manganese (II) binding variants of CcP (36, 61, 62) and native MnP (63). The difference between solution and solid-state structural features of the same protein demonstrates the need for structural characterization using NMR to complement x-ray structural analysis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Sample Preparation-- Chloroperoxidase was isolated from the growth medium of C. fumago according to the method established by Morris and Hager (64) with minor modifications using acetone rather than ethanol in the solvent fractionation step. Protein preparations with Rz values of 1.4 or higher were used in all experiments.

Protein samples for NMR experiments were prepared in either D2O buffer or 90% H2O, 10% D2O buffer solutions containing 100 mM potassium phosphate at pH 5.5 (direct meter readings from an Orion 720A pH meter using a standardized calomel combination microelectrode uncorrected for any isotope effects). Samples in D2O were prepared by at least five isotope exchanges of the protein solution in H2O with D2O buffered at pH 5.5. The isotope exchanges were carried out in either Centricon or Centriprep tubes (both from Amicon, Inc.) at 4 °C. All NMR samples contain ~1.5 mM protein as determined by electronic absorption spectroscopy for the Soret absorbance at 398 nm and the reported absorption coefficient of 91,200 M-1 cm-1 (65). The cyanide adducts of the protein were prepared by the addition of a 10-20% molar excess of cyanide from a freshly made 500 mM stock solution of KCN in 99.9% D2O.

UV-visible Titrations-- Manganese titration experiments were performed on a Hewlett-Packard 8453 diode array spectrophotometer. CPO was dialyzed against 100 mM KH2PO4, pH 5.5, and 10 mM EDTA twice overnight, followed by three dialyzes against 100 mM KH2PO4, pH 5.5, to remove EDTA from the sample. CPO (16 µM) is placed in both the sample and reference cuvettes. Under gentle stirring, aliquots of 50 mM MnSO4 solution were added to the sample with 2, 3, 4, 5, 10, 30, 40, 50, and 60 equivalents of Mn2+ per enzyme equivalent. At the same time, an equal amount of buffer was added to the reference cuvette to compensate for any dilution effects. UV-visible spectra were obtained in the range of 250-750 nm.

EPR-- EPR experiments were carried out on a 95-GHz (W-band) spectrometer at room temperature. Spectra were obtained on ~2 mM protein samples in 100 mM KH2PO4, pH 5.5. For Mn(II) binding studies, CPO was first dialyzed against buffer containing 10 mM EDTA twice overnight. After removal of excess EDTA, Mn(II) was titrated into CPO under gentle stirring at 4 °C. The samples were then subjected to repeated dilution and concentration in a Centriprep concentrator to remove any free and adventitiously bound Mn(II). Instrument settings used for the experiments were as follows: microwave frequency = 95 GHz, modulation amplitude = 32.4 G, and microwave power = 1.00 milliwatts.

NMR Spectroscopy-- Proton NMR spectra of both native and cyanide-bound forms of CPO were recorded at 25 °C on a Varian Unity 600 FT NMR spectrometer operating at a proton frequency of 599.97 MHz. The residual solvent signal was suppressed with either the super WEFT method (66) or presaturation during relaxation delay. Chemical shift values were referenced to the residual HDO signal at 4.76 ppm.

Variable temperature experiments were carried out on a Varian Unity-Inova 500 FT NMR spectrometer operating at a proton frequency of 499.77 MHz. The reference chemical shift of the residual HDO signal was calculated according to the relationship of delta T delta 25 - 0.012 (T - 25), where delta T is the chemical shift of HDO at temperature T in °C, and delta 25 is the chemical shift of HDO at 25 °C (67). A value of 4.76 ppm rather than 4.81 ppm (67) was used for delta 25 to match the previous NMR studies of CPO (54).

Phase-sensitive NOESY spectra for the cyanide-bound derivative of CPO were acquired at 25 °C with mixing times ranging from 1.5 to 35 ms. Typical NOESY spectra were collected with 256 experiments in the F1 dimension using the hypercomplex method of States et al. (68). In general, 400 scans were accumulated for each F1 experiment, which was acquired with 4096 complex points in the F2 dimension over a spectral width of 27 or 60 kHz. The residual solvent signal in all NOESY experiments was suppressed using a 200-ms presaturation with a weak decoupler power. NOESY spectra with mixing times of 3 ms or less were collected with the incorporation of the super WEFT sequence (66) to suppress the intense diamagnetic signals from the protein matrix and the residual signal from the solvent.

Clean TOCSY (69) spectra of CPOCN were recorded at both 500 and 600 MHz over different spectral windows using 4096 F2 points and 256 complex F1 points of 320-400 scans. Solvent suppression was achieved by a 200-ms direct saturation during the relaxation delay period. Various mixing times (2, 10, 30, and 40 ms) were used to allow effective spin lock for protons with different relaxation properties.

All two-dimensional data were processed on a Dell Dimension 8200 PC with a Pentium 4 processor using Felix 2001 (Accelrys, Inc.). Various apodization functions were employed to emphasize protons with different relaxation properties. For example, apodization over 256, 512, and 1024 points was used to emphasize fast relaxing broad cross-peaks at the expense of resolution, whereas apodization over 2048 points is necessary to emphasize slowly relaxing cross-peaks. All two-dimensional data were zero-filled to obtain 2048 × 2048 matrices as required by the large hyperfine shift dispersion exhibited by the paramagnetic nature of this protein.

The structure of CPO was examined on either a Silicon Graphics Indigo 2 Extreme workstation using Quanta (Accelrys) or a Dell Dimension 8200 computer using ViewerLite (Accelrys) to visualize the crystal coordinates supplied by the Brookhaven Protein Data Bank (6). Generally, atom separations are reported as distances between protons of interest with the exception of methyl protons, where methyl carbons are used for distance measurements.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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The proton NMR spectrum of the native ferric high spin CPO (data not shown) is essentially identical to the results reported previously (55, 57). Because of the high spin nature of the native CPO, considerably broad resonances are observed in the NMR spectra that provide only limited information about the structural properties of the enzyme. Therefore, no further efforts were made to analyze the spectra of native CPO in this study. We have focused on the NMR spectral properties of the cyanide-bound, ferric low spin derivative of CPO. This protein form, although not physiologically active, has been the most favorable system on which paramagnetic NMR investigations are carried out (35, 37, 70, 71). The short electronic relaxation times and large magnetic anisotropy of the low spin peroxidase cyanide complexes give much sharper and better resolved signals in their proton NMR spectra, providing much more information about the electronic, magnetic, and molecular structural properties of the heme pocket as compared with the native, high spin resting forms (27, 38, 50, 72). In addition, the cyanide adduct of heme peroxidases has been implicated as an important analogue for the active, oxidized low spin enzyme intermediates for which proton NMR spectroscopy is currently inapplicable due to the large resonance line widths (73).

Although the CPO preparations used in this study were spectrophotometrically homogeneous, the 1H NMR spectra of the cyanide-bound CPO complex are NMR spectroscopically inhomogeneous due to the presence of two isozymes. This is reflected by the pairwise pattern for most of the hyperfine-shifted signals as shown in Fig. 2. The approximately equal intensities of the two sets of signals suggest that the ratio of A and B isozymes is close to 1 in the current enzyme preparation. The spectral features are equivalent to that reported previously (54, 57). No noticeable solvent isotope effect on the chemical shifts of heme protons was observed when spectra were recorded in H2O (Fig. 2, lower trace). This is anticipated, since the distal acid-base catalyst in CPO is a glutamic acid (6) rather than a histidine as in other heme peroxidases (24, 74-76). The proton/deuteron exchange on the Nepsilon atom of the distal histidine has been shown to be responsible for the observed solvent effect on heme resonances in several heme peroxidases (36, 53, 72).


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Fig. 2.   600-MHz 1H NMR spectra of the low spin cyanide complexes of CPO in D2O (top trace) and H2O (lower trace). The spectrum is collected at 298 K in 0.1 M phosphate buffer at pH 5.5.

The spectral features of CPOCN display a high degree of similarity to that of other heme peroxidase cyanide derivatives (59). The four intense signals with integrated intensities of three protons each in the downfield region are typical of heme methyl groups. They have been tentatively assigned to the two heme methyls (5- and 1- or 8- and 3-CH3) for the two isozymes (54). Other resonances with intensities of one proton each in the downfield region represent protons from other heme substituents and those from amino acid residues in the proximal and distal heme pocket. The resolved upfield spectral region displays several single-proton resonances and a few multiproton signals. Previous work on both heme model compounds and a number of heme peroxidases have firmly concluded that this spectral region encompasses the resonances from beta -protons of the heme vinyl and propionate groups as well as those from some of the amino acid residues near the heme center (53, 72, 77-79). The chemical shifts and the corresponding diamagnetic shift values predicted from Curie plot as well as the spin-lattice relaxation times for the hyperfine shifted resonances and their assignments in CPOCN are compiled in Table I, along with the corresponding parameters in MnPCN (72, 80) and HRPCN (51) reported previously.

                              
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Table I
Proton NMR parameters and assignments of paramagnetically shifted resonances in CPOCN at 298 K, in 0.1 M phosphate buffer, pH 5.5 
Parameters for MnPCN and HRPCN are included for comparison. The intercepts (Int) are predicted from the Curie plots. NA, not assigned.

The assignment of the hyperfine shifted signals for the CPOCN complex was achieved through comparison with the assignments made for cyanide derivatives of other heme peroxidases (30, 34, 36, 53, 72) and examination of the active site structure of CPO revealed by its crystal structure (6) with confirmation through one- and two-dimensional NOE measurements as well as through bond connectivities (COSY). Shown in Fig. 3 is the NOESY spectrum of CPOCN collected in D2O buffer with a mixing time of 35 ms. The clear NOESY connectivities and the results from scalar (COSY; Supplemental Fig. S1) connectivities prove the validity of previous NOE connectivities observed in one-dimensional NOE experiments (54) and lead to the proposed assignments for all but three of the nonexchangeable hyperfine shifted protons (resonances A, B, and Z) from the heme active site in CPOCN. It should be noted that the observed cross-peaks in the COSY experiment are not necessarily true coherence peaks due to the large molecular weight of CPOCN (81, 82). Therefore, the suggested assignments are further verified by the Curie intercepts predicted from the temperature dependence of the resonances (Supplemental Fig. S2).


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Fig. 3.   600-MHz phase-sensitive 1H NOESY spectra of 1.5 mM CPOCN in D2O taken with a mixing time of 35 ms. Other conditions are identical to those in Fig. 2. Cross-peak assignments are as follows. 2, 8-CH3:7-Halpha (A); 3, 8-CH3:7-Halpha (B); 4, 8-CH3:7-Halpha '(A); 5-7, not assigned; 8, 8-CH3:Hbeta Leu97 (3.18 Å); 9, 8-CH3:7-Hbeta ; 10, 8-CH3:7-Hbeta '; 11, 8-CH3:delta -CH3 Leu97 (4.85 Å); 12, 3-CH3:4-Halpha ; 13, 3-CH3:He1 Phe57 (3.27 Å); 14, 3-CH3:Hd1 Phe57 (3.94 Å); 15, 3-CH3:Hgamma Ile63 (3.71 Å); 16, 3-CH3:beta -CH3 Ala31 (4.80 Å); 17, 3-CH3:Hbeta Phe186 (4.86 Å); 18, 3-CH3:gamma -CH3 Ile63 (4.05 Å); 19, 3-CH3:gamma -CH3 Ile68 (3.68 Å); 20, 3-CH3:gamma 1-CH3 Val67 (4.39 Å); 21, 3-CH3:2-Hbeta trans; 22, 3-CH3:4-Hbeta trans; 23, 3-CH3:4-Hbeta cis; 24, 4-Halpha :H-beta meso; 25, 4-Halpha :beta -CH3 Ala31 (3.76 Å); 26, 4-Halpha :beta -CH2 Phe186 (2.85 Å); 27 and 28, not assigned; 29, 4-Halpha :4-Hbeta trans; 30, 4-Halpha :4-Hbeta cis; 31, 7-Halpha :7-Halpha '(A); 32, 7-Halpha :7-Hbeta (A); 33, 7-Halpha :7-Hbeta '(A); 34, 7-Halpha :gamma -CH2 Pro28 (2.99 Å)(B); 35, 7-Halpha :7-Hbeta (B); 36, 7-Halpha :7-Hbeta '(B); 37, 7-Halpha ':7-Hbeta (A); 38, 7-Halpha ':7-Hbeta '(A); 39, 2-Halpha :2-Hbeta cis; 40, 2-Hbeta cis:2-Hbeta trans; 41, 2-Hbeta cis:beta -CH Ile68 (2.75 Å); 42, 4-Hbeta trans:4-Hbeta cis; 43, beta -CH2 Phe186:4-Hbeta cis (3.47 Å); 44, 2-Hbeta trans:beta -CH Ile68 (2.75 Å); 45, beta -CH:gamma -CH3 Ile68; 46, beta -CH2 Phe186:4-Hbeta trans (3.57 Å); 47, 2-Halpha :2-Hbeta trans. Cross-peaks 12, 37, and 38 can only be seen with lower contour levels. The numbers in parenthesis are the distances between protons that give rise to the cross-peak. The capitalized letters in parenthesis represent the isozyme forms of CPO.

Fig. 4 shows the NOESY spectrum collected with a mixing time of 1.5 ms. The clear NOE connectivity between the two fast relaxing, nonexchangeable, strongly hyperfine shifted resonances firmly establishes the geminal partner relationship of the two protons. Because of the extremely short T1 values (1.5 ms) of these two protons, previous efforts to correlate the two signals by one-dimensional NOE method were unsuccessful (54). Nonetheless, based on the relaxation properties, the large contact shift, and the expected distance to the heme iron center, these two resonances were tentatively assigned to the beta -CH2 protons of the heme iron ligand, Cys29 (54). The crystal structure of CPO (6) and the observed NOESY cross-peaks (Fig. 4) between these signals now confirm the earlier proposals (54). This is another example demonstrating that NMR spectroscopy can serve as an independent tool in characterizing the structural properties of large paramagnetic heme proteins.


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Fig. 4.   600-MHz phase-sensitive 1H NOESY spectra of 1.5 mM CPOCN in D2O taken with a mixing time of 1.5 ms. Other conditions are identical to those in Fig. 2. Cross-peak 1, beta H:beta 'H Cys29.

The structure of metal binding sites, such as Ca2+ and Mn2+ binding sites, within peroxidase molecules has received increasing attention in recent years due to its potential to modulate specific activities of this class of enzymes. This has stimulated great interest in designing metal binding sites in peroxidases (36, 61, 62). The crystal structure of CPO (6) has revealed a Mn(II) binding site near the heme group similar to the location of Mn(II) in MnP as shown in Fig. 5 (76). However, the exact role of Mn(II) in CPO is unclear. A careful NMR titration of the CPOCN preparation used in these experiments with Mn(II) failed to find any detectable effect of Mn(II) as shown in Fig. 6. The results are surprisingly different from that observed for the Mn(II) binding site in native MnP (63) and an engineered MnP mimic MnCcP (36, 61, 62). The absence of any detectable effect of the added Mn(II) on the spectral properties of CPOCN undoubtedly depends on the growth conditions used to produce the enzyme. C. fumago cultures grown in complex media bind one manganese ion per molecule (60). However, when the fungus is grown on synthetic media in the absence of manganese, the enzyme contains little or no manganese.


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Fig. 5.   The putative manganese(II) binding site in CPO and in the engineered manganese peroxidase model MnCcP.


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Fig. 6.   Effect of Mn(II) addition on Paramagnetic 1H NMR spectra of cyanide-bound CPO. The signals assigned to 7-propionate protons are indicated by arrows. a, no Mn(II) present. b, with 0.5 eq of Mn(II). c, with 1.0 eq of Mn(II). d, with 2.0 eq of Mn(II). e, with 5.0 eq of Mn(II). f, with 10 eq of Mn(II). Spectra were obtained in D2O solutions (100 mM phosphate) at pH 5.5. No specific broadening of paramagnetically shifted signals assigned to residues near the manganese binding site (as labeled by the arrows) was observed. At high Mn(II) concentration, nonspecific broadening of all signals is observed (f).

To confirm the NMR studies of Mn(II) binding, both UV-visible spectrophotometric and EPR spectroscopic Mn(II) titrations were performed (data not shown). These measurements supported the conclusion from NMR studies.

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Presence of Isozymes

Both one- and two-dimensional NMR spectra of CPOCN (Figs. 2 and 3) display pairwise resonances for most of the paramagnetically relaxed protons, indicating the presence of two structurally and magnetically closely related isozymes of CPO in solution. This is in complete agreement with previous one-dimensional NMR study of this protein (54, 57). The two isozymes are not due to heme disorder or chemical equilibrium between different forms of the same protein. Instead, the isozymes are chemically and structurally distinct, as judged by the observed NOE pattern and the lack of chemical equilibrium between them. This is different from the situation for recombinant CcP variants where multiple forms of the same protein were found to exist in equilibrium as reflected by exchange peaks in the NOESY spectrum and temperature-dependent NMR resonance intensity changes among them (28, 29, 36, 83).

It is interesting to note that not all of the hyperfine shifted resonances display corresponding partners in the NMR spectra. In fact, only signals assignable to the heme group and heme iron ligand show an observable doubled peak pattern as shown in Figs. 2 and 3. This fact, combined with the very similar chemical shifts of the two isozymes, suggests that there are no global structural differences between the two distinguishable protein forms. It seems that the magnetic inequivalence is particularly localized to a specific region of the heme active site, as will be further discussed under "Resonance Assignment."

Resonance Assignment

The Heme Substituents-- As shown in Fig. 3, the heme methyl resonances C at 24.0 ppm and D at 23.8 ppm give weak NOEs to peak J at 14.1 ppm, peak K at 12.9 ppm, and peak "O" at 11.6 ppm. Several other NOEs are also observed in the NOESY spectrum in the diamagnetic aliphatic region, attributable to amino acid residues close to this methyl group. Of critical importance to the assignment of these signals is the observation of the strong NOE connectivity between peaks J and O that are indicative of a geminal relationship between these two signals. Such NOE patterns are consistent with the results reported from the earlier one-dimensional work (54) and are typical of a methyl group near a propionate group as observed for CcPCN (31, 35), MnCcPCN (36), and HRPCN (40). Therefore, resonances C and D can be assigned to the 5- or 8-CH3 group of the two isozymes of CPO.

The other set of heme methyl signals E and F at 20.7 and 20.4 ppm produces an extremely weak NOE to resonance I at 14.8 ppm and weak NOEs to signals at -2.7 ppm (V) and -4.4 ppm (X). Clear NOEs are also observed between resonance I and the signals V and X. This is reminiscent of the NOE pattern observed for the engineered MnP model, MnCcP (36), and several other heme peroxidases (31, 34, 35, 72, 84). The lack of strong NOE between peak I and any other signals suggests the absence of geminal partners to this proton. Therefore, resonance I must be assigned to the alpha H of a vinyl group, and signals E and F must be assigned to a nearby methyl group of the two isozymes. Consequently, signals V and X must be assigned to the beta -protons of the same vinyl group. This assignment is further supported by the strong temperature dependence and the reasonable Curie intercepts of these resonances (Supplemental Fig. S2a and Table I). Furthermore, the proposed assignment is consistent with the expected chemical shift pattern of a heme vinyl group. The alpha -CH vinyl proton resonance is normally observed in the downfield region because of pi -spin delocalization of the unpaired spin density, whereas the beta -CH2 vinyl proton peaks are usually positioned between 0 and -5.0 ppm (36, 54, 71, 85).

Since signals E and F display NOE connectivity to signal I assignable to the alpha H of a vinyl group (see above), the methyl peaks E and F at 20.7 and 20.4 ppm must arise from either the 1-CH3 or the 3-CH3 group of the heme unit. The observed resonance E (F) for CPOCN is assigned to the 3-CH3 group, since peak E (F) also gives NOE connectivity to signal T at -1.4 ppm. The latter signal also displays a strong NOE to signal Y at -5.2 ppm. Both signals T and Y display clear NOEs to resonance P at 3.0 ppm. This NOE pattern and the observed shift positions coupled with the Curie behavior of these signals suggest the assignment of signals P, T, and Y to the protons of a vinyl group other than the vinyl group mentioned before. The presence of dipolar connectivities to two vinyl groups mandatorily assigns signal E (F) to 3-CH3, since it is the only candidate that can display such dipolar connectivities (Fig. 1). The observed NOE pattern between the 2-vinyl group (resonance T) and 3-CH3 (resonance E (F)) indicates that the alpha H of the 2-vinyl group is pointing away from the 3-CH3 group, and the beta H of the vinyl group is pointing toward this methyl group as shown in Fig. 1A. This is in complete agreement with the results observed for cyanide-bound low spin lignin peroxidase (84) and HRPCN (50, 51). This result is also in perfect agreement with the results obtained from crystallographic analysis of the CPO structure (5, 6). The position of the 2-vinyl group shown in Fig. 1B would produce observable NOE between the alpha H (usually under the crowded diamagnetic envelope region) and the 3-CH3 group as in the case of CcPCN (33, 35), MnCcPCN (36), and MnPCN (72). With resonance E (F) assigned, signals at 14.8 (I), -2.7 (V), and -4.4 (X) ppm observed in the NOESY spectrum can be readily assigned to 4-Halpha , 4-Hbeta -t, and 4-Hbeta -c, respectively, according to the magnitude of NOEs displayed (Fig. 3), the intensity of the scalar cross-peaks observed in the COSY experiment (Supplemental Fig. S1), and the temperature dependence displayed in the Curie plot (Supplemental Fig. S2a and Table I). Consequently, peak B (C) at 24.0 (23.8) ppm must originate from the 8-CH3 group, and the NOE observed at 14.1, 12.9, and 11.6 ppm must arise from the 7-propionate Halpha . The 7-propionate Halpha signals produce the expected reciprocal NOE to the assigned 8-CH3 as well as strong NOEs to signals at 2.0 (1.9) and 2.8 (2.7) ppm assignable to signals of the two beta -protons of the 7-propionate group. The temperature dependence behavior of these signals is strikingly similar to the corresponding protons in MnPCN (72) and HRPCN (51), further supporting the assignment proposed. This assignment is also supported by the observation of NOEs between 7-propionate group and Hepsilon 2 of His105 (see distal heme cavity assignment).

It is worth mentioning that although most heme resonances display a doubled-peak pattern due to the existence of two isozymes of this protein, the chemical shift of most protons differs only slightly from their counterparts between the two isozymes. For example, the chemical shift of the two resolved heme methyl resonances display differences of only 0.2 and 0.3 ppm for the two isozymes. However, the resonance positions of the 7-propionate alpha -CH protons (signals J and K at 14.1 and 12.9 ppm; signals O and K at 11.6 and 12.9 ppm) in the two isozymes exhibited a difference of ~1.2 ppm. The large chemical shift difference of the 7-propionate alpha -protons suggests that the protein modification differentiating the two isozymes is probably located in the vicinity of this propionate group or is communicated to the heme via this side chain. Further support for this implication comes from the fact that the two 7alpha -protons in one isozyme are observed as an essentially degenerate pair at 12.9 ppm (signal K), whereas the corresponding protons in the other isozyme are detected as chemically and magnetically distinctive groups at 14.1 and 11.6 ppm (signals J and O). It is interesting to note that the position of the degenerate 7alpha -protons in one isoenzyme (form B) is exactly at the middle of the distinctive 7alpha -protons in the other isoenzyme (form A). This could suggest that the degeneracy of the two 7alpha -protons is caused by a factor that averages the otherwise magnetically inequivalent protons.

Previous studies on CPOCN have suggested the assignment of signals G(H) and W to the heme mesoprotons based on their extremely short T1 values of 6-7 ms (54). The predicted distances of these signals to the heme iron center is about 4.5 Å (Table II) according to the relationship between T1 values of inequivalent protons in noncoordinating groups and their distances to the paramagnetic center (50), assuming that heme methyl protons have an average distance of 6.1 Å from the iron. Therefore, it is reasonable, in the absence of the crystal coordinates of CPO, to assign these signals to the heme mesoprotons, since they are the only remaining protons close enough to heme iron to experience such sufficient paramagnetic relaxation (54). Unfortunately, no NOEs are observed between these signals and any of the assigned heme signals in the current study, suggesting different identities for these protons. The relatively small diamagnetic intercepts of these signals as compared with that of the heme mesoprotons in MnPCN and HRPCN (Table I) also argue for different assignments for these protons. Furthermore, the crystal structure of CPO reveals that there are several nonheme protons within the heme cavity that can experience sufficient paramagnetic relaxation due to their close proximity to the iron (Table II).

                              
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Table II
Distances between heme iron and some selected protons of CPOCN
The predicted distances were obtained from the intrinsic spin-lattice relaxation times for the selected protons according to the relationship ri/rj = [T1i/T1j]1/6 (50). The average distance (6.05 Å) of the three protons of the heme 8-CH3 group to the iron was used as a reference for the predictions. The actual distance of the proton in question to the heme iron is determined from the crystal coordinates of CPO (6).

Careful examination of the NOESY map shown in Fig. 3 leads to the possible assignment of two heme mesoprotons in CPOCN. These are the beta - and delta -mesoprotons that resonate at 5.8 and 8.7 ppm, respectively. However, the essentially non-Curie behavior of the latter resonance (8.7 ppm) excludes the assignment of this signal to heme mesoprotons. The strong temperature dependence, the observed shift value, the extrapolated Curie intercept (8.4 ppm, Table I), and the NOE between the assigned 4-Halpha vinyl proton allow us to assign the signal at 5.8 ppm as the heme beta -mesoproton of CPOCN.

The Heme Thiolate Ligand Cys29-- The extremely broad, fast relaxing, strongly hyperfine-shifted peaks A (B) and Z at 39.0 (38.3) and -20.7 ppm are characteristic features of protons in close proximity to the heme iron. These peaks have been previously proposed to arise from the beta -CH2 protons of the coordinated cysteine, Cys29, based on their relaxation properties and shift positions (54). However, due to the considerably broad line widths and the extremely short T1 values of these resonances, no observable NOE was detected in the one-dimensional NOE work that could firmly relate the geminal relationship between the two protons (54). The clear NOE observed in the current two-dimensional study (Fig. 4) unequivocally confirms the earlier hypothesis and definitively establishes the geminal partner relation of these two resonances. The similar Curie intercepts of these signals as compared with that of the corresponding protons in MnPCN and HRPCN (Table I) also support the assignment of these signals to the beta -CH2 protons of the coordinated iron ligand, Cys29. The crystal structure of CPO (6) further supports this assignment. With distances of 3.13 and 4.10 Å from the heme iron, respectively, the two beta -CH2 protons of Cys29 are expected to display strong contact shift and effective paramagnetic relaxation as predicted previously (54). The firm assignment of the Cys29 alpha H is hindered by the unsymmetrical nature of the NOESY map obtained. Nonetheless, the NOE displayed between peak A (B) and a signal at 4.4 ppm (Fig. 4) suggests the assignment of the latter signal to the Cys29 alpha H. This is expected, since the extremely short mixing time needed to detect the NOEs between fast relaxing protons would fail to detect NOEs between nongerminal protons. The detection of NOE between Cys29 alpha H and signal A (B) but not Z suggests that resonance A (B) is cis to the alpha H of Cys29.

Pro28 and the Presence of Isoenzymes-- The downfield hyperfine shifted resonances that remain unassigned by now are signals L, M, and N, since they display no NOE connectivity to any other hyperfine-shifted resonances (Figs. 3 and 4). This situation adds uncertainties in assigning these signals. However, the extremely long nonselective T1 (220 ms) of signal L as compared with other hyperfine-shifted resonances suggests the assignment of signal L to a proton at a relatively remote distance to the heme iron. Although the identity of this signal cannot be determined from the current data, observation of NOEs between this signal and three other signals at 10.2, 8.7, and 7.9 ppm suggests the assignment of this signal to one of the CH2 protons in Pro28. This is based on the fact that the signal at 7.9 ppm displays an observed NOE to 7-Halpha proton (cross-peak 34 in Fig. 3). With a distance of 2.99 Å between gamma H of Pro28 and 7-Halpha , it is reasonable to expect observable NOEs between this proton pair (cross-peak 34 in Fig. 3). The temperature-independent nature of this signal (Table I) also suggests the remote position of this proton relative to the iron center. The distance of the proton (signal M) to heme iron predicted from the relaxation properties of this signal suggests the assignment of signal M to another gamma H of Pro28 (Table II). It is interesting to note that the proposed Pro28 protons also display a doubled peak pattern. This suggests that Pro28 is involved in protein modification that defines the two isozymes. This hypothesis complies with the conclusion that the protein modification that differentiates the two isozymes is located in the vicinity of the heme 7-propionate group or is communicated to the heme via this side chain. It is thus reasonable to propose that the existence of isoenzymes of CPO is a result of conformational change of Pro28. The isomerization of the prolyl imide bond has been recently attributed to the modulation of ligand recognition of proteins by controlling the relative orientation of protein-binding surfaces (86). However, the failure to observe any TOCSY connectivities among these signals makes it impossible to confirm the assignments proposed here. Difficulties for observing TOCSY connectivities have often been encountered for paramagnetic metalloproteins due to the spread of the relaxation properties and the chemical shifts of the resonances (87). Consequently, resonance assignments in paramagnetic metalloproteins are commonly achieved through analysis of NOE connectivities (35, 72, 87). Therefore, further studies are necessary to provide definitive assignment of this residue. This will in turn, allow the elucidation of the role this residue plays in defining the signature of the two isozymes of CPO.

Alternatively, the existence of isozymes of CPO may be attributed to post-translational modifications of the protein (88). The secreted form of CPO is processed from a precursor containing a 21-residue-long, moderately hydrophobic signal sequence, at an atypical Gln-Glu peptide bond. Following cleavage, the N-terminal glutamic acid readily cyclizes into pyroglutamic acid. Furthermore, CPO contains two high mannose N-glycosylation sites, identified as asparagine 12 and 213. Other modifications include deamidation of residues asparagine 13, asparagine 198, and glutamine 183 into the corresponding acids (88). Any difference in these modification processes could result in the formation of isozymes with similar global structural but distinct local environments.

The Distal Heme Cavity-- In contrast to most heme peroxidases that use a His at the distal heme pocket as the acid-base catalyst, CPO employs a more acidic amino acid, Glu183 for the same function. This makes it difficult to assign any signals from this residue, since it does not possess a characteristic resonance as in the case of a His. Our assignment for the distal heme environment was primarily achieved by analysis of the NOESY spectra collected in 90% H2O solution (Supplemental Fig. S3). The solvent-exchangeable signal "a" displays an observed hyperfine shift of 16.3 ppm, comparable with the position of the solvent exchangeable proton observed at 16.5 ppm in CcPCN and 16.3 ppm in HRPCN (34). In most of the heme peroxidases studied, the downfield shifted solvent-exchangeable signals have all been assigned to the residues at the distal side of the heme cavity (59). Therefore, we can assign signal "a" to a solvent-exchangeable proton of the distal residues in CPO. The corresponding proton has been assigned to the Hdelta 1 of the distal His in CcP and horseradish peroxidase (34). However, the distance of Hdelta 1 in His105 at the distal side of CPO is much further from the heme iron compared with that in CcP and horseradish peroxidase. On the other hand, the Hepsilon 2 is at a reasonable distance from heme iron and is hydrogen-bonded to the acid-base catalyst, Glu183. Since there are no other exchangeable protons at the distal heme cavity of CPO, signal "a" is assigned to the Hepsilon 2 of His105 in CPO. This assignment is further supported by the NOEs observed between "a" and the firmly assigned 7-propionate beta -protons. With distances of 3.22 and 3.45 Å between Nepsilon 2 and the two 7-propionate beta -protons, it is expected to observe NOEs between Hepsilon 2 and the 7-propionate protons (cross-peaks 50 and 51 in Supplemental Fig. S3). Observation of cross-peaks (48 and 49 in Supplemental Fig. S3) between signal "a" and two other protons assignable to the imidazole ring Cdelta and Cepsilon protons also favors the assignment of "a" as Hepsilon 2 of His105 in CPO. The long nonselective T1 (200 ms) of signal "a" predicts that this proton is 8.13 Å from the heme iron (Table II). This is in close agreement with the distance measured from the crystal structure of CPO (6).

The protonation of His105 might be critical for the catalytic activity of CPO. In most heme peroxidases, the concerted interaction of both the distal acid-base catalyst and a second polar residue is required to cleave the peroxide bond in the formation of compound I (89). The only polar residue at the heme distal side of CPO is His105, which is 3.5 Å above the 7-propionate group (6). Therefore, we propose that this His indirectly participates in the cleavage of the peroxide bond by hydrogen-bonding to and correctly positioning of the direct acid-base catalyst, Glu183, in CPO as shown in Fig. 7. This picture is in perfect agreement with the proposed reaction mechanisms of CPO reported previously (6, 90, 91).


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Fig. 7.   The proposed hydrogen bond network involving His105 that facilitates the cleavage of the peroxide O-O bond.

In this mechanism, the activation of hydrogen peroxide is initiated by its binding to the distal heme site of CPO that was occupied by a water molecule in the enzyme's resting state (Fig. 7, 1). The hydrogen bond between His105 (Nepsilon 2H) and Glu183 (Oepsilon 1) facilitates the abstraction of a peroxide proton by Glu183 through hydrogen bonding between the peroxide proton and the other carboxylate oxygen (Oepsilon 2) of Glu183 (2). The resulting singly ionized hydrogen peroxide covalently binds to the heme iron, leading to the formation of a short lived intermediate (3). This intermediate then brings the terminal oxygen of the activated peroxide closer to Glu183, which helps the formation of a strong hydrogen bond between Glu183 (Oepsilon 2H) and the terminal oxygen of peroxide (4). Formation of 4 dramatically promotes the subsequent proton delivery to the terminal oxygen from Glu183, resulting in the heterolytic cleavage of the O-O bond and the formation of the compound I oxoferryl center (Fe4+=O) and porphyrin pi  cation radical common to most heme peroxidases (5). Reaction of 5 with organic substrates regenerates the resting state enzyme (peroxidase activity). Alternatively, CPO compound I can be transformed into the resting state via evolution of dioxygen (catalase activity). Most importantly, the compound I of CPO can activate most halide ions to halogenate a broad range of organic acceptor molecules (halogenase activity). Although His105 is not directly involved in the reactions of CPO, it is critical to the normal function of CPO, because it helps to properly arrange the direct acid-base catalyst (Glu183) in its correct orientation.

The NOESY results also showed several cross-peaks between the firmly assigned heme signals and a signal that is assignable to the other distal residue, Phe186. The crystal coordinate of CPO indicated that the Hbeta of Phe186 is at a position capable of producing NOEs to four types of heme protons, 3-CH3, 4-Halpha , 4-Hbeta -t, and 4-Hbeta -c (cross-peaks 17, 26, 43, and 46 in Fig. 3). This is exactly what was observed. Therefore, the signal at 2.4 ppm (intercept at 2.2 ppm from Curie plot, Table I) could be assigned to one of the Cbeta protons of Phe186.

Signals from other distal residues, Phe103, a residue thought to be important in controlling substrate access to heme center, and especially Glu183, the residue that is directly involved in the catalytic process of CPO, cannot be clearly accessed from the current data. Therefore, further studies are needed to locate the NMR properties of these important residues. This can be achieved by either replacing these residues with other amino acids or selectively labeling these residues with an NMR active isotope such as 13C or 15N. This work is currently in progress in our laboratories.

Location of the Putative Mn(II) Binding Site-- The above assignments of paramagnetically shifted signals make it possible to locate the proposed Mn(II) binding site in CPO. Since the unpaired electrons of Mn(II) can interact with protons close to Mn(II) and broaden them, observation of broadening of specific signals indicates specific Mn(II) binding and helps to locate the Mn(II) binding site (36, 63). However, as pointed out earlier, essentially no specific spectral changes were observed upon the addition of up to 10 eq of Mn(II) to the CPO preparations (Fig. 6). This is in sharp contrast with the results reported for CPO crystals, native MnP (63), and the engineered Mn(II) binding variants of CcP (36, 62). As discussed earlier, CPO isolated from fungal cultures grown in the absence of manganese neither contains Mn(II) nor binds added Mn(II). Thus, we conclude that binding of Mn(II) to its specific site in CPO requires the presence of Mn(II) during folding of the enzyme into its native conformation. When CPO folds in the absence of Mn(II), the high affinity binding site remains vacant and is inaccessible to added Mn(II). This conclusion is supported by all of our NMR (Fig. 6), UV-visible, and EPR (data not shown) studies carried out on proteins isolated from cultures grown in the absence of Mn(II).

Other Inferred Assignments-- The NOESY data presented in this work contain several cross-peaks involving the hyperfine shifted heme protons. On the basis of the cross-peak intensities, the specific NOE pattern, the observed shift positions, the extrapolated Curie intercept, assignments in other heme peroxidases, and the distance measured from the crystal structure of CPO, most of these cross-peaks can be assigned to the amino acid side chains that are in close proximity to the heme protons in question. These assignments are given in the legend to Fig. 3 with proton distances (Å) given in the parenthesis after the assignments.

In summary, the results presented here allowed the first reasonable assignments for most of the hyperfine shifted resonances, including those of the heme iron ligand (Cys29) of CPO. The location of protein modification that differentiates the signature of the two isozymes of CPO is near the heme propionate group, possibly due to the conformational change of Pro28. The possible function of His105 at the distal heme cavity is to help the cleavage of the peroxide bond by hydrogen-bonding to and properly positioning the acid-base catalyst, Glu183, within the heme center. The high affinity Mn(II) binding site in CPO (in solution) is not accessible to externally added Mn(II). The results presented here provide a reasonable explanation for the discrepancies in the literature between spectroscopists and crystallographers concerning the Mn(II) binding site in this unique protein. Our study indicates that results from NMR investigations of the protein in solution can well complement the results revealed by x-ray diffraction studies in the crystal form and thus provide a complete and better picture of the actual structure of the protein.

    ACKNOWLEDGEMENTS

We are indebted to M. Feng, Dr. J. Chou, and Dr. G. Rai for technical help. We acknowledge the University of Southern Mississippi Polymer Science NMR facility (Varian UNITY-INOVA 500 MHz) and the Varian Oxford Instrument Center for Excellence in NMR laboratory (Varian UNITY 600 MHz) at the University of Illinois for use of the spectrometers.

    FOOTNOTES

* This research was sponsored by start-up support from the Chemistry Department of Jackson State University (to X. W.) and National Institutes of Health Grant GM07768 (to L. P. H.).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 on-line version of this article (available at http://www.jbc.org) contains three additional figures.

§ To whom correspondence may be addressed: Dept. of Chemistry, Jackson State University, 1400 J. R. Lynch St., Jackson, MS 39217. Tel.: 601-979-3719; Fax: 601-979-3674; E-mail: xwang@stallion.jsums.edu.

|| To whom correspondence may be addressed: Dept. of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-9686; Fax: 217-265-0385; E-mail: l-hager@uiuc.edu.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M209462200

    ABBREVIATIONS

The abbreviations used are: CPO, chloroperoxidase; CcP, cytochrome c peroxidase; CcPCN, cyanide-ligated low spin form of CcP; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser enhancement spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; CPOCN, cyanide-ligated low spin form of CPO; MnCcP, cytochrome c peroxidase containing Gly41 right-arrow Glu, Val45 right-arrow Glu, and His181 right-arrow Asp triple mutations; MnCcPCN, cyanide-ligated low spin form of MnCcP; MnP, manganese peroxidase; MnPCN, cyanide-bound low spin form of MnP; HRPCN, cyanide-bound low spin form of horseradish peroxidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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