Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase
2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês
10.1074/jbc.m209462200
ISSN1083-351X
AutoresXuemin Wang, Hiroyasu Tachikawa, Xianwen Yi, Kelath Murali Manoj, Lowell P. Hager,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoThe 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. 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. chloroperoxidase cytochrome c peroxidase cyanide-ligated low spin form of CcP nuclear Overhauser effect two-dimensional nuclear Overhauser enhancement spectroscopy two-dimensional total correlation spectroscopy cyanide-ligated low spin form of CPO cytochrome c peroxidase containing Gly41 → Glu, Val45 → Glu, and His181 → Asp triple mutations cyanide-ligated low spin form of MnCcP manganese peroxidase cyanide-bound low spin form of MnP cyanide-bound low spin form of horseradish peroxidase Chloroperoxidase is a monomeric glycoprotein (42 kDa) secreted by the mold Caldariomyces fumago (1Everse J. Everse K.E. Grisham M.B. Peroxidases in Chemistry and Biology. CRC Press, Inc., Boca Raton, FL1990Google Scholar). Like most members of the peroxidase superfamily, CPO1contains an iron protoporphyrin IX moiety (heme b; Fig.1) as its prosthetic group and shares major common reaction intermediates with other heme peroxidases (1Everse J. Everse K.E. Grisham M.B. Peroxidases in Chemistry and Biology. CRC Press, Inc., Boca Raton, FL1990Google Scholar). 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 (2Hollenberg P.F. Hager L.P. J. Biol. Chem. 1973; 248: 2630-2633Google Scholar, 3Sono M. Dawson J.H. Hager L.P. Biochemistry. 1986; 25: 347-356Google Scholar, 4Dawson J.H. Kau L.S. Penner-Hahn J.E. Sono M. Eble K.S. Bruce G.S. Hager L.P. Hodgson K.O. J. Am. Chem. Soc. 1986; 108: 8114-8116Google Scholar, 5Sundaramoorthy M. Mauro J.M. Sullivan A.M. Terner J. Poulos T.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1995; 51: 842-844Google Scholar). 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 (6Sundaramoorthy M. Terner J. Poulos T.L. Structure (Lond.). 1995; 3: 1367-1377Google Scholar). The unique active site structure of CPO dictates a broad spectrum of catalytic activities such as oxidation of organic substrates (peroxidase activity) (7Thomas J.A. Morris D.R. Hager L.P. J. Biol. Chem. 1970; 245: 3129-3134Google Scholar), dismutation of hydrogen peroxide (catalase activity) (8Hewson W.D. Hager L.P. Porphyrins. 1979; 7: 295-332Google Scholar, 9Ortiz de Montellano P.R. Choe Y.S. DePillis G. Catalano C.E. J. Biol. Chem. 1987; 262: 11641-11646Google Scholar), and monooxygenation of many organic molecules (monooxygenase activity) (10Allain E.J. Hager L.P. Deng L. Jacobsen E.N. J. Am. Chem. Soc. 1993; 115: 4415-4416Google Scholar, 11Allain E.J. Enantioselective Epoxidation of Alkenes by Chloroperoxidase and the Development of a Chloroperoxidase Expression System. Ph.D. thesis. University of Illinois, Urbana, IL1997: 27-38Google Scholar, 12Dawson J.H. Science. 1988; 240: 433-439Google Scholar, 13Dawson J.H. Sono M. Chem. Rev. 1987; 87: 1255-1276Google Scholar). 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 (14Libby R.D. Beachy T.M. Phipps A.K. J. Biol. Chem. 1996; 271: 21820-21827Google Scholar, 15Libby R.D. Rotberg N.S. Emerson J.T. White T.C. Yen G.M. Friedman S.H. Sun N.S. Goldowski R. J. Biol. Chem. 1989; 264: 15284-15292Google Scholar, 16Libby R.D. Shedd A.L. Phipps A.K. Beachy T.M. Gerstberger S.M. J. Biol. Chem. 1992; 267: 1769-1775Google Scholar). Most importantly, CPO is adept in catalyzing the stereoselective epoxidation of alkenes (10Allain E.J. Hager L.P. Deng L. Jacobsen E.N. J. Am. Chem. Soc. 1993; 115: 4415-4416Google Scholar, 17Hu S. Hager L.P. Tetrahedron Lett. 1999; 40: 1641-1644Google Scholar, 18Lakner F.J. Hager L.P. J. Org. Chem. 1996; 61: 3923-3925Google Scholar), hydroxylation of alkynes (19Hu S. Hager L.P. Biochem. Biophys. Res. Commun. 1998; 253: 544-546Google Scholar, 20Hu S. Hager L.P. J. Am. Chem. Soc. 1999; 121: 872-873Google Scholar), and oxidation of organic sulfides (21Colonna S. Gaggero N. Manfredi A. Casella L. Gullotti M. Carrea G. Pasta P. Biochemistry. 1990; 29: 10465-10468Google Scholar, 22Colonna S. Gaggero N. Pasta P. NATO ASI Ser. Ser. C Math. Phys. Sci. 1992; 381: 323-331Google Scholar, 23Colonna S. Gaggero N. Casella L. Carrea G. Pasta P. Tetrahedron Asymmetry. 1992; 3: 95-106Google Scholar). 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 (24Finzel B.C. Poulos T.L. Kraut J. J. Biol. Chem. 1984; 259: 13027-13036Google Scholar,25Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Google Scholar), 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 (26Satterlee J.D. Erman J.E. LaMar G.N. Smith K.M. Langry K.C. Biochim. Biophys. Acta. 1983; 743: 246-255Google Scholar, 27Satterlee J.D. Erman J.E. LaMar G.N. Smith K.M. Langry K.C. J. Am. Chem. Soc. 1983; 105: 2099-2104Google Scholar, 28Alam S.L. Satterlee J.D. Mauro J.M. Poulos T.L. Erman J.E. Biochemistry. 1995; 34: 15496-15503Google Scholar, 29Satterlee J.D. Alam S.L. Mauro J.M. Erman J.E. Poulos T.L. Eur. J. Biochem. 1994; 224: 81-87Google Scholar, 30Satterlee J.D. Russell D.J. Erman J.E. Biochemistry. 1991; 30: 9072-9077Google Scholar, 31Satterlee J.D. Erman J.E. Biochemistry. 1991; 30: 4398-4405Google Scholar, 32Satterlee J.D. Erman J.E. Mauro J.M. Kraut J. Biochemistry. 1990; 29: 8797-8804Google Scholar, 33Satterlee J.D. Erman J.E. DeRopp J.S. J. Biol. Chem. 1987; 262: 11578-11583Google Scholar, 34Banci L. Bertini I. Turano P. Ferrer J.C. Mauk A.G. Inorg. Chem. 1991; 30: 4510-4516Google Scholar). Compared with the extensive and in depth NMR studies on CcP (28Alam S.L. Satterlee J.D. Mauro J.M. Poulos T.L. Erman J.E. Biochemistry. 1995; 34: 15496-15503Google Scholar, 29Satterlee J.D. Alam S.L. Mauro J.M. Erman J.E. Poulos T.L. Eur. J. Biochem. 1994; 224: 81-87Google Scholar, 30Satterlee J.D. Russell D.J. Erman J.E. Biochemistry. 1991; 30: 9072-9077Google Scholar, 31Satterlee J.D. Erman J.E. Biochemistry. 1991; 30: 4398-4405Google Scholar, 32Satterlee J.D. Erman J.E. Mauro J.M. Kraut J. Biochemistry. 1990; 29: 8797-8804Google Scholar, 33Satterlee J.D. Erman J.E. DeRopp J.S. J. Biol. Chem. 1987; 262: 11578-11583Google Scholar, 34Banci L. Bertini I. Turano P. Ferrer J.C. Mauk A.G. Inorg. Chem. 1991; 30: 4510-4516Google Scholar, 35Savenkova M.I. Satterlee J.D. Erman J.E. Siems W.F. Helms G.L. Biochemistry. 2001; 40: 12123-12131Google Scholar, 36Wang X. Lu Y. Biochemistry. 1999; 38: 9146-9157Google Scholar) and horseradish peroxidase (34Banci L. Bertini I. Turano P. Ferrer J.C. Mauk A.G. Inorg. Chem. 1991; 30: 4510-4516Google Scholar, 37–53), relatively few NMR studies of CPO have been reported (54Dugad L.B. Wang X. Wang C.C. Lukat G.S. Goff H.M. Biochemistry. 1992; 31: 1651-1655Google Scholar, 55Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Google Scholar, 56Lukat G.S. Goff H.M. Biochim. Biophys. Acta. 1990; 1037: 351-359Google Scholar, 57Goff H.M. Gonzalez-Vergara E. Bird M.R. Biochemistry. 1985; 24: 1007-1013Google Scholar, 58Lukat G.S. Goff H.M. J. Biol. Chem. 1986; 261: 16528-16534Google Scholar). 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 (59Bertini I. Turano P. Vila A.J. Chem. Rev. 1993; 93: 2833-2932Google Scholar), 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 (54Dugad L.B. Wang X. Wang C.C. Lukat G.S. Goff H.M. Biochemistry. 1992; 31: 1651-1655Google Scholar) 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 (60Hollenberg P.F. Hager L.P. Blumberg W.E. Peisach J. J. Biol. Chem. 1980; 255: 4801-4807Google Scholar) and from manganese (II) binding variants of CcP (36Wang X. Lu Y. Biochemistry. 1999; 38: 9146-9157Google Scholar, 61Yeung B.K.S. Wang X. Sigman J.A. Petillo P.A. Lu Y. Chem. Biol. 1997; 4: 215-221Google Scholar, 62Wilcox S.K. Putnam C.D. Sastry M. Blankenship J. Chazin W.J. McRee D.E. Goodin D.B. Biochemistry. 1998; 37: 16853-16862Google Scholar) and native MnP (63Banci L. Bertini I. Bini T. Tien M. Turano P. Biochemistry. 1993; 32: 5825-5831Google Scholar). 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. Chloroperoxidase was isolated from the growth medium of C. fumago according to the method established by Morris and Hager (64Morris D.R. Hager L.P. J. Biol. Chem. 1966; 241: 1763-1768Google Scholar) 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 91,200m−1 cm−1 (65Gonzalez-Vergara E. Ales D.C. Goff H.M. Prep. Biochem. 1985; 15: 335-348Google Scholar). 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. 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 experiments were carried out on a 95-GHz (W-band) spectrometer at room temperature. Spectra were obtained on ∼2 mm protein samples in 100 mmKH2PO4, 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. 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 (66Inubushi T. Becker E.D. J. Magn. Reson. 1983; 51: 128Google Scholar) 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 δT = δ25 − 0.012 (T − 25), where δT is the chemical shift of HDO at temperature Tin °C, and δ25 is the chemical shift of HDO at 25 °C (67Pierattelli R. Banci L. Turner D.L. J. Biol. Inorg. Chem. 1996; 1: 320-329Google Scholar). A value of 4.76 ppm rather than 4.81 ppm (67Pierattelli R. Banci L. Turner D.L. J. Biol. Inorg. Chem. 1996; 1: 320-329Google Scholar) was used for δ25 to match the previous NMR studies of CPO (54Dugad L.B. Wang X. Wang C.C. Lukat G.S. Goff H.M. Biochemistry. 1992; 31: 1651-1655Google Scholar). 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.(68States D.J. Haberkorn R.A. Ruben D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar). 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 (66Inubushi T. Becker E.D. J. Magn. Reson. 1983; 51: 128Google Scholar) to suppress the intense diamagnetic signals from the protein matrix and the residual signal from the solvent. Clean TOCSY (69Griesinger C. Otting G. Wuethrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Scopus (1197) Google Scholar) 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 (6Sundaramoorthy M. Terner J. Poulos T.L. Structure (Lond.). 1995; 3: 1367-1377Google Scholar). 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. The proton NMR spectrum of the native ferric high spin CPO (data not shown) is essentially identical to the results reported previously (55Wang X. Goff H.M. Biochim. Biophys. Acta. 1997; 1339: 88-96Google Scholar, 57Goff H.M. Gonzalez-Vergara E. Bird M.R. Biochemistry. 1985; 24: 1007-1013Google Scholar). 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 (35Savenkova M.I. Satterlee J.D. Erman J.E. Siems W.F. Helms G.L. Biochemistry. 2001; 40: 12123-12131Google Scholar, 37de Ropp J.S. Sham S. Asokan A. Newmyer S. Ortiz de Montellano P.R. La Mar G.N. J. Am. Chem. Soc. 2002; 124: 11029-11037Google Scholar, 70Walker A.F. Simonis U. Berliner L.J. Reuben J. NMR of Paramagnetic Molecules. 12. Plenum Press, New York1993: 133-274Google Scholar, 71Dugad L.B. Goff H.M. Biochim. Biophys. Acta. 1992; 1122: 63-69Google Scholar). 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 (27Satterlee J.D. Erman J.E. LaMar G.N. Smith K.M. Langry K.C. J. Am. Chem. Soc. 1983; 105: 2099-2104Google Scholar, 38Asokan A. de Ropp J.S. Newmyer S.L. de Montellano P.R.O. La Mar G.N. J. Am. Chem. Soc. 2001; 123: 4243-4254Google Scholar, 39Chen Z. de Ropp J.S. Hernandez G. La Mar G.N. J. Am. Chem. Soc. 1994; 116: 8772-8783Google Scholar, 50Thanabal V. De Ropp J.S. La Mar G.N. J. Am. Chem. Soc. 1987; 109: 7516-7525Google Scholar, 72Banci L. Bertini I. Pease E.A. Tien M. Turano P. Biochemistry. 1992; 31: 10009-10017Google Scholar). 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 (73Satterlee J.D. Erman J.E. J. Biol. Chem. 1981; 256: 1091-1093Google Scholar). 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 (54Dugad L.B. Wang X. Wang C.C. Lukat G.S. Goff H.M. Biochemistry. 1992; 31: 1651-1655Google Scholar, 57Goff H.M. Gonzalez-Vergara E. Bird M.R. Biochemistry. 1985; 24: 1007-1013Google Scholar). 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 (6Sundaramoorthy M. Terner J. Poulos T.L. Structure (Lond.). 1995; 3: 1367-1377Google Scholar) rather than a histidine as in other heme peroxidases (24Finzel B.C. Poulos T.L. Kraut J. J. Biol. Chem. 1984; 259: 13027-13036Google Scholar, 74Edwards S.L. Raag R. Wariishi H. Gold M.H. Poulos T.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 750-754Google Scholar, 75Gajhede M. Schuller D.J. Henriksen A. Smith A.T. Poulos T.L. Nat. Struct. Biol. 1997; 4: 1032-1038Google Scholar, 76Sundaramoorthy M. Kishi K. Gold M.H. Poulos T.L. J. Biol. Chem. 1994; 269: 32759-32767Google Scholar). The proton/deuteron exchange on the Nε atom of the distal histidine has been shown to be responsible for the observed solvent effect on heme resonances in several heme peroxidases (36Wang X. Lu Y. Biochemistry. 1999; 38: 9146-9157Google Scholar, 53Thanabal V. De Ropp J.S. La Mar G.N. J. Am. Chem. Soc. 1988; 110: 3027-3035Google Scholar, 72Banci L. Bertini I. Pease E.A. Tien M. Turano P. Biochemistry. 1992; 31: 10009-10017Google Scholar). The spectral features of CPOCN display a high degree of similarity to that of other heme peroxidase cyanide derivatives (59Bertini I. Turano P. Vila A.J. Chem. Rev. 1993; 93: 2833-2932Google Scholar). 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 (54Dugad L.B. Wang X. Wang C.C. Lukat G.S. Goff H.M. Biochemistry. 1992; 31: 1651-1655Google Scholar). 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 β-protons of the heme vinyl and propionate groups as well as those from some of the amino acid residues near the heme center (53Thanabal V. De Ropp J.S. La Mar G.N. J. Am. Chem. Soc. 1988; 110: 3027-3035Google Scholar, 72Banci L. Bertini I. Pease E.A. Tien M. Turano P. Biochemistry. 1992; 31: 10009-10017Google Scholar, 77La Mar G.N. Walker F.A. Dolphin D. Porphyrins. 4. Academic Press, New York1979: 61-157Google Scholar, 78La Mar G.N. Viscio D.B. Smith K.M. Caughey W.S. Smith M.L. J. Am. Chem. Soc. 1978; 100: 8085-8092Google Scholar, 79La Mar G.N. Shulman R.G. Biological Applications of Magnetic Resonance. Academic Press, New York1979: 305-343Google Scholar). 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 TableI, along with the corresponding parameters in MnPCN (72Banci L. Bertini I. Pease E.A. Tien M. Turano P. Biochemistry. 1992; 31: 10009-10017Google Scholar, 80Banci L. Bertini I. Kuan I.C. Tien M. Turano P. Vila A.J. Biochemistry. 1993; 32: 13483-13489Google Scholar) and HRPCN (51Thanabal V. DeRopp J.S. La Mar G.N. J. Am. Chem. Soc. 1987; 109: 265-272Google Scholar) reported previously.Table IProton NMR parameters and assignments of paramagnetically shifted resonances in CPOCN at 298 K, in 0.1 m phosphate buffer, pH 5.5SignalCPOCNMnPCNaTaken from Ref.72.HRPCNbTaken from Ref.51.Assignment (isozyme)ShiftT 1IntShiftT 1IntShiftT 1IntppmmsppmppmmsppmppmmsppmhemeC24.0330.120.492−1.229.944−0.88-CH3(A)D23.8350.4cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.8-CH3(B)E20.730−0.730.767−18.225.157−1.03-CH3(A)F20.434−0.6cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.cCorresponding isozyme does not exist in MnPCN and HRPCN.3-CH3(B)G17.463.1dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.NAH17.063.0dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exist in MnPCN and HRPCN.dCorresponding resonance does not exis
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