Biochemistry 1986, 25, 5844-5849
5844
Articles
Structural Characterization of Lactoperoxidase in the Heme Environment by Proton NMR Spectroscopyt Yoshitsugu Shiro and Isao Morishima* Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606, Japan Received December 19, 1985: Revised Manuscript Received May 12, 1986
ABSTRACT: T h e heme environmental structures of lactoperoxidase (LP) have been studied by the use of hyperfine-shifted proton NMR and optical absorption spectra. The NMR spectra of the enzyme in native and cyanide forms in H 2 0 indicated that the fifth ligand of the heme iron is the histidyl imidazole with an anionic character and that the sixth coordination site is possibly vacant. These structural characteristics are quite similar to those of horseradish peroxidase ( H R P ) , suggesting that these may be prerequisite to peroxidase activity. The p H dependences of the spectra of LP in cyanide and azide forms showed the presence of two ionizable groups with pK values of 6 and 7.4 in the heme vicinity, which is consistent with the kinetic results. T h e group with pK = 7.4 is associated with azide binding to LP in a slow N M R exchange limit, which is in contrast to the fast entry of azide to HRP.
Lactoperoxidase (LP) is a constituent of mammalian milk, saliva, and tears. In common with other peroxidases, the enzyme catalyzes the oxidation of a large number of substrates by hydrogen peroxide and is therefore a component of the biological defense system of mammalians. It also relates to the biosynthesis of the hormon thyroxine through an iodination reaction (Morrison & Schonbaum, 1976). The early works have shown that the prosthetic group of LP is not protoheme IX but is a mesoheme IX in conjunction with the porphyrin skeleton and hydroxyl groups attached to the side chain (Hultquist & Morrison, 1963; Morrison et al. 1970), or the heme is covalently bound to the protein through an alkalinelabile chemical bond such as an ester or amide linkage (Morel1 & Clezy, 1963). The spectral properties of LP suggested that a group ionizing with pK = 11.2, which may be either the guanidine of Arg or the eamino of Lys, is probably the coordinating group to the heme iron (Morrison et al., 1966, 1970). These proposed structures of the active site of LP are much different from those of plant peroxidases such as horseradish peroxidase (HRP), although both enzymes essentially have the same catalytic function. Recently, however, Sievers (1979) showed the heme of LP to be protoheme IX by isolating the prosthetic group from a pronase hydrolysate of the enzyme and indicated that it is buried in a crevice of the protein molecule. On the basis of his comprehensive studies of LP, he also suggested that the proximal ligand of the heme iron might be the histidyl imidazole (Sievers, 1980; Sievers et al., 1983, 1984). Although some information on LP has been accumulated in the past decade (Kimura et al., 1981), a knowledge of the structure of L P in the heme vicinity has not yet been sufficient for understanding its characteristic function and the heme environmental structure. Proton N M R studies of hemoproteins in paramagnetic forms can provide us with some structural details in the heme vicinity relevant to their functions. Recently, La Mar and his 'This work was supported by grants from the Ministry of Education, Japan (60540285 and 60790122). * Author to whom correspondence should be addressed.
0006-2960/86/0425-5844$01.50/0
co-workers (La Mar et al., 1977, 1979, 1980; Cutnell et al., 1981) showed the proton N M R spectra of some hemoprotein derivatives in H 2 0 and assigned an exchangeable proton resonance located in the paramagnetically shifted region as the histidyl imidazole N , H . We have studied here proton NMR spectra of LP in H 2 0 and 2H,0 solutions in comparison with those for H R P and myoglobin (Mb) and assigned the proximal histidyl N , H proton N M R resonance of LP. The pH dependences of the N M R spectra of LP derivatives show the presence of the ionizable groups in the heme vicinity responsible for regulating the external ligand binding to the heme iron. These spectral data are also discussed in relation to the structural specificities of peroxidases for their function and their ligand-binding properties (Morishima et al., 1977, 1978). MATERIALS AND METHODS Lactoperoxidase purchased from Sigma (type L-2005) as a salt-free lyophilized powder was further purified by dialysis at pH 7 and following application to CM-52. The purity index, R Z value, of the enzyme was 0.7. The concentration of the enzyme was determined spectrophotometrically at 412 nm by using an absorptivity of 114 cm-' mM-' at pH 7. Sperm whale myoglobin (type 11) was purchased from Sigma, and horseradish peroxidase (type G-I-C, R Z = 3.3) was obtained from Toyobo Co. (Osaka). The cyanide or azide complexes of the proteins were prepared by adding a 5- to 10-fold excess amount of the ligand to the enzyme solution. The pH titrations were performed by directly adding 0.1 N N a O H or HC1 to the enzyme solution. The pH values were measured with a Radiometer Model PHM-100 pH meter, equipped with an Ingold microcombination glass electrode. Proton N M R spectra were recorded at 300 M H z on a Nicolet NTC-300 spectrometer equipped with a 1280 computer system. Typical spectra of the enzyme consisted of 40000-100000 transients using 8K data points and 5.7-ps 90' pulse after the strong solvent resonance in H 2 0 solution was suppressed by a 500-1s low-power 180' pulse. Proton chemical shift is referenced with respect to the proton signal of the water in the enzyme solution, assigning a positive value for low-field resonance.
0 1986 American Chemical Society
HEME ENVIRONMENT IN LACTOPEROXIDASE
VOL. 25, N O . 20, 1986
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1: (A) Proton NMR spectra of aquometmyoglobin, horseradish peroxidase, and lactoperoxidase in H 2 0 at pH 7 and 23 O C The downfield region of the hyperfine-shifted spectra is illustrated. Chemical shift is referenced with H 2 0 signal. (B) Temperature dependence of the heme peripheral proton signals of LP at pH 7. FIGURE
The spin-lattice relaxation time, T I ,of the water proton was measured a t 100 MHz with a JEOL FX-100 pulsed spectrometer by using a 18Oo-7-9O0 pulse sequence. The frequency dependence of the T , between 0.01 and 20 MHz was measured with an originally designed N M R spectrometer at IBM, Thomas J. Watson Research Center, Yorktown Heights, N Y (Fabry & Koenig, 1966; Gupta & Koenig, 1971; Fabry et al., 1971). The paramagnetic relaxivity of the enzyme was analyzed with Solomon and Bloembergen, Luz and Meiboom, and Swift and Connik equations (Dewk, 1973; Mildvan & Cohn, 1970). The visible absorption spectra were recorded with a Union Giken SM-401 spectrometer by using a cell with a 1-cm path length. RESULTS The hyperfine-shifted portions of the 300-MHz proton N M R spectrum of ferric lactoperoxidase (LP) in H 2 0 are compared with those of aquometmyoglobin (aquometMb) and ferric horseradish peroxidase (HRP) at 22 OC and neutral pH in Figure 1A. In the spectrum of LP, the poorly resolved proton peaks, probably arising from the heme peripheral groups, are observed at 61.5, 55.7, 50.7, 38.3, and 30.6 ppm. The spectral characteristics are quite different from those for the two other hemoproteins, where four well-resolved heme methyl signals are observed in the 50-90 ppm region. However, a broad single-proton resonance that was not detected in 2 H 2 0solution is observed at 104.1 ppm. The resonance is close in position and width to the proximal histidyl N , H signals for aquometMb and H R P (La Mar & de Ropp, 1979; La Mar et al., 1980). The temperature dependence of the paramagnetic shifts strictly follows the Curie law over a temperature range of 17-37 OC as is shown in Figure 1B. In order to obtain the structural implication of the heme sixth site of LP, we have studied the temperature dependence of the water proton magnetic relaxivity of L P (Mildvan & Cohn, 1970; Dewk, 1973). The spin-lattice relaxivities (T,-I) measured at pH 7 and 100 MHz are plotted in Figure 2A against inverse absolute temperature between 9 and 40 OC (Gupta & Mildvan, 1975; Lanir & Schejter, 1975). TI-' of L P monotonically increased with decreasing temperature (Morishima & Ogawa, 1982), showing the presence of a rapidly exchanging water molecule in and out of the para-
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