Eur. J. Biochem. 210,337-341 (1992)

0FEBS 1992

The Soret magnetic circular dichroism of ferric high-spin myoglobins A probe for the distal histidine residue Ariki MATSUOKA, Nagao KOBAYASHI and Keiji SHIKAMA

Biological Institute and Pharmaceutical Institute, Tohoku University, Sendai, Japan (Received July 2/August 28, 1992) - EJB 92 0935

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.

In studies of myoglobin and hemoglobin molecules, much attention has been directed to the possible roles of the distal (E7) histidine residue. It has been suggested to act as a gate (Karplus and McCammon, 1986) or a swinging door (Johnson et al., 1989) for ligand entry into the heme pocket, and to stabilize the bound dioxygen by hydrogen-bond formation (Wittenberg et al., 1965; Phillips and Schoenborn, 1981). Furthermore, to facilitate the effective movement of a catalytic proton from the solvent to the coordinated dioxygen via the imidazole ring, it participates in a proton-relay mechanism for the autoxidation of oxymyoglobin to its met form (Sugawara and Shikama, 1980; Shikama, 1985,1988; Shikama and Matsuoka, 1986). However, it is not a simple task to determine unequivocally whether or not a myoglobin or a hemoglobin contains the usual distal histidine residue, even if the amino acid sequence is known. This is particularly true for the proteins isolated from lower organisms, since their globins show no notable degree of sequence similarity with mammalian myoglobins and hemoglobins. Another criteria is therefore needed to identify the distal (E7) histidine residue in these proteins in solution. Shikama and Matsuoka (1989) have examined a dozen myoglobins from various species for their spectrophotometric properties, and found that the proteins can be divided into two groups by a significant difference in the positioning of the Soret band. The oxymyoglobin peaks are in very close positions, whereas the acidic met forms have separate peak Correspondence ta K . Shikama, Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan 980 Abbreviation. MCD, magnetic circular dichroism.

positions in two different ranges depending on the presence or absence of the distal histidine residue. The myoglobin from Aplysia kurodai shows a Soret peak that is profoundly blue shifted and accompanied by a marked intensity decrease, which may be due to a broadening of the spectrum. So far in all cases we have examined, this spectral feature is unique for myoglobins lacking the distal histidine residue, and a possible explanation seems to be as follows. In the acidic metmyoglobins, the sixth-(distal)-coordinate position of the ferric heme iron is usually occupied by a water molecule, which is hydrogen bonded to the distal histidine. In the case of Aplysia myoglobin, the distal position is vacant in its acidic met form, and the water molecule is found near Val63 at position E7, far from the heme iron (Giacometti et al., 1981 ; Bolognesi et al., 1989). This seems to explain the different spectral patterns, since the Soret band originates from a n -+n* transition of the porphyrin ring and since this chromophore is likely to be open to more vibration, as well as to more doming toward the proximal side, due to lack of the axial-water ligand. In this respect, the magnetic circular dichroic (MCD) signal in the Soret region (350-450 nm) has been used as a more direct probe for the spin states and iron-coordination geometry in a wide variety of hemoproteins (Vickery et al., 1976; Holmquist, 1978; Bracete et al., 1991). In the present study, we evaluate the MCD spectra of ten native myoglobins and correlate the positions and intensities of their Soret peaks with the presence or absence of an axial water molecule, indicative of the distal (E7) histidine residue. On the basis of the MCD signals, we also examine a protozoan myoglobin (or monomeric hemoglobin) isolated from Paramecium caudatum for its unique structure.

338 MATERIALS AND METHODS Chemicals Sephadex G-50 (fine) was a product of Pharmacia. DEAEcellulose (DE-32) and carboxymethyl-cellulose (CM-32) were purchased from Whatman. All the chemicals used were of reagent grade and from Wako Pure Chemical. Solutions were made with deionized and glass-distilled water. Myoglobin preparation According to our standard procedures, native metmyoglobins were isolated from sperm whale skeletal muscle (Suzuki and Shikama, 1983), bovine heart muscle (Gotoh and Shikama, 1974), bigeye tuna fish (Kitahara et al., 1990), and chicken gizzard smooth muscle (Matsuoka et al., 1987). Specimens of A . kurodai and A . juliuna were collected along the Onahama coast, Fukushima Prefecture, and in the Mutsu Bay area, Aomori Prefecture, Japan. The fresh buccal masses were freed from radula and mucous membrane, washed well with cold distilled water, and used for the preparation of myoglobin as described (Shikama and Katagiri, 1984). P. cuudutum (syngen 3, stock StGl) was cultivated for 7 days at 25°C in a bacteria-free Dryl's solution (2 mM trisodium citrate, 2 mM sodium phosphate, 1.5 mM CaCl,, pH 7.0) containing 0.4% (massjvol.) reddish bean broth. The cells were harvested and kept frozen at -80°C until used for the preparation of myoglobin as described (Tsubamoto et al., 1990). Samples of the longissimus muscle of the African elephant (Loxodonta ufricana) were kindly supplied by the Yagiyama Zoo Park (Sendai), obtained from an animal that died from acute cardiomyopathy. The buccal masses of Dolabella auricularia and the crude myoglobin extract of Galeus nipponensis were gifts from Dr. T. Suzuki (Kochi University, Kochi, Japan). The essential step for the myoglobin preparation was the chromatographic separation of metmyoglobin from oxymyoglobin (Mb02) on a DEAE-cellulose or carboxymethylcellulose column. The concentration of myoglobin was determined after conversion into cyanometmyoglobin, using the millimolar absorption coefficient at 540 nm, obtained on the basis of the pyridine hemochromogen method (De Duve, 1948). Spectrophotometric measurements Spectrophotometric measurements were carried out in an Hitachi spectrophotometer (model 557 or U-3210) equipped with a thermostatically controlled (within 0.1 "C) cell holder. Magnetic-circular-dichroic measurements MCD spectra were recorded at 20°C in a Jasco model J500 spectropolarimeter equipped with a 1.09-T electromagnet. To minimize the experimental error, the spectra were measured twice, first with the electromagnetic field parallel, then antiparallel to the direction of light propagation. The differences in magnitude at each wavelength were divided by twice the strength of the magnet. In the Soret region, recordings were made with myoglobin of 50- 100 pmol/l in a 0.1-cm cell and at a scale setting of 0.5-5.0 x l o p 3 deg/cm on the chart. The MCD magnitude is thus expressed by the molar ellipticity/ unit magnetic field, [el,, in deg . cm2 . dmol- . T - or in deg . M-' . c m p l . T - I . Another parameter used in the literature is the differential molar absorption coefficient, A E M , in M-'

'

I

1

-40

200

300

400

500

600

700

wavelength ( nm )

Fig. 1. MCD spectra of the cyano derivativesof sperm whale (-) and A . kuvodui (---) myoglobins. The concentration of each protein was 50 pM in 0.01 M sodium phosphate, pH 7.1 in the presence of 0.01 M KCN.

. cm-I

[el,

. T-', which is connected to [el, by the relationship

= 3300

AEM.

RESULTS AND DISCUSSION MCD measurements, applied to hemoproteins, provide spectra sensitive to the direct environment of the heme iron, including the oxidation and spin state of the metal, the kind of axial ligands and the metal-ligand bond distances (Holmquist, 1978). Fig. 1 shows the MCD spectra of the cyano derivatives of sperm whale and A . kurodai metmyoglobins recorded in 10 mM sodium phosphate, pH 7.1. Although these proteins have quite different primary structures, the curves are essentially identical, indicating that the coordination spheres of the heme irons strongly resemble each other. In these derivatives, the sixth ligand is a cyano group of a very strong ligand field strength, thereby producing the same spectra for both myoglobins within an experimental error, regardless of the large difference in their globin moieties. Normally, in the acidic met form, the sixth coordinate position is occupied by a water molecule, a weak ligand which is usually stabilized by hydrogen-bond formation to the distal (E7) histidine residue. In the case of Aplysia myoglobins that lack the distal histidine (Takagi et al., 1984), the sixth coordinate position is vacant in its acidic met form (Bolognesi et al., 1989). We therefore examined the MCD spectra of native sperm whale and Aplysia metmyoglobins in 10 mM sodium phosphate, at pH 6.1 and 5.8, respectively. As shown in Fig. 2, both spectra are characteristic of the ferric high-spin species (Dawson and Dooley, 1989; Kobayashi et al., 1983, 1985; Kobayashi, 1985), but are different from each other, especially in the Soret region (350 - 450 nm). This is likely to be a reflection of the difference in the sixth-coordinate sphere. A distinct and symmetric dispersion-type curve is observed for sperm whale metmyoglobin, whereas A . kurodai metmyoglobin reveals a relatively weak and unsymmetric band pattern, which seems to be a superimposition of two dispersion curves, each having minus to plus signs from longer wavelength. By the differences in shape and intensity of the Soret MCD pattern for the acidic met form, myoglobins can be divided into two groups. Specifically, if we pay attention to the inten-

339 200

-

-

J

I

I

r

1

I

I

-4

-5

-6

180

160

2

-E

140

z

v)

120

200

400

300

500

700

600

wavelength ( nm )

Fig.2. MCD spectra of the acidic met forms of sperm whale (-) and A . kuvodui (- - -) myoglobins. The concentration was 100 pM for each protein in 0.01 M sodium phosphate; the pH was 6.1 for sperm whale and 5.8 for Aplysia myoglobin.

100

80 0

Table 1. Magnetic circular dichroism and absorption characteristics of the Soret peak in the acidic met form of myoglobins from various sources. His(E7) denotes the presence (+) or absence (-) of the distal histidine residue. Source

Sperm whale Bovine Chicken Bigeye tuna P. cuudatum Aplysiu kurodai Aplysiu juliana D. auriculariu Africanelephant G. nipponensis BrCN-modified sperm whale Mb a

His(E7) MCD i

[el,

Absorption An,,

8

nm

deg.M-' . c m - l . T-1

nm

mM-' . cm-'

(+) (+) (+) (+) (+)" (-) (-) (-) (-) (-)

416 417 418 415 416 418 419 421 418 417

-45900 -47700 -49200 -51400 -58300 - 15100 -17600 -21100 -14700 -15400

409 409 409 406 407 395 395 402 408 399

168 188 164 151 163 97 94 99 117 100

(-)b

420

-12200

393

107

As revealed by the data presented in this paper. The cyanation of the distal histidine imidazole NH site was

carried out with sperm whale metmyoglobin by the addition of BrCN in a 10-fold molar excess at pH 6.0 and 4°C (Shiro and Morishima, 1984; Morishima et al., 1989).

sity of the negative MCD extremum of the Soret band, one group has the values close to that of sperm whale myoglobin, and the other shows values similar to that of A . kurodai myoglobin, as summarized in Table 1. In the myoglobins from the gastropodic sea molluscs A . kurodai, A . juliana and D. auriculuria, the distal (E7) histidine is replaced by Val63 (Takagi et al., 1984; Suzuki 1986). The shark myoglobin from G. nipponensis has a distal glutamine at position 59 (Suzuki et al., 1988), as is also the case for the myoglobin from the African elephant ( L . africana), containing Gln64 in place of the distal histidine (Romero-Herrera et al., 1981). All these substituted residues seem to be incapable of hydrogen bonding to the axial water ligand, thereby making the ferric myoglobins

I

I

-1

-2

I

-3

-7

Fig. 3. Different positioning of acidic metmyoglobins in an absorptivity versus MCD map for the Soret peak. The intensities separate myoglobins into two groups; one with (0)and the other lacking ( 0 ) the distal histidine residue. The sources were sperm whale (S), bovine heart (B), chicken gizzard (C), bigeye tuna (T), a ciliated protozoa P. caudatum (P), African elephant (E), a shark G . nipponensis (G); three sea molluscs A . kurodai (Ak), A . juliunu (Aj) and D.auricularia (D). BrCN-modified sperm whale metmyoglobin (CN) was also plotted (see Table 1).

pentacoordinate with a substantially less-negative MCD signal in the Soret region (Table 1). In a previous paper, we have reported that myoglobins can be divided into two groups on the basis of the different positionings of the Soret absorption bands (Shikama and Matsuoka, 1989). For comparison with the MCD signals, we have also measured and listed in Table 1 the spectrophotometric parameters of the Soret peak. As is illustrated in Fig. 3, there is a good correlation between the molar absorptivity of the Soret peak and the MCD intensity. It can thus be concluded that either from absorption or MCD spectra of the Soret band for the acidic met form, one can predict with high accuracy whether or not the distal histidine residue is present in myoglobin molecules in solution. Among the proteins listed in Table 1, the myoglobin (or monomeric hemoglobin) from P. cuudatum is of particular interest, since its amino acid sequence (Iwaasa et al., 1989) provides us with an insight into the myoglobin-hemoglobin chemistry as well as the evolution of these molecules from protozoa to higher animals. Paramecium myoglobin consists of 116 amino acid residues with a molecular mass of 12 565 Da, including the heme moiety. It is smaller than the mammalian myoglobins by 37 residues, and even smaller than a bacterial hemoglobin subunit from Vitreoscillu by 30 residues (Wakabayashi et al., 1986). The protein contains two histidine residues at positions 68 and 84 (Iwaasa et al., 1989), but reveals no notable degree of sequence similarity with other known hemoproteins. In relation to the oxygen-binding property of Paramecium myoglobin having P50 = 80 Pa (0.6 Torr) for the

340 I

0 ,

-6

‘

200

I 300

400

500

600

700

wavelength ( nm )

Fig. 4. MCD spectra of the acidic met forms of Puvarnecium (-) and elephant (---) myoglobins in 0.01 M sodium phosphate. The pH was 5.0 for Paramecium and 6.1 for elephant myoglobin. Protein concentrations werc 10-40 pM.

half-saturation pressure (Smith et al., 1962), of primary concern is the positioning of the two histidine residues. The distance between them, however, is shorter than that of the usual myoglobins by a dozen residues or so. Fig. 4 shows the MCD spectra of the acidic met forms of Paramecium myoglobin and elephant myoglobin, the latter lacking the distal histidine residue. Judging from a strong and symmetric dispersion-type Soret MCD signal, Paramecium myoglobin falls unequivocally within the group ofmyoglobins that has a water molecule at the sixth-coordinate position in the acidic met form. Consequently, its axial water molecule is stabilized, probably by hydrogen-bond formation to a distal histidine residue at position 68, while His84 represents the proximal heme-binding histidine residue. These structural features, deduced from MCD spectroscopy, are in full accord with our previous observations (Tsubamoto et al., 1990)of the stability of Paramecium oxymyoglobin (MbOz), involving the distal histidine as the catalytic residue (via its imidazole ring) for its autoxidation reaction to metmyoglobin (Shikama and Matsuoka, 1986; Shikama, 1988). The Soret absorption band of ferric myoglobin complexes is regarded as a superimposition of a high-spin and a low-spin component present in equilibrium, although the corresponding peaks are unlikely to be resolved (Smith and Williams, 1970). In this respect, MCD spectroscopy has a more ‘fingerprint’ character than absorption spectroscopy, since MCD spectra show the positive and negative-sign features which are sensitive to degenerate electronic-energy levels of the molecule (Dawson and Dooley, 1989). For this advantage, many metmyoglobin derivatives have been examined for their MCD properties, and the intensity of the negative extremum to long wavelength of the Soret band has been shown to increase linearly in proportion to the low-spin content of each derivative (Vickery et al., 1976; Browett and Stillman, 1979). For the acidic metmyoglobins having a water molecule at the sixthcoordinate position, it is clear that the negative MCD is much less deep as compared with that of the cyano-derivatives shown in Fig. 1. In fact, the low-spin content has been estimated to be about 12% for sperm whale aqua-metmyoglobin, if the value of 100% is assigned for its cyano-derivative (Smith and Williams, 1968). However, for the myoglobins with the

sixth-coordinate position being vacant due to lack of the distal histidine residue, the Soret MCD signal appears to be somewhat complicated, presumably with superimposition of a highspin component appearing at slightly shorter wavelength. Nevertheless, it is clear that the intensity of the negative extremum is significantly lower than that of the usual aqua-met species. In conclusion, from MCD spectra of the Soret band for the acidic met form, we can predict with high accuracy the presence or absence of the distal histidine residue in myoglobin (or monomeric hemoglobin) molecules in solution. This finding is reinforced by recent studies (Morikis et al., 1990; Egeberg et al., 1990; Rajarathnarn et al., 1991) using sitedirected mutagenesis of sperm whale myoglobin. They clearly show that replacement of the distal (E7) histidine residue can discriminate against water molecule as a sixth ligand and favors a pentacoordinate structure. We are greatly indebted to Professor R. J . P. Williams, Inorganic Chemistry Laboratory, University of Oxford, for his valuable comments on the Fe(lT1) spectra. This work was partly supported by grants from the Ministry of Education, Science and Culture of Japan (04454022), and from the Japan Society for the promotion of Science (02954047).

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