1H NMR study of dynamics and thermodynamics of acid–alkaline transition in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi)

Biochimica et Biophysica Acta 1385 Ž1998. 89–100

1

H NMR study of dynamics and thermodynamics of acid–alkaline transition in ferric hemoglobin of a midge larva ž Tokunagayusurika akamusi /

Kunihiro Koshikawa a , Yasuhiko Yamamoto a,) , Satoshi Kamimura b, Ariki Matsuoka b, Keiji Shikama b a

b

Department of Chemistry, UniÕersity of Tsukuba, Tsukuba 305-8571, Japan Biological Institute, Graduate School of Science, Tohoku UniÕersity, Sendai 980-77, Japan Received 16 December 1997; revised 27 February 1998; accepted 19 March 1998

Abstract One of the components of hemoglobin from the larval hemolyph of Tokunagayusurika akamusi possesses naturally occurring substitution at the E7 helical position ŽLeu E7. wM. Fukuda, T. Takagi, K. Shikama, Biochim. Biophys. Acta 1157 Ž1993. 185–191x. Its oxygen affinity is almost comparable to those of mammalian myoglobins and it exhibits Bohr effect. Both acidic and alkaline forms of the ferric hemoglobin have been investigated using 1 H NMR in order to gain insight into molecular mechanisms for relatively high oxygen affinity and Bohr effect of this protein. The NMR data indicated that the acidic form of the protein possesses pentacoordinated heme, and that the alkaline form possessing OHy appears with increasing the pH value. pH titration yielded a p K value of 7.2 for the acid–alkaline transition, and this value is the lowest among the values reported so far for various myoglobins and hemoglobins. The kinetic measurements of the transition revealed that the activation energy for the dissociation of the Fe-bound OHy, as well as the dissociation and association rates, decrease with increasing the pH value. These pH dependence properties are likely to be related to the Bohr effect of this protein. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Acid–alkaline transition; Monomeric hemoglobin; NMR; Paramagnetic relaxation; Paramagnetic shift; Saturation transfer

1. Introduction

Abbreviations: Hb, hemoglobin; HbV, Tokunagayusurika akamusi hemoglobin component V; Mb, myoglobin; metHb, ferric hemoglobin; metMb, ferric myoglobin; metHbVŽOHy ., met-hydroxyl form of T. akamusi hemoglobin component V; metMbŽOHy ., met-hydroxyl form of myoglobin ) Corresponding author. Fax: q81-298-53-7369; E-mail: [email protected]

Hemoglobin Ž Hb. from the 4th-instar larva of Tokunagayusurika akamusi, a midge Ž Diptera. commonly found in eutrophic lakes in Japan, is composed of at least 11 different components w1,2x. Among these components, hemoglobin V Ž HbV. is one of the two components of which primary structures have been determined and functional properties have been measured w1x. HbV possesses a single peptide chain

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 5 1 - X

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that consists of 152 amino acid residues. Its primary structure is completely different from those of mammalian Mbs, except that Phe and His occupy at the CD1 and F8 helical positions, respectively. A comparison of the sequence between HbV and the monomeric Hb components from other midge larva such as Chironomus thummi thummi w3–9x and C. thummi piger w10x, on a basis of a matrix analysis, indicated the homology of less than 30% w1x. Furthermore, the mean residue ellipticity at 222 nm, obtained from the circular dichroism spectrum, indicated that HbV possesses considerably reduced helical contents, compared with mammalian myoglobins ŽMbs. Ž Shikama et al., unpublished results. . In addition, it has been suggested that Leu occupies at the E7 helical position of HbV. In spite of the lack of an amino acid residue capable of being a hydrogen-bond donor, at the E7 helical position, for stabilizing Febound oxygen, HbV exhibits relatively high oxygen affinity Ž P50 s 0.79 mmHg at 258C and pH 7.4. ŽShikama et al., unpublished results. . Another characteristic feature of its function is that it exhibits alkaline Bohr effect ŽShikama et al., unpublished results.. In most vertebrate Mbs and Hbs, His E7 is highly conserved and acts as a proton donor to stabilize the Fe-bound ligand w11–17x. In fact, genetic replacement of His E7 by a residue with a non-polar side-chain leads to a considerable decrease in the ligand affinity w18,19x. However, from the studies of Mbs of mollusc Aplysia limacina w20–22x and Dolabella auricularia w23x, it has been shown that functional properties of Mb and Hb are also controlled by a residue other than the residue at the E7 helical position w24–28x. The mollusc Mbs exhibit relatively high oxygen affinity, although they possess Val E7 w27,29x. X-ray w24,25,28x and NMR w26,27,30x structural studies of the mollusc Mbs have revealed that the guanidyl group of Arg E10 serves as a hydrogen-bond donor to the bound ligand, and that Arg E10 in the mollusc Mb plays similar roles in controlling the stabilization of Fe-bound ligand to what His E7 does in most vertebrate Mb and Hb. Both the structural and functional properties of Arg E10 in the mollusc Mb were mimicked by the double mutation, His E7 ™ Val and Thr E10 ™ Arg, of sperm whale Mb w31,32x. HbV possesses Arg E11 w1x, and this Arg residue may contribute to functional properties of this protein, as Arg E10 does in the mollusc Mb.

The acid–alkaline transition in ferric Mb Ž metMb. and Hb ŽmetHb. sharply reflects characteristics of ligand binding properties of the active sites in hemoproteins w31–41x. The acidic form of metMb and metHb generally possesses H 2 O as Fe-bound ligand and, upon the transition to alkaline form, the bound ligand is changed to OHy, with the concomitant change of the spin state from S s 5r2 to S s 1r2. In the case of Mbs possessing His E7, in which the sixth ligation site is usually occupied by H 2 O, the interconversion between acidic and alkaline forms is quite fast because the protonationrdeprotonation process of the His E7 imidazole bound to the Fe-bound ligand via a hydrogen bond facilitates the transition w42x. On the other hand, in the case of metMbs in which the sixth ligation site is vacant, the transition occurs in a relatively slow time scale because exogenous ligand OHy has to diffuse intorout of the heme pocket w34,37,39–41x. Recent studies revealed that the presence of an amino acid residue capable of being a proton acceptor is essential for stabilization of Fe-bound H 2 O w41x. Hence, H 2 O coordination at the sixth ligation site of heme iron in acidic form of metMb and metHb provides a diagnosis of the presence of a proton acceptor in the proximity of the binding site. Thermodynamics of the acid–alkaline transition has been analyzed using a variety of techniques such as 1 H NMR w33,36x, optical spectroscopy w35,43x, EPR w44x and magnetic susceptibility measurement w43x. Paramagnetic 1 H NMR saturation transfer experiment allows quantitative characterization of the kinetics of the transition, provided that the transition rate is comparable to paramagnetic relaxation rate of heme peripheral side-chain proton w32,40,41x. We report herein the results of 1 H NMR study in dynamics and thermodynamics of the acid–alkaline transition of ferric T. akamusi Hb component V ŽmetHbV.. The present study revealed that H 2 O is not coordinated to heme iron of acidic form of metHbV, as in acidic form of the mollusc Mbs. The high affinity of metHbV to OHy is reflected in the value of 7.2 for the p K value of the transition, and this value is the lowest among the values reported for various metMbs and metHbs w31,34,39–41,45,46x. Furthermore, the reaction rate of the dissociation of Fe-bound OHy was found to decrease with increasing the pH value, and this result is possibly related to

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alkaline Bohr effect of this protein. A unique heme active site in HbV is discussed on the basis of the analyses of the acid–alkaline transition and heme peripheral methyl proton hyperfine shifts.

ening. The chemical shifts are given in parts per million Žppm. relative to sodium 2,2-dimethyl-2silapentane-5-sulfonate with the residual H 2 HO as internal reference.

2. Materials and methods

2.3. Determination of the kinetic parameter for acid–alkaline transition Õia saturation transfer factor

2.1. Protein preparation Hb from the 4th-instar larva of T. akamusi was prepared as met form using the reported procedure w1,2x. The Hb solution was concentrated to about 1 mM, and the solvent was exchanged to 2 H 2 O in an Amicon ultrafiltration cell. Sperm whale Mb was purchased from Biozyme and used without further purification. The p 2 H value of the sample was adjusted using 0.2 M NaO 2 H or 0.2 M KH 2 PO4 in 2 H 2 O solution, and the p 2 H value was measured using a Horiba model F-22 pH-meter with a Horibatype 6069-10 c electrode. The isotope effect was not considered to correct the p 2 H value. 2.2. NMR spectroscopy 1

H NMR spectra were recorded using Bruker AC400P FT-NMR spectrometer operating at a 1 H frequency of 400 MHz. A typical spectrum consisted of 3 k transients with 8 kbyte date points over 50 kHz band width, and a 4.6 m s 458 pulse. Solvent signal suppression, when necessary, was achieved by direct saturation during the relaxation delay. Intrinsic spin– lattice relaxation time Ž T1intr . was measured using the saturation-recovery method with a selective saturation pulse. Saturation transfer experiments were carried out by selectively saturating a desired peak for a variety of time and steady-state value of the saturation transfer factor Ž IrI0 ; I and I0 are the signal intensities of peak A without and with the saturation of peak B which is connected to peak A by dynamic process, respectively. was achieved for the saturation time G 80 ms. The spectra that resulted from the saturation transfer experiments were presented in a form of difference spectrum. The saturation transfer factor was obtained from measuring the signal intensity. The signal-to-noise of the spectra was improved by apodization, which introduced a 50-Hz line broad-

The acid–alkaline transition in metHbV is expressed as, k AB

metHbVq OHy ° metHbV Ž OHy . k BA

where k AB and k BA are the association and dissociation rates, respectively. In the saturation transfer experiments, the pair of spectra recorded without and with the saturation of a heme methyl proton resonance of metHbV yields I0 and I for the signal intensity of the corresponding heme methyl proton resonance of metHbVŽ OHy., respectively. The saturation transfer factor Ž IrI0 . is related to k BA and T1intr of the heme methyl proton resonance of metHbVŽOHy. by the formula w47x, k BA s Ž T1intr .

y1

Ž 1 y IrI0 . r Ž IrI0 .

Ž1.

k AB is related to equilibrium constant of the reaction, K, by the following equation, K s k ABrk BA s metHbV Ž OHy . r metHbV OHy Ž2. where wmetHbVŽOHy.x, wmetHbVx and wOHyx are the concentration of corresponding species. Therefore, the k AB value can be calculated from the k BA value, wOHyx and wmetHbVŽOHy.xrwmetHbVx, which is determined from the intensity of the heme methyl proton resonance arising from metHbVŽOHy. and metHbV. 2.4. Spectrophotometric measurements Spectrophotometric measurements were carried out using a Hitachi two-wavelength double-beam spectrophotometer Ž model U-3210. equipped with a thermostatically controlled cell holder. The acid–alkaline transition of metHbV was monitored at 577 nm band in 0.1 M KCl at 258C.

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3. Results 3.1. 1H NMR spectra of metHbV The 400 MHz 1 H NMR spectra of metHbV at 258C and pH 5.10 is illustrated in trace A of Fig. 1, and is compared with that of sperm whale metMb, pH 7.10, shown in trace B. Four heme methyl proton resonances in trace A are clearly identified below 60 ppm. It has been demonstrated that heme methyl proton hyperfine shift pattern in ferric high-spin form of b-type hemoprotein is independent of the coordination structure of heme iron, as long as heme is inserted into the heme pocket with its usual orientation relative to protein moiety w39,41,49x. Consequently, the heme methyl proton resonances of metHbV are assigned to 8-, 5-, 3- and 1-Me in order of decreasing chemical shifts. The heme methyl proton signals of metHbV are compared with those of sperm whale metMb, which possesses hexacoordinated heme with the Fe-bound water at the sixth ligation site, in Table 1. The comparison reveals some characteristic differences. First, the line width of heme methyl proton signals in TA metHbV Ž; 300 Hz. is much smaller than that of sperm whale metMb Ž ; 600 Hz. . Since the line

Fig. 1. The 400 MHz 1 H NMR spectra of T. akamusi metHbV, pH 5.10, ŽA. and sperm whale metMb, pH 7.10, ŽB. in 2 H 2 O at 258C. Heme methyl proton assignments w48x are given with the spectra. Heme meso-proton signals are observed at about y25 and 38 ppm in ŽA. and ŽB., respectively.

width of these resonances is influenced by T1e , which is primarily determined by zero-field level of the complex w50,51x, relatively short T1e of metHbV, reflected by smaller line width of the signals, can be attributed to lower symmetric nature of the heme coordination structure. Furthermore, heme meso-proton signal of sperm whale metMb is observed at about 38 ppm. On the other hand, an extremely broad signal is observed at about y25 ppm in the spectrum of metHbV, and its line width strongly suggests that this signal arises from heme meso-protons of this protein. It has been demonstrated from the studies of model compounds and ferric high-spin Mbs that the meso-proton signals of hexacoordinated and pentacoordinated ferric high-spin heme complexes resonate at downfield and upfield hyperfine shifted regions of the spectra, respectively w52x. Therefore, upfieldshifted meso-proton signal in the spectrum of metHbV also supports that this protein possesses the pentacoordinated heme in its active site. In addition, the average heme methyl proton shift of metHbV is larger than that of sperm whale metMb Žsee Table 1. . It has been reported that the average heme methyl proton shift of hexacoordinated heme is larger than that of pentacoordinated heme possibly due to the difference in zero-field splitting between the two complexes w49x. Hence, the present result is consistent with the pentacoordinated heme in metHb. Furthermore, the spread of heme methyl proton shift of metHbV is smaller than that of sperm whale metMb, indicating that the in-plane asymmetry of the heme electronic structure in the former is smaller than that of the latter. Finally, it has been reported that the shift and the line width of the hyperfine shifted heme methyl proton resonances of ferric high-spin hemoprotein are influenced by solvent isotope composition, i.e., H 2 Or2 H 2 O, through hydrogen bond between the coordinated water molecule and a distal amino acid residue w53x. The fact that the resonances of metHbV are independent of the solvent isotope composition Žresult not shown. also supports the absence of the coordinated water molecule in this protein. Therefore, all these NMR results are consistent with the pentacoordinated heme in metHbV. The absorption spectrum of metHbV at pH 5.5 exhibits the Soret band at 396.8 nm with the extinction coefficient of 106.5 mMy1 cmy1. A slight hypochromicity and a large hypsochromicity of the

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Table 1 Assignment and chemical shift of heme methyl proton signals in both acidic and alkaline form of T. akamusi metHbV and sperm whale metMb at 258C Acidic form metHbV 8-Me 5-Me 3-Me 1-Me Average g Spread h

a

90.22 Žy33.06. e 83.15 Žy31.95. 80.09 Žy35.12. 67.47 Žy33.06. 80.23 22.75

Alkaline form metMb

b

90.83 84.29 72.71 52.85 75.17 37.98

metHbVŽOHy . c

metMbŽOHy . d

26.82 wq0.79x f Ž29.84. e 30.80 wq0.65x Ž17.17. 20.78 wq1.10x Ž53.61. 25.32 wq0.52x Ž16.18. 25.93 wq0.76x Ž29.84. 10.01

38.12 Ž15.59. e 38.12 Ž15.59. 33.61 Ž20.77. 26.78 Ž12.24. 34.16 Ž16.19. 11.34

a

pH 5.10. pH 7.10. c pH 9.95. d pH 11.03. e The numbers in parentheses are the shifts to Ty1 ™ 0, obtained from the Curie plot. f Shift difference, in ppm, 2 H 2 O and H 2 O is given in brackets; positive shifts indicate downfield bias in H 2 O relative to 2 H 2 O. g Average of heme methyl proton shifts. h Spread of signals. b

Soret band of metHbV, compared with those of mammalian metMbs possessing the usual His E7, also strongly suggested the absence of Fe-bound water in this monomeric metHb w1,54x. 3.2. pH dependence of 1H NMR spectra of metHbV The pH dependence of the 400 MHz 1 H NMR spectra of metHbV is shown in A of Fig. 2. The resonances arising from alkaline form appear in the chemical shift region 15–35 ppm. The shifts of heme methyl proton signals of alkaline form are similar to those of met-hydroxyl form Ž metHbVŽ OHy.. . With increasing pH, the signal intensity for the resonances of alkaline form increases at the expense of the signals of the acidic form. The fact that the two sets of the signals from the acidic and alkaline forms are separately observed in the spectra indicates that the acid–alkaline transition in this protein is slower compared with the NMR time scale. This result is in sharp contrast to the rapid acid–alkaline transition in sperm whale metMb Žsee B of Fig. 2.. The analysis of the pH dependence of the signal intensity for the resonances arising from the two forms of metHbV yielded the p K value of 7.2 " 0.1. The acid–alkaline transition of ferric hemoprotein is conveniently monitored by spectrophotometry w45x. A pH titration curve of metHbV monitored using the absorption at 577 nm in 0.1 M KCl at 258C is illustrated in Fig. 3. The analysis of the titration curve based on the Hender-

son–Hasselbalch equation yielded a p K value of 7.22. Thus, the p K values obtained from NMR and absorption spectra are essentially identical. This p K value is the lowest among the values reported for various ferric hemoproteins w31,34,39–41,45,46x. Since the acidic form of metHbV possesses pentacoordinated heme, its low p K value reflects a considerable stabilization of Fe-bound OHy with a protondonor in this protein. 3.3. Heme methyl proton signal assignment of metHbV(OH y) Õia saturation transfer The saturation transfer difference spectra resulted from the saturation of individual heme methyl proton signal of metHbV at 358C, pH 7.12, are shown in Fig. 4. The traces B–E exhibit the saturation transfer connectivities between the corresponding heme methyl proton resonances in the two forms. The heme methyl proton signals of metHbVŽ OHy. can be assigned from the known assignments of metHbV using the connectivities. The assigned heme methyl proton signals of metHbVŽOHy. are compared with those of sperm whale metMbŽOHy. in Table 1 and their Curie plots, shift vs. the reciprocal of absolute temperature, are illustrated in Fig. 5. At ambient temperature, the met-hydroxyl form of hemoprotein has shown to exhibit a thermal equilibrium between the high-spin, S s 5r2, and low-spin, S s 1r2, states, due to the intermediate field strength of OHy ligand w43,55–57x.

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Fig. 2. pH dependence of the 400 MHz 1 H NMR spectra of T. akamusi metHbV ŽA. and sperm whale metMb ŽB. in 2 H 2 O at 258C. In ŽA., with increasing the pH value, the signals, at 15–35 ppm, arising from the alkaline form gain intensity at the expense of the signals of the acidic form, indicating that the acid–alkaline transition in this Hb is slower than the NMR time scale. A p K value of 7.2 " 0.1 was estimated from the analysis of the signal intensity for the two forms. On the other hand, in ŽB., the progressive pH dependence shift of the signals shows that the transition is much faster than the NMR time scale.

The presence of the thermal spin equilibrium in this complex is manifested in the large deviation of the shift to Ty1 ™ 0 for the Curie plot from the diamag-

Fig. 3. A spectrophotometric titration curve for the acid–alkaline transition of T. akamusi metHbV monitored at 577 nm in 0.1 M KCl at 258C. A p K value of 7.22 was obtained as the midpoint by the Henderson–Hasselbalch plot.

netic shift of 3.1 ppm w58x Ž see Table 1. and the anti-Curie behavior of the plot for the average of heme methyl proton shifts. Since the shift decreases with increasing low-spin contents, the fact that the average heme methyl proton shift of metHbVŽ OHy. is about 10 ppm smaller than that of metMbŽOHy. indicates the larger low-spin contents in the former protein than in the latter, although the spread of the signals are roughly the same with each other. The spin equilibrium is influenced by the strength of axial ligand field; therefore, larger low-spin contents in metHbVŽOHy. strongly suggests that OHy coordination to heme iron in this protein is stronger than that in metMbŽOHy.. Furthermore, the heme methyl proton shift pattern of 5-, 8-, 1- and 3-Me, in the order of decreasing chemical shift, for metHbVŽOHy. is different from that for metMbŽ OHy., i.e., 5-, 8-, 3and 1-Me, in order of decreasing chemical shift. The downfield hyperfine shifted regions of the 400 MHz 1 H NMR spectra of metHbVŽ OHy. in H 2 O and 2 H 2 O are compared with each other in Fig. 6. An exchangeable proton signal is observed at about 43

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Fig. 5. Curie plot, the observed shift vs. reciprocal of temperature, for the individual heme methyl proton signal Žopen circles. and the average of all the heme methyl proton shifts Žfilled circles. of T. akamusi metHbVŽOHy . in 2 H 2 O, pH 9.95. The anti-Curie behavior for the plot of the average shift clearly demonstrates the thermal spin equilibrium in this protein.

Fig. 4. Saturation transfer difference spectra of T. akamusi metHbV in 2 H 2 O, pH 7.12, at 358C. ŽA. Reference spectrum. ŽB–E. Saturation of the heme proton signal of the acidic form exhibits the saturation transfer to the corresponding signals of the alkaline form. The peak indicated by an arrow is saturated. The heme methyl proton signal assignments of the alkaline form are indicated in the spectra.

ppm in the spectrum of metHbVŽ OHy.. Its line width is about 1000 Hz, and this value is close to three times as much as the line width Žabout 350 Hz. of heme methyl proton signal. The inverse sixth power dependence of paramagnetic relaxation rate on ironnucleus distance indicates that this exchangeable proton is located at about 0.51 nm from the iron. Therefore, this signal is assigned to His F8 imidazole Nd H proton. The observation of His F8 Nd H proton signal strongly suggests that this proton is hydrogen-bonded to a proton acceptor. Heme methyl proton signals for the H 2 O sample are shifted toward downfield, by 0.5–1.1 ppm, relative to the corresponding resonances for the 2 H 2 O sample and the line width of the signals are smaller in H 2 O than in 2 H 2 O. The effects of solvent isotope composition on heme methyl proton shifts have been interpreted as indicative of the formation of a hydrogen bond between coordinated

Fig. 6. The downfield hyperfine shifted portions of the 400 MHz 1 H NMR spectra of T. akamusi metHbVŽOHy . in H 2 O Žlower trace. and 2 H 2 O Župper trace., pH 8.10, at 258C. An exchangeable proton signal is observed at about 43 ppm. In addition, there is an isotope effect on the heme methyl proton signals such that the heme methyl proton signals in the lower trace are shifted toward downfield relative to the corresponding signals in the upper trace.

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ligand and an amino acid residue w16,32,53x. Although larger paramagnetic shifts in 2 H 2 O than H 2 O have been reported for ferric high-spin w53x and lowspin w16,32x complexes of sperm whale Mb, upfield bias in 2 H 2 O to H 2 O was observed for the present Hb. 3.4. Determination of acid–alkaline transition rate using saturation transfer experiments The rate of the acid–alkaline transition of some Mbs have been determined using the saturation transfer method. The T1intr value of 5-Me resonance of metHbVŽOHy. at 258C and pH 9.95 is 2.7 " 0.4 ms and was found to be essentially constant, within experimental error of "15%, in the temperature range from 20 to 358C. The absence of the acidic form in the sample at the pH value used in the measurement ensures that the obtained T1intr value is free from the acid–alkaline transition. The substitution of both the T1intr value and the saturation transfer factor, measured with the saturation of the corresponding signal in the acidic form, of 5-Me signal on metHbVŽOHy. into Eq. Ž1. yields the k BA value. Since the ratio between the concentration of both the acidic and alkaline forms can be calculated from their NMR signal intensities, the k AB value is obtained form the k BA value, and the equilibrium constant using Eq. Ž2.. The k AB and k BA values for metHbV were determined at various pH and temperatures values, and the results are summarized in Table 2. Interestingly, not only the k AB value, but also the k BA value, are influenced by the pH change in a fashion that the k AB value increases with increasing the pH value, whereas the k BA value decreases. These results reflect the enhanced affinity of metHbV to OHy with increasing the pH value.

Fig. 7. Arrhenius plot of k BA for T. akamusi metHbV at the indicated three pH values. The activation energy Ž D E . for the dissociation of Fe-bound OHy increases with increasing the pH value.

The activation energy Ž D E . for the dissociation of the bound OHy ligand in metHbV is obtained from the Arrhenius plots shown in Fig. 7. As expected from the pH dependence of the k AB value, the obtained D E value increases with the pH value as summarized in Table 2. Such pH dependence of D E may be related to the Bohr effect of this Hb. 4. Discussion 4.1. Ligation sate of metHbV The NMR shift data of metHbV are consistent with the absence of Fe-bound water in this protein, as

Table 2 Kinetic parameters for acid–alkaline transition in T. akamusi metHbV Temperature Ž8C. 20 25 30 35 D E ŽkJ moly1 .

pH 6.64

pH 7.12

pH 7.73

k AB Ž=10 8 moly1 sy1 .

k BA Žsy1 .

k AB Ž=10 8 moly1 sy1 .

k BA Žsy1 .

k AB Ž=10 8 moly1 sy1 .

k BA Žsy1 .

4.4 " 0.7 7.3 " 1.1 13.4 " 2.1 18.0 " 2.7

129 " 20 213 " 32 354 " 54 478 " 72 68 " 11

1.6 " 0.2 3.0 " 0.5 5.2 " 0.7 8.9 " 1.3

26 " 5 50 " 8 85 " 13 147 " 22 88 " 13

0.4 " 0.2 0.8 " 0.2 1.4 " 0.3 3.3 " 0.4

8"2 15 " 3 26 " 6 60 " 9 103 " 12

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described above. In addition, relatively slow acid–alkaline transition in metHbV also strongly supports the pentacoordinated heme in this Hb. Therefore, we conclude that the acidic form of metHbV possesses the pentacoordinated heme. The absence of an amino acid residue capable of being a proton acceptor for forming a hydrogen bond in close proximity to Fe-bound H 2 O is common to all the metMbs and monomeric metHbs that have been shown to be pentacoordinated w39,41,49,59x. Since Leu is thought to occupy at the E7 helical position in HbV, the side-chain of this residue does not form a hydrogen bond with Fe-bound ligand. The side-chain of Arg E11 in this Hb is likely to act as a proton donor for Fe-bound OHy in the alkaline form. But its side-chain cannot be a proton acceptor for Fe-bound H 2 O. 4.2. Heme electronic structure of metHbV(OH y) Heme methyl proton hyperfine shift pattern of metHbVŽOHy. is 5-, 8-, 1- and 3-Me, in the order of decreasing chemical shift. This shift pattern is completely different from that reported for sperm whale metMbŽOHy., i.e., 8-, 5-, 3- and 1-Me, in the order of decreasing chemical shift w60x. Additionally, the value of 25.93 ppm for the average shift of the four heme methyl proton resonances of metHbVŽ OHy. is smaller by almost 10 ppm than the corresponding value of sperm whale metHbVŽOHy., i.e., 34.16 ppm. This result indicates the presence of a significant difference in the delocalization of unpaired electron density from the heme iron to the porphyrin p-system between the two proteins. Since contact shift dominates the paramagnetic shift of these resonances, the difference in the hyperfine shift pattern between the two can be interpreted in terms of heme electronic structure. Since contact shift of heme methyl proton resonance is proportional to the quantity SŽ S q 1. w61x, smaller average shift of the four heme methyl proton resonances of metHbVŽOHy. can be attributed to smaller fraction of high-spin state in this protein, relative to that in sperm whale metHbVŽOHy.. The heme methyl proton hyperfine shift pattern of ferric low-spin Mb is modulated by the interaction of the highest d p orbital of the iron, where the unpaired electron resides, with pp orbital of the imidazole of His F8. But the shift pattern of

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ferric high-spin Mb is consistently 8-, 5-, 3- and 1-Me, in order of decreasing chemical shift, with significantly large magnitude of shifts. Therefore, with increasing the fraction of high-spin state in the complex, the average shift of the four heme methyl proton resonances increases, and the heme methyl proton hyperfine shift pattern tends to become similar to that in ferric high-spin form. Smaller fraction of high-spin state in metHbVŽOHy., relative to that in sperm whale metMbŽ OHy., at ambient temperature should result in decelerating T2 relaxation through the contact hyperfine interaction w62x, as well as the Curie spin relaxation w63,64x. In fact, heme methyl proton signals of metHbVŽ OHy. are much narrower than those of sperm whale metMbŽ OHy.. The spin state of heme iron depends crucially on the strength of axial ligand field. Relatively large fraction of low-spin state in metHbVŽOHy. strongly suggests that the bonding interaction between the heme iron and both His F8 imidazole and exogenous ligand in this protein is much stronger than that in sperm whale metMbŽOHy.. The strength of the ironHis F8 bond is modulated by steric interaction or the hydrogen bond of the Nd H proton with a proton acceptor. The former interaction simply compresses or extends the Fe–N´ bond. The latter affects the Fe–N´ bond through the alteration of the imidazole basicity. Due to the lack of the knowledge about molecular structure of the active site of this protein, the effects of the steric interaction between heme iron and His F8 imidazole on the Fe–N´ cannot be estimated at present. But the observation of His F8 Nd H proton signal in metHbVŽ OHy. indicates that this proton is hydrogen-bonded to a proton acceptor. In addition, in the spectra of met-cyano Mb, His F8 Nd H proton signal is generally observed at ; 20 ppm w14x. The observation of His F8 Nd H proton signal of metHbVŽ CNy. at ; 14 ppm Žunpublished result. suggests the partial deprotonation of His F8 Nd H proton by a strong hydrogen bond with a proton acceptor. Furthermore, Fe-bound OHy is stabilized by a hydrogen bond with a proton donor w28,42x. The formation of this hydrogen bond enhances the localization of electron density at O atom directly bonded to the heme iron, which in turn strengthens the Fe–O bond. The increased axial ligand field strength in metHbVŽOHy. could be partly attributed to these electronic interactions.

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4.3. pK Õalue of acid–alkaline transition in metHbV metHbV exhibits a remarkably low p K value of 7.2 for the acid–alkaline transition. Since acidic form of metHbV possesses the pentacoordinated heme, p K value is thought to be primarily controlled by the affinity of OHy to the pentacoordinated heme. Therefore, it can be concluded that there is a molecular mechanism for considerable stabilization of Febound OHy in this protein. Fe-bound OHy demands a proton-donor to form a hydrogen bond. Considering the primary structure of HbV w1x, it is conceivable that the side-chain NH proton of Arg E11 is hydrogen-bonded to the Fe-bound ligand in this protein. Similar functional properties of Arg residue in the E helix has been reported for Aplysia w25,26,28x and Dolabella w27,41x metMbs, which exhibit the p K values of 7.6 and 7.8, respectively. Cutrruzzola´ et al. w31x had demonstrated that the replacement of His E7 by Val in sperm whale Mb alters the p K value from 9.0 to 10.2, and that the further replacement of Thr E10 by Arg reduces the p K value to 8.8. Thus, the guanidino NH proton of the side-chain of Arg in these proteins acts as a strong proton donor for Fe-bound OHy. The change of the k AB andror k BA values influences the p K value. In the case of Dolabella metMb w41x, its k BA is one order of magnitude larger than that of metHbV, whereas the k AB values are similar with each other. Therefore, the difference of 0.6 pH unit between the p K values of these proteins is attributed to the difference in the k BA value. On the other hand, the difference in the p K value between Aplysia metMb w30x and metHbV arises from the fact that the k AB values of Aplysia metMb is almost one order of magnitude smaller than that of metHbV, because their k BA values are similar with each other. These results indicate that both k AB and k BA values contribute to control the p K value for the acid–alkaline transition of ferric hemoproteins that possess pentacoordinated heme in the acidic form. 4.4. Kinetics of acid–alkaline transition As described above, the k BA value of metHbV is considerably smaller than that of Dolabella metMb w40,41x at a given temperature and pH value. This result indicates that Fe-bound OHy in the former

protein is more strongly stabilized by the hydrogen bond with Arg E11 than that in the latter, which is stabilized by the hydrogen bond with Arg E10. Furthermore, the pH dependence of the k BA value indicates that the affinity of metHbV to OHy increases with increasing the pH value. The pH dependence of the OHy affinity of metHbV is also clearly manifested in the activation energy Ž D E . for the dissociation of the bound OHy ligand, which also increases with increasing the pH value. This result is in a sharp contrast with those obtained for Dolabella and Mustelus Mbs w41x, in which the D E value are essentially independent of the pH value. Furthermore, the D E values of 68–103 kJ moly1 for metHbV are considerably larger than the values of 58 " 9 and 27 " 3 kJ moly1 obtained for Dolabella and Mustelus metMbs, respectively. The kinetic data in Table 2 demonstrate that both the k AB and k BA values become smaller with increasing the pH value. The difference of about three orders of magnitude in the k BA value observed between Dolabella and Mustelus metMbs w41x is likely to arise from differences in intrinsic structural and dynamic properties of the active site between the two. Hence, the decrease in the k AB and k BA values with increasing the pH value would be attributed to the effects of pH on molecular structure and dynamic properties of the heme active site in this protein. The pH dependence of the k BA and D E values indicates that the stabilization of Fe-bound OHy is enhanced at higher pH value. These results may be related to alkaline Bohr effect of this protein Ž Shikama et al., unpublished result. . Since the acidic form of metHbV possesses pentacoordinated heme in its active site, the binding of OHy to heme iron in alkaline form of the protein, i.e., metHbVŽ OHy., exerts the changes in both the coordination and spin states of the iron with its oxidation state unchanged. Therefore, the acid–alkaline transition in metHbV resembles oxygenation process of the protein, although there is a difference in the oxidation state of heme iron between the two processes. Gersonde et al. w65x demonstrated that the Bohr effect of the monomeric C. thummi thummi Hb is primarily controlled by dissociation rate constant of ligand. The present results show that the enhanced affinity of metHbV to OHy at higher pH stems from the decrease of the k BA value with increasing the pH value. Consequently, both alkaline Bohr effect and

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the enhanced affinity of metHbV to OHy at higher pH are controlled by similar molecular mechanisms. A detailed structure determination of HbV at different pH values is needed to determine such mechanisms.

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