Biochimica et Biophysica Acta, 427 (1976) 28-37

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37267 I N F R A R E D M A G N E T I C C I R C U L A R D I C H R O I S M OF M Y O G L O B I N DERIVATIVES

TSUNENORI NOZAWA, TAKAO YAMAMOTO and MASAHIRO HATANO Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, 980 (Japan)

(Received August 18th, 1975)

SUMMARY By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the M C D spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (% (dn)). Since the fraction of A term estimated was ~ 8 0 % for myoglobin fluoride and ~ 3 5 % for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low st~in derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive M C D spectra which are very similar to the electronic absorption spectra. These M C D spectra were assigned to the charge transfer transitions from porphyrin 7r to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the M C D magnitude indicated that the M C D spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive M C D around 760 nm with an anisotropy factor (A e/e) of 1.4.10- 4. It shows some small M C D bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable M C D in the infrared region while oxymyoglobin seemed to have significant M C D in the range from 700 to 1000 nm.

INTRODUCTION In the course of studies [1-4] it has become evident that the magnetic circular dichroism (MCD) provides much valuable information about electronic states of hemoproteins which play many important roles in living systems. Except for a few studies [5-7], the measurements of the M C D have been restricted to a wavelength range below 700 nm where most of the electronic transitions in the hemoproteins originate from porphyrin n electrons [8, 9]. Although experimental results show that the M C D in this region is still sensitive to the spin and oxidation states of the heme

29 iron [1, 2, 4] it was expected that more direct evidence on the electronic structures of heme might be obtained from the MCD spectra in the infrared region where charge transfer bands associated with the heme iron or d-d transitions in iron will be observed [7]. Using a newly constructed infrared CD instrument, we could obtain MCD spectra for the typical myoglobin derivatives as basic data for various hemoproteins. The results not only reinforced the infrared band assignment which is of vital importance to relate MCD spectra with the structures and functions of hemoproteins, but also gave some direct information on the excited electronic states of the hemoproteins. MATERIALS AND METHODS Muscle whale myoglobin used was purchased from Miles-Servac [PTY] Ltd. (Maidenhead, Berkshire, U.K.). All chemicals are guaranteed grade reagents and used without further purification. 2H20 phosphate buffer (1/15 M) (pH 6.98) was used throughout the study unless otherwise noted. Potassium ferricyanide was used to oxidize myoglobin completely, and the reduction was affected by a slight excess of solid sodium dithionite on deaerated solutions. Various myoglobin derivatives were prepared by adding respective solid salts until the absorption spectral changes were saturated [1]. MCD spectra beyond 1000 nm were obtained by use of a JASCO J-100 CD instrument equipped with an electromagnet which produces a magnetic field up to 11.4 kG. The magnetic field was determined with freshly prepared ferricyanide using the established value [0]ra z 1.0 (molar ellipticity per gauss) [1]. The JASCO model J-100 CD instrument has been built in cooperation with us. It incorporates a CaF2quartz photoelastic modulator (Morvue Electronic System's 50 kHz photoelastic modulator) and an InAs photovoltaic cell (Hamamatsu Television Co. Ltd.). The instrument has a sensitivity of 2 mdegrees/cm. Calibration was done with an Ni complex whose molar ellipticity at 1000 nm was determined with JASCO model J-20c CD apparatus calibrated using the natural CD of 10-[ZH]camphorsulfonic acid as a standard with [0]290 = 7260 [1]. The details of the instrument will be reported elsewhere in a separate paper. A typical run was performed with a spectral band width of 10 nm, time constant of 0.25 s and scan rate of 50 nm/min. The CD and MCD in the wavelength region from 700 to 1000 nm were obtained by use of a JASCO model 1-20c CD instrument with an S-1 type photomultiplier (Hamamatsu Television Co. Ltd.). The magnitude of the MCD is expressed as the molar ellipticity per gauss ([0]M). The quartz cuvettes with optical path lengths of 10, 5, 2 and 1 mm were used in response to the wavelengths and sample concentrations. A 2 mm quartz cell with an AuCo-Cu thermocouple was subjected to a stream of cold nitrogen gas in a dewar for low temperature measurements. Potassium glycerophosphate and glycerol were used for the solid glass solvent in the ratio of 2:1:1 (potassium glycerophosphate: glycerol:ZH20 phosphate buffer). RESULTS

Ferri-(Met-) myoglobin high spin complexes Metmyoglobin derivatives of fluoride, water and hydroxide are categorized as

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Fig. 1. M C D and absorption spectra of the high spin complexes o f ferri-(met-)myoglobin. Concentrations approx. 3 m M in 1/15 M 2H20 p h o s p h a t e buffer (p2H 7.0), (a) n o addition (2H20 - - - - ) , (b) with 1.5 M fluoride ( F ) or p2H 11.5 (OZH - - - - - - - ) ; p a t h = 2 m m ; field = 11.4 k G ; near 25 °C. T h e bars indicate the average noise levels.

high spin complexes from magnetic susceptibility data [1, 8, 10]. Fig. la shows MCD and absorption spectra for metmyoglobin (metMbZH20). MetMbZHzO shows MCD troughs at 1210 and 850 nm and peaks at 1010 and 770 nm. The relatively resolved MCD spectra are noted in comparison with the broad absorption spectra. Fig. lb illustrates the MCD and absorption spectra for metmyoglobin fluoride (metMbF) and metmyoglobin hydroxide (metMbOZH). MbF shows an MCD trough at 890 nm and a peak at 810 nm, which compose a Faraday A term. Besides an MCD trough at 830 nm and a peak at 780 nm, MbO2H exhibits an extra MCD band (around 1000 nm) which may be attributable to the low spin component of MbO2H as described below.

Ferri- (Met-) myoglobin low spin complexes Fig. 2 shows MCD spectra for low spin metMb complexes. MbCN and MbIm exhibit the well-resolved MCD spectra which are very similar to the absorption spectra. They have three MCD peaks at 1530, 1330 and 1030 nm corresponding with the absorption peaks at 1560, 1280 and 1060 nm. MbN3 exhibits, on the whole, a similar MCD spectrum, that is, the longest wavelength absorption peaks gave the

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strongest positive M C D . H o w e v e r , it shows extra M C D troughs at 990 a n d 800 nm, a n d p e a k s at 909 a n d 714 nm, which c o m p o s e a similar M C D p a t t e r n to metMb2HzO.

Ferrous myoglobin high spin complex D e o x y m y o g l o b i n belongs to this category. A strong positive M C D is observed a r o u n d 760 n m associated with the a b s o r p t i o n peak. The t e m p e r a t u r e variation exp e r i m e n t s (Fig. 3b) revealed that the M C D at 760 n m is a B term. A t least four o t h e r M C D b a n d s are o b s e r v e d (Fig. 3a). However, since they are as w e a k as the limit o f the sensitivity o f the a p p a r a t u s , the M C D spectra in the wavelength region over 1000 n m are tentative results. W e are n o w reconstructing the C D a p p a r a t u s with higher sensitivity a n d p r e p a r i n g to use a s u p e r c o n d u c t i n g m a g n e t which affords up to 50 k G magnetic field.

Ferrous low spin complexes A l t h o u g h M b C O has neither M C D n o r a b s o r p t i o n b a n d in the n e a r infrared region, MbO2 a p p e a r s to have some M C D b a n d s below 1000 nm. The M C D trough at 830 n m a n d p e a k at 990 n m can be observed. (Fig. 4). T h e M C D a b o v e 1000 n m was t o o small to be observed f r o m the sensitivity for the a p p a r a t u s .

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Fig. 3. (a) M C D , C D and absorption spectra of deoxymyoglobin. Concentration 0.9 m M in 1/15 M ZH20 phosphate buffer (p2H 7.0). p a t h = 5 m m ; field -- I 1.4 k G ; near 25 °C. The bars indicate the average noise levels. (b) The effect of temperature on the 760 n m (13.2.10acre -1) M C D band of deoxymyoglobin. The sample was 2.2 m M in a solvent of potassium glycerophosphate, glycerol, 1/15 M 2H20 phosphate buffer (2:1 :l, v/v/v) at p2H 7.0. p a t h -- 2 m m ; field -- 11.4 kG. Temperature = 300 ° K ( - - ) , 209 ° K ( - - -- --), 149 ° K ( - ). The inset is the plot of zeroth moment ( < 0 > 0 = J" ([O]M/v)dv)against 1/kT (k is the Boltzmann constant, and T is the absolute

temperature). DISCUSSION

Ferric high spin derivatives The MCD spectra for the ferric high spin Mb derivatives not only reinforced the assignment of the near infrared absorption band [8], but also yielded information about the excited electronic states. The near infrared bands for the high spin ferric derivatives have been assigned to a charge transfer band which is likely to be of the ligand-to-metal type [8]. Braterman et al. [11] showed that Eu (in-plane polarized) and A2. (out-of-plane polarized) charge transfer states are possible. Single crystal spectra [12, 13] of ferrimyoglobin derivatives are polarized in the heme plane, so that the high spin near infrared band has been assigned to one of the in plane polarized charge transfer transition. The transitions allowed in plane in D4h symmetry [9] are: a2, (n) al. (n) Eu(x,y) bzu (n) ÷ e~ (an). azu, (n)

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Fig. 4. MCD and absorption spectra of the low spin complexes of ferrometmyoglobin. Concentration approx. 3 mM in 1/15 M 2H20 phosphate buffer (pH 7.0). Oxymyoglobin ( ), Carbonmonoxymyoglobin (-- -- --). path = 2 ram; field = 11.4 kG; at 0 °C. The bar indicates the average noise level. Usually a2u (z0 and al, (zt), and b2u (~) and a2u' (~) have been considered to be accidentally degenerate and the former group is higher in energy than the latter [14]. As has been discussed in detail by Smith and Williams [8], the near infrared charge transfer band (CT1) is the lower energy one. Therefore it can be assigned to be the transition from a2u (~), alu (z0 to eg (dr0. Hence the charge transfer band in the visible region (CT2) should correspond with the transition b2u (z0, a2u (~) --->eg (dz0. Although CT2 has complex configuration interactions with a,fl states, the resolved CT2 bands have a linear relation with the CT1 bands [8]. This was experimental evidence in favour of the bands being charge transfer transitions from absorption spectra. This assignment can be confirmed by the MCD results in the infrared region with the comparison to those in the visible region as follows. Figs. la and lb tell that the MCD for the predominantly high spin derivatives can be further separated into two groups according to the band positions. MbF and MbOD exhibit the absorption peak around 800 nm (12.5 kK (kK = 10a cm-a)); while MbZH20 at about 1050 nm (9.5 kK). Though the visible MCD spectra for high spin derivatives [I] are very complex because of strong configuration interactions between the a-fl (Q) and charge transfer states, the MCD bands with predominant CT character were determined to be at 500 nm (20.0 kK) for MbF and MbOH, and at 640 nm (15.6 kK) for MbH20 from the MCD shape and temperature dependence [1]. The similar energy separation for the two groups of CT bands both in the near infrared and in visible region strongly supports the assignment proposed by Smith and Williams [8] as described above. Except for some shifts in the wavelength, the ferric high spin derivatives commonly exhibit characteristic MCD spectra in the near infrared region. As typically

34 seen for MbF, the ferric high spin myoglobin derivatives exhibit A terms associated with the strongest electronic absorption peak. This indicates that the excited state is degenerate, and is consistent with the assignment proposed by Williams et al. [8] that the near infrared bands for ferric high spin derivatives are the CT transitions from non-degenerate porphyrin orbitals to the degenerate ion eg (dz0 orbital. The essentially pure A term observed for MbF implies that the effective symmetry of the iron is as close as D4h. Therefore the degeneracy in the eg excited state is not practically lifted at all in MbF. This situation is compared with that for MbDaO. As seen in Fig. la, it is apparent that the main MCD spectra around 9 kK is non-symmetric. So that, the Faraday parameter other than A term (plausibly B term) significantly contributes to the MCD spectra around 9 kK. Apparently the B term contribution to the MCD spectra for MbZH20 is greater than that for MbF. (The fractions of B terms were approximately estimated to be 65 and 20~o for MbZHzO and MbF, respectively, at room temperature (near 25 °C)). Hence the excited state degeneracy, i.e. the effective symmetry of the iron for the MbZHzO is lower than that for MbF at room temperature (near 25 °C). A similar conclusion has resulted from the analysis of the charge transfer transition in the visible region, and the existence of hydrogen bond between H20 and the distal histidine has been considered to lower the symmetry [15]. The MCD spectra for MbOEH exhibit a positive MCD band corresponding with the small electronic absorption peak around 1000 nm. Since, as we will see later, the ferric low spin derivatives show positive MCD, and MbOZH is known to have low spin fraction of ~ 3 5 ~ [10], this positive MCD around 1000 nm can be considered to be due to the low spin component. The high spin MCD bands left for MbO2H constitute a non-symmetric, S-shaped MCD band. Because of the large B term present, the effective symmetry of the heine iron in MbOEH can be estimated to have a significantly lower symmetry component than D4h. It is noted that, though both MbF and MbOZH show shorter wavelength side shoulders around 720-750 nm in the electronic absorption spectra, there is practically no MCD corresponding to these absorption shoulders. The absorption shoulders are considered to be due to one of three possibilities [8]: (1) the split component of % (d~) excited state; (2) the low spin component; (3) the vibrational component. Possibilities 1 and 2 are not compatible with the MCD results and magnetic susceptibility data for MbF. Thus, the pure A term MCD for MbF indicated the complete degeneracy of e~ (d~) state, and moreover MbF has been estimated to have very few low spin components (as low as 2~o) from the magnetic susceptibility data [10]. Therefore, Possibility 3 is the only promising candidate. Then how can we explain the lack of MCD for the absorption shoulder? This is probably because the MCD for the various vibrational bands have canceled one another. A similar explanation has been employed for the small MCD magnitude for the Qv band in comparison with Qo-0 band [1]. The MCD for MbZH20 in the wavelength region from 700 to 900 nm may be due to an incomplete cancellation of the vibrational MCD bands. Hence, the incomplete cancellation seems to relate closely to the kind of axial ligands, and the symmetry of the heme vicinity. Ferric low spin derivatives The characteristic MCD spectra were observed for the ferric low spin myo-

35 globin derivatives, i.e. MbCN, Mblm and MbN3. The result for MbCN is in good agreement with that for hemoglobin cyanide reported by Cheng et al. [5]. A direct contribution of the ferric low spin iron to these transitions is suggested from the fact that the three ferric low spin myoglobin derivatives exhibit similar MCD spectra which are not observed for other type of myoglobin derivatives. The possibility that the near infrared band could be a charge transfer transition from the axial ligand to metal can be ruled out on the ground that the absorption wavelengths are not as different as expected from the relative optical electronegativities of ligands [16]. Accordingly, the main possibilities for the low spin infrared band found here are porphyrin to metal charge transfer or intrametal d-d transitions. Since the d-d bands in low spin ferric hemoproteins are expected to appear at rather higher energies [8, 9, 17], the charge transfer from porphyrin to metals is strongly suggested for the low spin infrared band. The extended Hfickel calculation by Zerner et al. [9] implies that the near infrared low spin bands belong to the transition with Eu symmetry described in the high spin section, from the calculated relative energies of the CT bands. The energy differences between the two accidentally degenerate pairs of a2u (7/7) and alu (zr) and of b2~ (~r) and a2u, (Yt) were estimated to be 6 kK [8] (CT1 = 9.62 kK, CT2 = 15.63 kK for Mb2H20). Since the energies' separation between the longest and shortest wavelength bands are ~ 3 kK, the possibility that both degenerate pairs contribute to the infrared band can be ruled out. Hence, the three peaks observed in the MCD spectra for MbCN should be assigned to the band from one of the accidentally degenerate pairs, probably from the pair of b2u (~r) and aEu" (er). As evident from the fact that ESR g values have three different components (gx = 1.72, gy ---- 2.22 and gz = 2.80 for MbNa), the effective symmetry of the iron has lower symmetry component than Dgh [18, 19, 20]. Therefore b2g (dxy) and eg (d~r) are mixed to give three Kramer's doublets. Therefore three MCD peaks are possibly assigned to the transition from porphyrin to the three Kramer's doublets. The spacing of three Kramer's doublets determined from ESR g values is 1.1 kK (between first and second), and 1.6 kK (between second and third) [18, 19, 20]. This spacing is not so different from that of the observed MCD peaks. (See Table I). Another possibility is to assign the MCD peaks to a type of vibrational progression with an interval of 1.5 kK which is about the same as the usual a-fl band separation in a typical metalloporphyrin. Cheng et al. [5] assigned the near infrared MCD spectra for hemoglobin cyanide to the Faraday C term originated from the spin degeneracy of the low spin ferric iron [5]. However, the temperature dependence of MCD for MbCN showed that there is essentially no increase in the MCD magnitude for the peak at 1530 nm (not shown), therefore indicating that the MCD for MbCN is the Faraday B term rather than C term. It is of interest that the MCD peaks for MbCN and MbIm exist around 1530 nm, but MbNa and MbOZH showed the MCD peaks at 1230 nm and 990 nm, respectively. Although it is difficult to explain the observed energy shift of the band in terms of a charge transfer transition because many factors are involved, the most important terms will be (i) the spin pairing energy for the reduction d s -~ d 6, (ii) the energy change of d orbital on the charge transfer, and (iii) the electrostatic repulsion in the excited state [8]. At present we cannot determine specific reasons for these phenomena, which may, however, be worthy of further study.

36 The M C D bands of MbNa from 700 to 1100 nm look like high spin bands at first sight. However, the high spin fraction of MbN3 ( ~ 2 0 %) is too small to give this M C D magnitude. MbNa has been known to give also the high spin band in the visible region (around 630 nm). These two phenomena might be attributable to a similar reason.

A ferrous high spin derivative In deoxyMb, the 760 nm band showed strong positive MCD. This band has been reported to be very sensitive to the conformation of myoglobin or hemoglobin [21] and considered to be due to either a d-d transition or a charge transfer band. The low anisotropy factor (Ae/e) of 1.4" 10 -4 favors the assignment as a charge transfer transition or a magnetically forbidden d-d transition. The rest of the M C D troughs and peaks are very small and almost of similar order to the noise level. We should leave further discussion until more definite spectra are obtained. However, the M C D peak at 1330 nm is very likely to be a magnetically allowed d-d transition because of high anisotropy factor (Ae/e), as large as 0.01. The bands at 970 nm and 760 n m have anisotropy factors of 0.001 and 0.0001, respectively. If we could assume the D4h symmetry in the first approximation, the d-electron energy states would be like the inset in Fig. 3 [17]. The transitions allowed in the CD spectra a r e 5B2g --~ 5Eg and SB2g -+ 5Big. Since 5B2g ~ 5Eg should exist in vibrational infrared, the observed CD at 7.4 kK may be assigned to 582g - + 5Blg. If this is the case, the 760 nm band could not be a d-d transition. Hence, it should be assigned to some charge transfer transition.

Ferrous low spin derivatives Because of the instrumental limit, the M C D spectra is available only under 1000 nm for the ferrous low spin derivatives of myoglobin with the very low M C D magnitude. Yet, the critical difference between MbO2 and M b C O is prominent, since both belong to the same ferrous low spin group. Several reports recently proposed that Mb(Fe2+)O2 has actually the structure of Mb(Fea+)O2 - [22-24]. However, neither high nor low spin ferric derivatives have given M C D spectra similar to that of MbO2, so that the infrared M C D spectra do not formally support the structure Mb(Fe3+)O2 - . In other regions, especially in the Soret region near 400 nm, the M C D spectra of MbO2 are fairly different from M b C O [1]. For this reason, a contribution of some charge transfer transition directly associated with the 02 molecule has been proposed [25]. Therefore, another charge transfer band can be a possible origin for the MbO2 band near 1000 nm. REFERENCES

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37 7 Cheng, J. C., Csborne, G. A., Stephens, P. J. and Eaton, W. A. (1973) Nature 241,193-194 8 Smith, D. W. and Williams, R. J. P. (1970) Structure and Bonding, 7, 1-45 9 Zerner, M., Gouterman, M. and Kobayashi, H. (1966) Theoret. Chim. Acta. 6, 363-400 10 Iizuka, T. and Kotani, M. (1969) Biochim. Biophys. Acta 181,275-286 11 Braterman, P. S., Davies, R. C. and Williams, R. J. P. (1964) Adv. Chem. Phys. 7, 359-407 12 Day, P., Smith, D. W. and Williams, R. J. P. (1967) Biochemistry 6, 1563-1566 13 Day, P., Smith, D. W. and Williams, R. J. P. (1967) Biochemistry 6, 3747-3750 14 Weiss, C., Kobayashi, H. and Gouterman, M. (1965) J. Mol. Spect. 16, 415-450 15 Yoshida, S., Iizuka, T., Nozawa, T. and Hatano, M. (1975) Biochim. Biophys. Acta 405, 122-135 16 Jergensen, C. K. (1965) Adv. Chem. Phys. 5, 33-146 17 Eaton, W. A. and Charney, E. (1971) in Probes of Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T. and Mildvan, A. S., eds.), Vol. I, pp. 155-165, Academic Press, New York 18 Weissbluth, M. (1967) Structure and Bonding 2, 1-125 19 Griffith, J. S. (1957) Nature 180, 30-00 20 Kotani, M. (1961) Prog. Theoret. Phys. Suppl. 17, 4-13 21 Iizuka, T., Yamamoto, H., Kotani, Y. and Yonetani, T. (1974) Biochim. Biophys. Acta 371, 126-139 22 Weiss, J. J. (1964) Nature 202, 83-84 23 Barlow, C. H., Maxwell, J. C., Wallace, W° J. and Caughey, W. S. (1973) Biochem. Biophys. Res. Commun. 55, 91-95 24 Yamamoto, T., Palmer, G., Gill, D., Salmeen, I. T. and Rimai, L. (1973) J. Biol. Chem. 248, 5211-5213 25 Nozawa, T., Hatano, M., Yamamoto, H. and Kan, T. (1975) Bioinorg. Chem., in press