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HIGH-RESOLUTION XANES STUDY OF THERMAL SPIN EQUILIBRIUM IN MYOGLOBIN
H. Oyanagi, E. Svastits, T. Iizuka, T. Matsushita, S. Saigo, R. Makino, Y.
Ishimura
To cite this version:
H. Oyanagi, E. Svastits, T. Iizuka, T. Matsushita, S. Saigo, et al.. HIGH-RESOLUTION XANES STUDY OF THERMAL SPIN EQUILIBRIUM IN MYOGLOBIN. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1147-C8-1150. �10.1051/jphyscol:19868223�. �jpa-00226136�
JOURNAL D E PHYSIQUE
Colloque C8, supplkment au n Q 12, Tome 47, decembre 1986
HIGH-RESOLUTION XANES STUDY OF THERMAL SPIN EQUILIBRIUM IN MYOGLOBIN
H. OYANAGI, T. IIZUKA*, T. MATSUSHITA", S. SAIGO'**, R. MAKINO' and Y. ISHIMURA"
Electrotechnical Laboratory, Sakuramura, Niiharigun, Ibaraki 305, Japan
*Department of Biochemistry, School of Medicine, Keio University, Shinanomachi, Shinjuku, Tokyo 160, Japan
""National Laboratory for High Energy Physics, Ohomachi, Tsukubagun, Ibaraki 305, Japan
"""Department of Physics, Jichi Medical School, Yakushiji, Minamikawachi, Tochigi 329-04, Japan
ABSTRACT
The local s t r u c t u r e of heme-iron of alkaline met-myoglobin during the thermal spin equilibrium between high and low spin s t a t e s has been studied by a high resolution XANES. The spin-state sensitive near e d g e features have been found in t h e near threshold region, which a r e related t o the distorted ligand field. The spin-state sensitive near-edge structures w e r e also found for other myoglobin derivatives. From a systematic high resolution XANES study on MbCO, MbCN, and oxy-Mb, near-edge features which a r e sensitive t o t h e angle between t h e diatomic ligand molecule and h e m e normal were found. These characteristic features c a n provide t h e elaborate local s t r u c t u r e of heme-iron such a s t h e displacement of iron atom from t h e heme plane or the bond angle of ligand molecule.
INTRODUCTION
Some f e r r i c hemoproteins such as hydroxide complexes of ferric hemoglobin and myoglobin show t h e intermediate values of spin susceptibilities between those characteristic of 5- and I-unpaired d electrons. T h e intermediate paramagnetic susceptibilities and light absorption s p e c t r a of these complexes have been interpreted on t h e basis of thermal spin equilibrium between two magnetic isomers, one in high spin and t h e o t h e r in low spin s t a t e s [1,2].
Althogh t h e thermal spin equilibrium h a s been extensively studied for a variety of hemoproteins and their model compounds from t h e thermodynamical viewpoints, t h e relation between t h e local s t r u c t u r e of heme-iron and spin s t a t e s has not been established yet. Heme-iron is expected t o b e ~ u t of hemeplane in high spin s t a t e s due t o extended distribution of d-electrons.
The magnitude of this displacement is difficult t o determine from EXAFS which is insensitive t o s i t e symmetry. Therefore we have studied t h e temperature dependence of F e K-XANES of alkaline metmyoglobin (MbOH) which shows a thermal spin equilibrium between 80 K where heme-irons a r e purely in low spin s t a t e s and 300 K where most of them a r e in high spin s t a t e s [2].
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868223
C8-1148 JOURNAL DE PHYSIQUE
EXPERIMENTAL
Efforts have been taken to measure Fe K-XANES spectra with a high resolution (<2 eV) t o resolve fine structures. A sagittally bent crystal monochromator [3] and a fluorescence-detection spectrometer [41 were used. A typical energy resolution a t the F e K-edge (7.11 keV) is 1.5 eV using silicon (111) monochromator crystals. A scintillation counter array consisting of 9 NaI detectors covering 18 % of the total solid angle and a Si(Li) detector were used for the concentrated (>5 mM) and dilute (<2mM) samples, respectively. Mn filters were used t o reduce t h e background. The use of a solid s t a t e detector (SSD) is essential t o record weak preedge features with a high signal-to- background ratio.
MbOH solution (2-24 mM) was rapidly frozen in an aluminum cell with a Kapton window by liquid nitrogen and immediately transferred t o a cryostat which was cooled down to 80 K with a closed cycle helium refrigerator. The temperature dependence of Fe K-XANES and EXAFS was made between 80 K and 300 K. Myoglobin derivatives such a s MbCN, MbCO and oxy-Mb were also measured a t 80 K.
RESULTS AND DISCUSSION
Heme-iron of metmyoglobin is coordinated with four nitrogen atoms of pyrrole rings and another nitrogen atom of proximal histidine as indicated in Fig. 1.
The 6th ligand of MbOH is a hydroxy ion. In myoglobin (Mb), heme-iron is, displaced from t h e heme plane by 0.40 h; and from nitrogen plane by 0.27 A according t o the structure analysis by Takano [5]. Because of a doming of pyrrole ring, the iron-nitrogen distance is slightly shorter than the iron-
hemeplane distance. The displacement of heme-iron from the mean hemeplane was reported for various high-spin porphyrin compounds [6]. On the other hand, heme-iron in low-spin myoglobin derivatives such a s cyanide is expected t o be close t o the hemeplane. Figure 2 shows the temperature dependence of F e K- XANES for MbOH. Solid and dotted lines indicate the raw spectra and their first derivatives. At 80 K where MbOH is purely in low spin states, several characteristic features a r e observed which a r e denoted a f t e r Bianconi e t al.
171. Peak P observed a t a t 7112 eV is a quadrupole-allowed transition from F e ks t o empty 3d states. This weak peak broadens on going t o high temperature.
As indicated in Fig. 1, 3d orbitals split into two e g orbitals and three t2g orbitals in an octahedral ligand field. In high spin states, there are empty s t a t e s in both e g and tpg s t a t e s whereas only one out of five empty states is in t2: orbital in low spin states. Therefore the width of 1s-3d transition peak expected t o increase in high spin states.
HIGH SPIN LOW SPIN
S;5/2 S= 1/2
eg +
Fig. 1 Local structure of heme-iron in Fe-protopophyrin IX.
7.10 7.1 7.13 7.14 7.16 7.17 PHOTON ENERGY ( k e V )
I I
7100 7120 7140 7160 Fig. 2 Temperature dependence of F e K-XANES spectra of MbOH.
PHOTON ENERGY ( e V )
In Fig. 2, A indicates the position of a shoulder structure observed for low-spin MbOH a t 7122 eV, which appears a s an inflection point in the first derivative. This shoulder disappears with the increase of high spin content and a t 300 K, no shoulder structure is observed. After a careful normalization, a difference spectrum was obtained by subtracting 80 K data from spectra a t higher temperature. In a difference spectrum, a sharp positive peak with the FWHM (full width a t half maximum) of 5 eV is observed a t 7122 eV.
At 80 K, a broad bump structure Cp is observed a t 7148 eV. This peak decreases i t s intensity on going t o high spin states and it disappears a t 300 K. This bump appears a s a broad negative peak in a difference spectrum with a FWHM of 14 eV. The magnitude of C 2 and A in a difference spectrum is correlated with a high spin content. These spin-state sensitive features are expected t o be related with a distortion of octahedral coordination resulting from the displacement of heme-iron from the hemeplane in high spin states. The axial movement of heme-iron also causes the stretching of other Fe-N distances.
Full multiple scattering calculations a r e necessary t o further clarify the local structure, for which bond distances should be determined by EXAFS.
F e K-XANES spectra for various low-spin myoglobin derivatives a r e shown in Fig. 3. A and Cp are present in all spectra indicating that these "spin s t a t e markers" can be applied t o other myoglobin derivatives. The absorption maximum for MbCN denoted as C l is located a t 7129 eV. On going from CN t o 02, C1 decreases its intensity while C2 and A a r e not affected. For MbCN, Fe-C-N angle is 180°while Fe-C-0 and Fe-0-0 angles are 150°and 120' , respectively.
As suggested by Bianconi e t al. [7], C 1 originates from a strong focusing scattering in the linear chain consisting of heme-iron and a diatomic molecule.: Therefor the intensity of C 1 can be used t o determine the a n i l e between the ligand molecule and the heme normal.
A broad bump denoted a s P' is observed 5.4 eV above the 1s-3d transitin peak P for MbCN. On the other hand, a doublet structure separated with each other by 2.3 eV is observed in the prepeak region for MbCO while only a single peak is observed for oxy-Mb.
C8-1150 JOURNAL DE PHYSIQUE
-.
LLFig. 3 Fe K-XANES spectra of various low-spin myoglobin derivatives.
7100 7110 712% 7130 7140 7150 7160 PHOTON ENERGY ( e V 1
The 1s-3d transitions are allowed between 1s states and x2-y2, z2 and xy orbitals for all three complexes in low spin states. According to the polarized absorption experiment for MbCO [7], I? and P' a r e polarized along the heme normal and heme plane, respectively, suggesting that P and P' correspond to the transitions t o 22 and x2-y2 orbitals with e g symmetry, respectively.
Because of a crystal field splitting, x2-y2 orbital is expected to be shifted toward higher energy in MbCN. This large crystal field split is due t o a large pdb overlap integral for Fe-CN. The magnitude of split decreases on going to weak ligand such as dioxygen where two levels merge into a single peak. In all cases, the distortion of Oh symmetry seems t o involve only axial movements of ligands since other distortion would affect the p-d hybridization and intensity of preedge peak. If the above assumption is correct, the prepeak should broaden its width in the lower energy region in high spin states since the transitions t o both e g and t i g states a r e allowed in high spin states. In Fig. 2, t h e prepeak for MbOH a t 300 K is assymetric and broadening in the lower-energy region is confirmed. Because of a weak crystal field for oxy-Mb or MbOH, the energy difference between e g and t2g orbitals is expected t o be small as well as t h e split between x 2 - ~ 2 and 22 orbitals.
ACKNOWLEDGEMENT
The authors express their greatest thanks t o Prof. A. Bianconi and Dr.
Fujikawa for valuable discussions and encouragements.
REFERENCES
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5 T. Takano, J. Mol. Biol. 110 (1977) 537.
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