MÖSSBAUER STUDIES ON THE IRON-LIGAND BINDING IN HEMOPROTEINS AND THEIR RELATED COMPOUNDS

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MÖSSBAUER STUDIES ON THE IRON-LIGAND BINDING IN HEMOPROTEINS AND THEIR

RELATED COMPOUNDS

Y. Maeda

To cite this version:

Y. Maeda. MÖSSBAUER STUDIES ON THE IRON-LIGAND BINDING IN HEMOPROTEINS

AND THEIR RELATED COMPOUNDS. Journal de Physique Colloques, 1979, 40 (C2), pp.C2-514-

C2-522. �10.1051/jphyscol:19792180�. �jpa-00218557�

JOURNAL DE PHYSIQUE Colloque C2, supplPment au n

"

3, Tome

40,

mars

1979,

page C2-5

14

MOSSBAUER

STUD1 ES ON THE I R3N-LI GAND BINDING IN HEMOPROTE INS AND THE1

R

RELATED COMPOUNDS

Y. Maeda

Research Reactor Institute, Kyoto Miversity, Kwnatori-cho, Sennan-gun, Osaka, 590-04, Japan

RBsum6.- La structure 61ectronique du fer dans les hCmoprot6ines est caractdrisde par ses dtats variables de valence et de spin. Ces points sont discutds pour les hgmoprotlines et les composBs similaires. La liaison fer-ligand, en particulier la liaison rhersible avec l'oxiggne dans la myoglobine et l'hdmoglobine est analysde en ddtail B partir de donndes de la spectromdtrie Miissbauer.

La structure Blectronique des composds ddsoxygdnds est aussi discutde, ainsi que la cindtique des liaisons de ligand dans les hdmoprot6ines.

Abstract.- The electronic structure of the heme iron in hemoproteins is characterized by its changeable valence and spin states. These features are discussed in hemoproteins and their related model compounds. The iron-ligand binding, especially the reversible dioxygen binding in myoglobin and hemoglobin is discussed in details by using the f4;issbauer spectroscopic knowledge obtained from the experiments with Mb single crystals and model compounds. A comparison of recent experimen- tal evidences with calculations has improved the knowledge of the iron-dioxygen binding structure.

The electronic structures in the deoxygenated state and the CO compound are also deduced. In addition, kinetics of ligand-binding to hemoproteins is reported.

1. Introduction.- The prosthetic group of hemopro- teins is an iron-porphyrin complex which is called heme. In order to interpret the biological activi- ties of hemoprotein in terms of the electronic structure of the heme iron, Msssbauer spectroscopy has been extensively applied to all members of hemoproteins. Since ~Essbauer spectroscopy has no limitations as to valence and spin states of the iron ion, it has already become an essential tech- nique in the study of hemoproteins 11-61.

The electronic structure of the heme iron is characterized by its easily changeable valence state and the small energy difference between the high spin and low spin states 171. The ~sssbauer studies have revealed these features and have pro- vided more detailed information on the iron-ligand binding. Especially for the dioxygen binding in myoglobin and hemoglobin, the Msssbauer spectros- copy has revealed important evidences through the isomer shifts and quadrupole splittings and allow- ed us to discuss the proposed iron-dioxygen bindine structures.

Recent studies of synthesized oxygen carrier have much improved our understanding of the iron- dioxygen binding 18-101. The X-ray structural ana- lysis have revealed a close relationship between the spin state of the iron and its relative posi- tion to the porphyrin. The presence of the inter- mediate spin states 111-121 and the evidence for conformational excitation 1101 have been success- fully confirmed by the Xsssbauer studies of model

compounds.

One of the recent developments of ~Essbauer spectroscopy of hemoproteins is found in the deter- minationof the electric field gradient tensor in myoglobin single crystal 113-15,621. The results would provide new experimental data for the calcu- lations which are intended to analyze the iron- ligand binding in hemoproteins.

Another successful application was recently performed to the photo-dissociation of ligands at low temperatures 116-181. Msssbauer spectra of photo-dissociated materials may contain useful in- formation on the oxygen binding, and it should be discussed in conjunction with the dynamical studies 119-21,74,75/ of ligand binding to the heme iron.

This paper will attempt to summarize the pre- sent status of Msssbauer studies of hemoproteins and their related compounds and to discuss the iron- ligand binding, especially the reversible oxygena- tion in oxygen carriers.

2. The valence and s ~ i n states of the heme iron in hemoproteins and their model compounds.-

2.1.- _H_ep~p_r~~rj~~.- The heme contains an iron atom on the center of protoporphyrin IX. In the native molecules, iron ions are ferrous in myoglobin and hemoglobin and ferric in peroxidase and catalase.

The four N atoms of the porphyrin coordinate to the iron in the plane. The two coordinate positions above and below the plane are available for compound formation. While the one is occupied by a proximal base such as the imidazole of histidine, the other

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19792180

is open for a small ligand such as 0 2 , CO, F-, OH-, N B , and CN

- .

Since the Fe ion is subjected to a li- gand field of rather high symmetry, its electronic structure has been successfully explained by the ligand field theory. It is worthwhile noting here that the ligand field around the heme iron is chars- cterized by a small energy difference between the high spin and the low spin states for both ferrous and ferric ions. This character is easily observed by MEssbauer spectroscopy.

In figure 1, isomer shifts and quadrupole spli- tting~ are given for several hemoproteins in the native and derivative states. The valence and spin states of hemoproteins are easily deduced and clas- sified by the Mgssbauer parameters. For the most part the classification is in good agreement with other information obtained from optical and magnetic measurements 137-391.

Fig. I : Isomer shifts (I. S.) and qucdrupole split- tings (Q.

s.)

for several hemoproteins in the native and derivative states. The isomer shifts are relati- ve to metallic iron at room temperature.

: myoglobin /13,15,22-251,

0

: hemoglobin 126- 281.

I

: catalase 1291,

n

^: peroxidase 130-331,

A

: cytochrome c 1341,

A

: cytochrome c ~eroxi- dase 135-361.

Generally speaking, the spin state is governed by the spectro-chemical series of ligands, and in some cases the crossover of spin states takes place. To discuss the electronic structure in details, such a simple analysis is inadequate and Mgssbauer mea- surements of magnetic hyperfine interactions are required.

The most striking result of Miissbauer measure- ments is found in the oxygen-carrying molecules : myoglobin (Mb) and hemoglobin (Hb). It is well known from the magnetic measurements that the fer- rous ion in the deoxygenated state is in a high spin state (5'12) and the binding with 0 2 (S=1) or CO

(S=O ) molecules changes the electronic state into

a diamagnetic ferrous low spin state (S=O). The Xgssbauer studies, however, provided three signifi- cantly different spectra for the deoxy-, 02-and CO- compounds / 1 3,15,23,26- 281. The deoxy-?ib and -Hb show large isomer shifts and large quadrupole split- tings with temperature dependence, which are charac- teristic of ferrous high spin ions. In the diamagne- tic state both compounds have small isomer shifts, which are to be expected for ferrous low spin ions.

However, the quadrupole splittings in the 02 cGm- pounds are fairly large and strongly temperature d e pendent, whereas those in the CO compounds are very small as can be expected for a spherical charge dis- tribution in a ferrous low spin state ( ' ~ 1 :t ').

2g As recognized in figure I, the Y6ssbauer para- meters in the 02 compounds are very close to those obtained in ferric low spin states rather than in ferrous low spin states. From the measurements with an applied magnetic field of 45 kOe at temperatures ranging from 4.2 to 130 K , however, the 02 compounds have been confirmed to be diamagnetic because of the absence of an induced internal magnetic field 1151.

These facts could be easily understood with the met- superoxide [~e(111)-02-1 model of Weiss /40/ in which all electrons are pair-coupled, producing dia- magnetism.

2.2. M~dgh-mmp~unds.

-

Recently several synthetic porphyrins have been prepared as models for the ac- tive center of hemoproteins and their electronic structure has been studied in details by ~gssbauer spectroscopy. As previously mentioned, only the high spin (S=2 or S=5/2) and the low spin (S=0 or S=1/2) states are found in hemoproteins as the eLectronic structure of the heme iron. In the model compounds, however, the intermediate spin states (S=l or S=3/2) can be observed in addition to these states.

In a cubic ligand field iron d-orbitals are split into the t2g (dxy,dxZJd and the e

YZ (dZ2,

d 2- 2) orbitals. The addition of an axial ligand

? =

freld along the zaxis brings further splittings of the t ande orbitals. In the case of square-planar

2g $7

complexes, the d 2 orbital comes close to the t orbitals and the d 2 orbital becomes higher rn 25'

x2-y

the energy level because of the spatial distribution of the electrons. Under such circumstances interme- diate spin states (S=1 or 5 ~ 3 1 2 ) may be stabilized according to the Hund's rule filled to the t and

dZz orbitals. 2g

In figure 2, the isomer shifts and the quadru- pole splittings are plotted for several model C O ~

pounds. With these Mgssbauer parameters a classi- fication of valence and spin states is also possi- ble for model compounds.

JOURNAL DE PHYSIQUE

Fig. 2 : Isomer shifts (I. S.) and quadrupole split- tings (?.

s.)

for model compounds. The isomer shifts are relative to metallic iron at room temperature.

I: Fe(III)TPP.CI 141,421, 2: Fe(II1)OEP-CI 1421, 3: Fe(III)TPP.OMe 1421, 4: Fe(I1I)OEP-OMe 1421, 5: Fe(II1)TPP'NCS 14 I/, 6: Fe(111)~PP-Br 1411, 7: Fe(III)TPPeI 1411, 8: [F~(III)TPP]ZO 142,431, 9: [F~(III)OEP]~O /42/, 10: F ~ ( I I I ) O E P ~ P ~ . C I O I I / ~ ~ / , I I: Fe(III)OEP.PyCN.CIO~ /45/,

12: Fe(II1)OEP .PyCHO.CIO+ 1451, 13: Fe(III)OEP.CIOk 145,461,

14: F~(III)OEP.~THF.CIOI, 1451,

15: Fe(II1)OEP' (Im)2'CI0,, 1451. 16: F~(III)OEP'S~BU 1451,

17: F~(III)OEP.O~BU 1451, 18: Fe(III)OEP.PhOMe 1481, 19: F~(III)PP. (1m) 2~1/47/, 20:Fe(III)PP. (Py) 2C1

1471,

a: Fe(I1)TPP Ill/, b: Fe(1I)OEP 1121, c: Fe(II)Pc 1491,

d: Fe(II)TPPe (Py)2 /SO/, e: Fe(II)OEP.(Py)2 1121, f: Fe(II)Pce ( p y ) ~ 15 I/, g: Fe(II)PPe (Py)2 1471, h: Fe(II)OEP.(NH3)2 1121, i: Fe(II)TPP'(ZMeIm) /II/, j: F~(II)OTBP- (THF)2 1121, k: F~(II)PFP.( 1-MeIm)-02

181,

1: Fe(11)PFP.(l-MeIm)2 /8/, m: Fe(II)PFP.(l-MeIm).

CO 181,

n: Fe(II)PFP.(THF)2.02 181, o: Fe(1I)PFP-(THF)2 181, p: Fe(1I)PFP' (1-MeIm) 181. A:*CO(II)OEP 1521, B: *Co(II)OEP.Py.02 152.1, C:x*~o(II)PPD.Py-02 1531, D: *Co(II)OEP.Py2 1521, E: Co(1I)PPD-Py2 1531.

Square-planar complexes Fe(I1)-tetraphenyl porphine (TPP) /II/ and Fe(I1)-octaethylporphyrin (OEP) 1121 are suggested to be in intermediate spin states (S=l). The magnetic moments in these complex-

es are very close to the spin-only value for high spin states. Furthermore, the quadrupole splittings of I .5 mm.s-' in the Fe(1I)TPP and Fe(I1)OEP complex- es are much smaller than 2.7 mm s-l in Fe(11)-?htha- locyanine (PC) 1491. However, Collman et aZ.11 I/

have concluded from a similarity of magnetically perturbed Mb;ssbauer spectra between Fe(I1)TPP and Fe(I1)Pc that these complexes are in the S-1 state.

In this case the electronic configuration is regard- 2 2

ed as fxy) (ZZ) (yz)lfz2)l. In the ferric state the existence of such an intermediate spin state (S=3/2), (~z)~f~z)~fxycyl~fz~)~, has been also reported for Fe(II1) OEP-CIOs and Fe(II1)-tetraazamacrocyclic

complexes 146,541. In both ferrous and ferric com- plexes with intermediate spin, the sign of the qua- drupole interactions is positive and the asymmetry parameter q is close to zero. For these complexes except for Fe(I1) PC, further studies are strongly required, since any reasonable electronic configura- tion is not available to explain all experimental data obtained from Mgssbauer and magnetic measure- ments. Here it should be noted that the iron sits in the plane of the complexes and has no axial li- gands in the complexes which are suggested to be in the intermediate spin state.

In the pyramidal complexes with an axial ligand such as C1-, the iron atom moves to the out- of-plane position and its electronic state becomes high spin. In the hexa-coordinated complexes with both axial ligands such as pyridine and imidazole, the dZz orbital becomes antibonding and the low s p n state is stabilized by using the tzg orbitals.

The most interesting model compound is an ana- logue of ?Iand Hb. Fe(I1)-picket % fence porphyrin (PFP) 18-101 is the only model found in iron compounds, capable of reversible binding of molecular oxygen.

The observed quadrupole splittings were quite simi- lar to those of m02 and HbOz in the temperature dependence and the sign of the quadrupole interaction.

Considering a relaxation between the two possible conformational states determined from X-ray studies, Spartalian et aZ. /lo/ succeeded in explaining the peculiar temperature dependence of the quadrupole splitting. Other analogues have been obtained in the

electron capture decay products of 5 7 ~ o ( ~ ~ ) ~ ~ ~ - ~ y - 0 2 1521 and 5 7 ~ o ( ~ ~ ) p r o t o p o r p h y r i n ( ~ ~ ) .Py - 0 2 1531

complexes. The Ikssbauer spectra of these compounds are very similar to Mb02 and HbOz.

3. Oxygen binding in hemoproteins.

-

3.1. -zro_pogeA models for-rh_e-bonding structure.- The oxygen bind-

ing on ^Mb and Hb has been the main subject of nu- merous studies in hemoproteins. Several models have been proposed for the bonding of the 0 2 molecule

to the heme: the linear model [FenO-0 and Fe-0=0I[of Pauling andCorye11 1551, the bent model

[F~=\I[

of Pauling 156,581. the equidistance model

[

Fe#

7

of Griffith 1591, the met-superoxide model [F~(III)-0;

7

of Weiss 1401 and the two- electron oxidative addition model

[F~(IV)-0:-1

of Gray 1601.

As already mentioned, the binding of triplet oxygen molecule (S=I) to paramagnetic Hb and Hb

(S=2) gives rise to a diamagnetic oxygenated state (SO). It is clear that this reaction violates a

r u l e o f t h e s p i n c o n s e r v a t i o n f o r a c h e m i c a l r e a c - oxygen b i n d i n g t o t h e heme b r i n g a b o u t a n e g a t i v e t i o n . T h e r e f o r e , o t h e r b i n d i n g s t r u c t u r e s s u c h a s l a r g e q u a d r u p o l e i n t e r a c t i o n i n good agreement w i t h t r i p l e t - t r i p l e t c o u p l i n g h a v e b e e n proposed f o r t h e t h e e x p e r i m e n t a l r e s u l t s . T h i s i s a s i m p l e d i s c u s - iron-oxygen b i n d i n g . Among them, a n ozone model i s s i o n f o r t h e b i n d i n g s t r u c t u r e o f t h e 02 m o l e c u l e p r o p o s e d by Goddard and O l a f s o n 1611. o n t h e heme i r o n . However, even i n compounds s u c h 3.2

.-

~ ; i s s b a ~ ~ ~ - ~ ~ _ u _ d j e s . - As p r e v i o u s l y m e n t i o n e d , 111

a s [ F e 1 I ( C ~ ) 6 1

'-

and [Fe (CN) 6 1 3 - , t h e Xb and Hb show a l a r g e q u a d r u p o l e s p l i t t i n g w i t h a c t u a l b i n d i n g may b e n o t s o s i m p l e . The r e a l s i t u - t e m p e r a t u r e dependence i n t h e o x y g e n a t e d s t a t e , a t i o n may b e a b a l a n c e between a d o n a t i o n o f e l e c - and t h e s i g n o f t h e q u a d r u p o l e i n t e r a c t i o n (e&vzZ) t r o n s from O2 i n t o t h e e o r b i t a l o f Fe ( a i n t e r a c - i s d e t e r m i n e d t o b e n e g a t i v e from measurements w i t h t i o n ) and a b a c k - d o n a t i o n o f e l e c t r o n from t h e 9 t

a n a p p l i e d m a g n e t i c f i e l d / 1 5 , 2 7 , 2 8 / . I n o r d e r t o 2g

o r b i t a l o f Fe i n t o t h e pn o r b i t a l of 02 ( T i n t e r - e x p l a i n t h e o b s e r v e d l a r g e n e g a t i v e q u a d r u e o l e i n - a c t i o n ) a s shown i n f i g u r e 3.

t e r a c t i o n , Lang p r o p o s e d two p o s s i b i l i t i e s / I / .

One i s b a s e d o n t h e ~ r i f f i t h ' s model 1 5 9 1 , i n which S = l S = 2 S = O t h e O2 m o l e c u l e l i e s p a r a l l e l t o t h e heme p l a n e .

By f o r m i n g a n b o n d i n g between a t o r b i t a l ( d n ) 2g

o f t h e i r o n and an a n t i b o n d i n g n o r b i t a l o f t h e 02 m o l e c u l e , e l e c t r o n i c c h a r g e i s p a r t l y t r a n s f e r r e d from t h e s p h e r i c a l ( t ) c o n f i g u r a t i o n o f t h e i r o n

29

i n t o t h e O2 m o l e c u l e . The d e f i c i t o f e l e c t r o n c h a r g e i n a d n o r b i t a l would b e l a r g e , and i t would b e a r e a s o n f o r t h e l a r g e q u a d r u p o l e s p l i t t i n g i n MbO2 a n d Hb02. The s e c o n d p o s s i b l e b o n d i n g a r r a n g e m e n t is t h e l i n e a r model o f P a u l i n g and C o r y e l l 1551,

i n which t h e 02 m o l e c u l e l i e s p e r p e n d i c u l a r t o t h e F i g . 3 : S c h e m a t i c r e p r e s e n t a t i o n o f t h e e f f e c t s of heme p l a n e . A s t r o n g a b o n d i n g is formed between d o n a t i o n and b a c k - d o n a t i o n i n i r o n - d i o x y g e n b i n d i n g . a n e f d z 2 ) o r b i t a l o f t h e i r o n a n d a n a n t i b o n d i n s F u r t h e r m o r e , t h e geometry o f t h e O2 m o l e c u l e t o t h e

a o r b i t a l o f t h e 02 m o l e c u l e . If t h e l o s s o f e l e c - 9 i r o n c o u l d a l s o b e o f a b e n t F e = q O t y p e p r o p o s e d t r o n c h a r g e from t h e d n o r b i t a l s i s s m a l l , t h e i r o n by P a u l i n g 156-581. I n f a c t , s u c h a b e n t geometry would h a v e a d d i t i o n a l e l e c t r o n c h a r g e o n t h e dZ2 h a s b e e n c o n f i r m e d i n t h e Fe(II)PFP'(N-Met-1mid)'Os o r b i t a l i n a d d i t i o n t o t h e s p h e r i c a l f t J 6 c o n f i - complex which h a s a n e g a t i v e q u a d r u p o l e i n t e r a c t i o n g u r a t i o n . As g i v e n i n t a b l e I, b o t h models f o r t h e 2g w i t h rl=0.23 / l o / .

T a b l e I : Components o f e l e c t r i c f i e l d g r a d i e n t t e n s o r

(Fpd

i n t h e heme c o o r d i n a t e s y s t e m ( z , y , z ) f o r t h e v a r i o u s e l e c t r o n i c s t a t e s i n deoxygenated and o x y g e n a t e d compoundsa.

-

e l e c t r o n s f r o n t h e Pe n u c l e u s .

S t a t e s o r models

i

~ d < r - ~ >

i

^V / e < r - 3 >

i

vZz/e<re3>

: YY

Deoxygenated s t a t e

5 ~ z -217 -217 + 4 / 7

I

+ 4 / 7 -21 7 -217

-217 + 4 / 7

5 E Y -217

-217 + 4 / 7

3 ~ x -217

+ 4 / 7 - 2 / 7

3 ~ Y -217

' ~ 1 0 0 0

Oxygenated s t a t e

G r i f f i t h ' s model -417 + 2 / 7 + 2 / 7

i

P a u l i n g and C o r y e l l ' s j +2/7 + 2 / 7 -417

model

Ozone model *4/ 7 -217

r 2 1 7 +4/7 -217 -217

Only t h e c o n t r i b u t i o n o f 3d e l e c t r o n s i s i n v o l v e d i n t h e EFG, and t h e c o n t r i b u t i o n I

o f t h e l a t t i c e i s n e g l e c t e d . < F 3 > i s t h e e x ~ e c t a t i o n v a l u e o f F3 where r i s t h e d i s t a n c e o f 3d

JOURNAL DE PHYSIQUE

In order to discuss the bonding structure of ligands to the heme iron, it is very valuable to know the charge distribution at the iron atom. The quadrupole interaction of the iron nucleus provides such information through the electric field gradient

(EFG) (-Vii). For our purpose the full knowledge of the EFG tensor is necessary ; the largest principal component (-V ) , the asymmetry parameter q and the

9.2

orientation of the principal axis system (PAS) (Euler angle a, 6 , y) should be determined. From usual measurements with polycrystalline samples or in frozen solution the values of Vg9 and 17 cannot be determined separately. Only in favorable cases, for instance in a diamagnetic substance, the deter- mination of Vc2 (including the sign) and 17 becomes

possible by applying a high magnetic field exter- nally to the sample. In order to get the full know- ledge of the EFG tensor, it is necessary to measure the quadrupole interaction with a single crystal.

Since the intensity of quadrupole split lines de- pends on the angle between the propagation direction of y-rays and the PAS of the EFG tensor, it is possi- ble to determine not only the magnitude of the EFG but also the orientation of the PAS by measuring the

change of line intensities at various crystal orien- tations relative to the y-rays.

Recently such measurements have been success- fully performed on Mb single crystals in the three different forms, deoxy-Mb, MbOz and MbCO, at 77 K 113-15, 62/. The crystals were monoclinic type A , and the structure is fully known from X-ray analysis.

The crystal contains two hemes within one unit cell.

The cne is transformed into the other by 180' rota- tion around the b axis. Unfortunately this symmetry makes it impossible to determine the local EFG ten- sor at each heme iron from the experimentally obtained macroscopic EFG tensor 1141. For monocli- nic crystals manifold solutions are usually obtained in the determination of the local EFG tensor 1631.

With the knowledge of the sign of VSS and the q value obtained from other measurements, the EFG tensors at the heme irons in deoxy-Mb, XbOz and MbCO have been determined in details as follows :

I) In deoxy-% the largest principal axis of the EFG tensor is nearly in the heme plane dis- regarding the sign of V.2z and the 17-value.

2) In Mb02 the largest principal axis of the EFG lies nearly in the heme plane and

n

is limited to the range between 0.3 and 0.4.

3) In MbCO the largest principal axis of the EFG is nearly perpendicular to the heme plane and

q is limited to the range between 0.2 and 0.4.

3.3.- galcu1ations.- The electronic structure of the neme iron in Mb and Hb has been intensively interpreted in the ligand field approximation. In the tetragonal ligand field with the symmetry axis perpendicular to the heme plane, the 5 ~ 2 , 'A*, 'E and 3~ states are the low-lying states in the 3d6 configuration. From the magnetic measurement, the ground state of paramagnetic deoxygenated compounds has been ascribed to the ' ~ 2 or 'E state, whereas those of diamagnetic 02 and CO compounds have been ascribed to the 'A, state. If we assume the heme plane of the EFG tensor and the heme normal as the z axis of the PAS, the components of the EFG tensors in the 'B2, 'E, 3~ and 'A, states are given in ta- ble I.

3.3. 1.

-

_Dgmygenated state.- A well-isolated 'B2 or 'E state will give a quadrupole splitting of about 4.5 mm. s-', which is nearly twice the experi- mental value observed in deoxy-Mb and -Hb at 4.2 K.

Since the axis of the largest principal component of the EFG tensor has different directions for the

' ~ 2 , 'E and 3~ states, a mixing of these many-elec- tron terms should be taken into account in explain- ing the quadrupole splitting and its temperature dependence. Along this line, the analyses of the EFG and magnetic susceptibility tensors have been tried by Huynh et a l . 1641 and Eicher et a l . 1651.

However, it turned out that further splittings of the tetragonal many-electron terms due to the per- turbation of a rhombic distortion and spin-orbit couplings should be considered in getting good agreement with the experimental results. Their con- clusions can be summarized as following : 1) in the tetragonal field the 'A, and 3~ levels lie close to the ground state '~7. ; 2) with a rhombic pertur- bation one component of the 'E level becomes lower than the 'B2 level ; and 3) the axis of the largest principal component of the EFG tensor is in the heme plane and the sign of Vgz is positive as sup- posed in table I. Both calculations strongly supprt the experimental results of single crystal ~gssbauer spectroscopy in deoxy-Xb / 13,14 /.

On the other hand, Kent et aZ. 1661 have analyzed magnetically perturbed 'iiissbauer spectra in deoxy-Mb frozen solution, and reported that the sign of VS9 is negative and the axis of the largest principal component is nearly in the heme plane.

Their results do not reconcile with the analyses based on the ligand field theory.

Recently, Lumpkin and Dixon /67/ have measured

a proton NMR relaxation time and proposed a consis- tent interpretation of all the experiments inclu- ding Sssbauer studies. Considering a motional nar- rowing between the ' ~ 2 state and one of the 5~

states, they are suggesting that the averaged EFG tensor has a negative VZg and its largest principal axis lies in the heme plane.

3.3.2.

-

Diamagnetic state.- As already mentioned, the large quadrupole splitting in oxygenated hemo- proteins is qualitatively interpreted by both models of Pauling and Corye11/55/ and Griffith 1591. How- ever, more detailed analyses are necessary to fur- ther characterize the nature of reversible 02-bind- ing to Mb and Hb. After the fruitful success in synthesizing a reversibly 02-binding model compound (picket fence porphyrin), a number of calculations have been carried out. Using the oxygen binding conformation obtained from X-ray structure analysis, the EFG tensor at the iron nucleus was calculated and compared with the Miissbauer results. These results are summarized in table 11.

/lo/. Furthermore. a fair amount of charge is trans- ferred from the iron to the ligands due to strong covalent binding, even without the consideration for a charge transfer ~Fe(111)-0;~ configuration

(Weiss model 1401).

On the other hand, Huynh et aZ. I691 used the extended SCF-CI-Pariser-Parr-Pople method and the Xa multiple scattering method, obtaining consistent EFG tensors of Fe(I1)PFP .(N-l4et-Imid). 02.

As shown in table 11, these two calculations are in good agreement with the experimental results obtained from the model compound / 101 and Mb02 sin- gle crystals 1151.

Goddard and Olafson I611 proposed an ozone model for the 02-binding to heme iron and concluded that the ground states of %On and Hb02 are singlet states formed by one component of the 3~ state of deoxy-Mb and -Hb coupled with the

3 ~ g

state of an 0 2

molecule. The electron configuration of the iron ion should be (t )'(e ) I . As indicated in table I,

29 g

Table 11 : Comparison between calculated and observed quadrupole interaction parameters in oxygenated hemoproteins.

Method

i

Q.S. (nun s-l) rl

i

Orientation of Vg2 Ref.

;

Calculation

ab initio SCF

i

^{+0.40 1.0}

i i

1791

GVB-CI

i

^+3.85 0.0

i

in plane 1611

Ext

.

SCF-CI-PPP -2.24 0.84

i

in plane

i

1691

Xa Nulti. Scattering

i

-1.52 0.60

i

^{in plane}

i

I691

Iter. Ext. ~ G c k e l

i i

^I681

Model Compound

i

-2.67(+1.80)

i

0.37(0.02): in plane(perpend.)

i

Hemoprotein

i

-2.67(-2.40)

j

0.37(0.57); in plane(in plane)

-2.96 0.38

i

Bent Off-Axis Bind.

i

in plane

Experimental

Model Compound

i

-2.10(?1.34)

i

^0.23( ) ;

1 ^{Ilol i}

Myoglobin

i

-2.31(-1.96) 0.4 (0.4

) i

inplane(inp1ane) 1151

Hemoglobin

i

-2.24(-1.89)

i i

1271

( ) : High Temperature Values Kirchner and Loew 1681 applied the iterative extended ~ i c k e l method to twenty-two binding confor- mations of an oxygen molecule. A bent, end-on struc- ture (Pauling model) is found to be favored over a doubly coordinated structure (Griffith model) and further stabilized by an off-axis displacement of the 0 2 ligand. They obtained z large negative V

ZZ only in a totally paired Fe(I1)-02 configuration and succeeded in explaining the temperature dependence of quadrupole splitting with a rotation about the iron-oxygen bond as proposed by Spartalian et a l .

the sign of the largest Vii (Vmor V I is positive

YY

in the 'E state, although the axis of the largest principal component of the EFG is in the heme plane.

As an essential conclusion of the valence bond cal- culation, however, they report that the ozone model leads to a large negative V which is in agreement

ZZ

with ~Essbauer results. This argument is clearly incorrect, because they deal with the sign of V z z instead of the largest component VS2. Since a magne- tically perturbed spectrum allows us to determine the sign of the largest principal component of the

c2-520 JOURNAL DE PHYSIQUE EFG tensor, Vm or V is Vgz in this case.

YY

In the Heitler-London calculation, however, Seno et aZ. /7,70-721 indicated the importance of triplet-triplet coupling in the heme iron-oxygen binding.

In contrast to the 02 binding, the CO binding to the heme does not result in a large amount of charge transfer. The electronic state is the 'AI state, in which the charge distribution is almost spatially uniform. As molecular orbital calculations 123,731 suggested, however, a small EFG may be ex- pected through a relatively strong n bonding of the Fe dn's orbital with the unoccupied n orbital of

g

a CO molecule. The observed positive value of eQV 22 and the largest principal axis perpendicular to the heme plane 115,621 are in good agreement with cal- culations.

4. Kinetics of ligand-binding to hemoproteins.- As previously mentioned, penta-coordinated deoxy-Mb and-Hb with high spin change to hexa-coordinated low spin states when the binding to an 02 mlecule takes place. Simultaneously the iron atom moves into the heme plane from the out-of-plane position. The ligand binding to hemoproteins should be accompanied by a large amount of change in the electronic struc- ture of the iron atom and the O2 molecule.

Recent studies with laser-phoyolysis allow us to observe the photo-dissociation and subsequent recombination of the ligand in hemoproteins. By these experiments information concerning the kinetics of ligand-binding has been obtained. The kinetics of the rebinding reaction has been analyzed by the classical barrier model /19,20/ and the quantum- mechanical tunneling model /21,74,75/.

Recently Mgssbauer spectroscopy has been also applied to this field. The compounds of Mb and Hb with 02, C and NO were photo-dissociated at low

temperatures, and the resulting spectra were compa- red with those of the deoxygenated form 116-181.

The photo-dissociated compounds are in a high spin state having infinite lifetime at low temperatures.

Maeda et a l . 1171 have investigated the thermal con- version of the photoproduct back to the original compound and suggested the existence of a very sta- ble photoproduct below 50 K which has not been observed in earlier works,as shown in figure 4.

One should notice an emission ~gssbauer spectrum of 5 7 ~ o -substituted Hb 1761. In the case of 5 7 ~ o - ~ ~ , vitamin B12 and S7~o-porphyrins, the emission Mijssbauer spectra were very similar to the Fe analogues. In contrast, the 7~o-substituted

deoxy-Hb showed a spectrum of an intermediate spin which is different from that of the deoxy-Hb(Fe).

I ' 1

..: \-- ^//.

^;*: -<-+-*.--: ..*,-*

MbCO

L .

I

-

¹ 0 1 I 3 V E L O C I T V I m m % - ( I

Fi&. 4 : Mgssbauer spectra of myoglobin-carbomono- oxide (?lbCO) compound and its photo-dissociated form.

-. mco~ ---. .

photo-dissociated form.

a) MbCO measured at 20 K before photo-irradiation, b) XbCO irradiated from both sides with 650W halogen lamp for 5 minutes at 20 K and measured at 20 K.

c)---e) thermal-annealed and measured at 30,40 and 50 K, respectively.

As discussed in sec. 2-2. an iron atom of interme- diate spin sits in the heme plane and a cobalt atom also sits in the heme plane of CoHb. It is therefore suggested that the daughter "Fe atom is frozen almost in the same position as that of the parent 5 7 ~ o . This finding does not support Perutz's trigger mechanism for cooperative binding of oxygen /77,78/.

Acknowledgements.- The author is indebted to Dr. Y.

Morita, Dr. U. Gonser, Dr. A. Trautwein, Dr. H.

Sakai and Mr. T. Harami, since the results described in this paper were obtained through collaboration with them. He wishes to express his sincere thanks

to Dr.T. Higashimura for his' continual interest and encouragement.

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