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MÖSSBAUER SPECTRA OF DEOXYGENATED
HEMOGLOBIN IN R AND T STATES
A. Merli, I. Ortalli, E. Papotti, G. Rossi
To cite this version:
A. Merli, I. Ortalli, E. Papotti, G. Rossi. MÖSSBAUER SPECTRA OF DEOXYGENATED
JOURNAL DE PHYSIQUE Colloque C6, supplément au n° 12, Tome 37, Décembre 1976, page C6-181
MOSSBAUER SPECTRA OF DEOXYGENATED HEMOGLOBIN
IN R AND T STATES
A. MERLI, I. ORTALLI, E. PAPOTTI and G. L. ROSSI
Institutes of Physics and Molecular Biology, University of Parma, 43100 Parma, Italy
Résumé. — L'Hémoglobine humaine normale, non oxygénée (Hb), présente une faible affinité
pour l'oxygène et coopérativité dans la liaison du ligand (Etat T). Les propriétés fonctionnelle sont perdues par la suppression de deux acides aminés terminaux de la chaîne fi (Hb-CPA) ou de l'acide aminé terminal de la chaîne a suivi par une réaction avec NEM (NES-des-Arg-Hb) (état R). Les dernières espèces retrouvent ses propriétés fonctionnelles normales en se liant avec l'IHP.
Les valeurs obtenues à 78 K pour le déplacement isomérique et le gradient du champ électrique sont pour Hb et HbCPA très proches et différentes de celles relatives au NES-des-Arg Hb et
IPH + NES-des Arg Hb. Ainsi les paramètres Môssbauer indiquent des variations dans la
struc-ture électronique des ions ferreux qui ne sont pas directement corrélées à l'état conformationnel de la molécule.
Abstract. — Normal human deoxygenated hemoglobin (Hb) exhibits low oxygen affinity and
cooperativity in ligand binding (T state). These functional properties are lost upon enzymatic removal of the two C-terminal amino acids of the P chains (Hb-CPA) or of the C-terminal amino acid of the a-chain followed by reaction with N E M (NES des-Arg-Hb) (R state). This last species recovers normal functional properties upon binding of IHP. The isomer shift and quadrupole splitting values at 78 K of Hb and HbCPA are very close and distinct from those relative to NES-des-Arg Hb and IPH + NES-NES-des-Arg Hb. Thus Mossbauer parameters reflect changes in the ferrous ions electronic structure that cannot be directly related to the conformational state of the molecule.
The cooperative behaviour of normal human hemo-globin may be explained on the basis of the two state model. According to this model, the ligand affinity is determined by the equilibrium between two quater-nary states of the protein respectively with high (R) and low (T) ligand affinity [1-4]. Perutz has suggested that the equilibrium between R and T states is governed primarily by the displacement of the iron atom from the plane of the porphyrin ring [5-7]. This interpreta-tion, however, has been criticized by Gibson [8-9] who
excludes a linking between spin state of the iron and conformation or ligand binding and suggests that the functional difference in R and T states can be related to stereochemical difference in accessibility imposed by the polypeptide chain of the globin.
We have analysed the Mossbauer spectra at 78 K of native and variously modified deoxygenated hemoglobins.
The measurements were taken in transmission geo-metry using an acceleration spectrometer, connected TABLE I
Mossbauer parameters of hemoglobin in R and T states. (*)
Hb NbCPA (NES-des-Arg) Hb IHP + (NES-des-Arg) Hb C (mg/ml) 298 298 296 270 S (mm/s) ± 0.03 + 0.73 + 0.76 + 0.70 + 0.70 AE (mm/s) ± 0.03 2.27 2.27 2.41 2.43 % 0.92 0.93 1.19 0.90 I2 % 1.10 0.84 1.30 0.94 (mm/s) 0.35 0.32 0.26 0.32 C1) The hemoglobins studied are : a) Normal deoxyhemoglobin (Hb) which is in T state ; b) Deoxyhemoglobin in which the C-terminal aminoacids histidine 146 /? and tyrosine 145 ft have been removed by digestion with carboxipeptidase A [des-(His-Tyr) Hb] which is in the R state ; c) Deoxyhemoglobin in which the Arginine 141 a has been removed by digestion with carboxipeptidase B and the sulfhydryl group of Cysteine /J-93 has been blocked by reaction with N-ethylmaleimide to form the N-ethyl-succinimide derivative [NES-des-Arg-Hb] which is in the R state ; d) Same as c) switched to the T state by addition of inositol hexaphosphate (IHP).
C6-182 A. MERLI, I. ORTALLI, E. PAPOTTI AND G. L. ROSS1 with a multichannels anafyser operated in multiscaler
mode. The source was a Co57 in Pd (10 mCi) and a proportional counter was used as detector. The expe- rimental data were analysed using a Laben 701 compu- ter program assuming a Lorentzian line shape and were corrected for the background assuming a parabolic velocity dependence.
The materials and methods are reported in (10). The electronic absorption spectra before and after recording Mossbauer spectra show that the sample has not been oxidized and that the sample is fully deoxygenated
.
The Mossbauer spectra are shown in figure 1 and the value of isomer shifts (6) and quadrupole splittings
( A E ) are listed in table I.
The values of 6 and A E are typical of high spin ferrous ions and are in good agreement with those reported for deoxygenated hemoglobin [ll-141. The data reported in table I show that only minor, if any, differences exist in the iron electronic structure a t 78 K in molecules that in solution at room temperature have different conformations.
Perutz suggested that the low oxygen affinity of the T structure may be related to an increased tension a t the heme which pulls the iron atom further away from the plane of the porphyrin ring. Eicher [15] has calcu- lated a slight different iron-porphyrin plan distance in deoxy-Hb and Mb. The mechanism proposed by Eicher, although different from that proposed by Perutz, assumes that the difference in ligand affinity arises from the difference in out of plane distance and electronic state of the iron atom.
On the other hand Huynh et al. [16] have reported that no difference exists in the Mossbauer spectra of deoxyhemoglobin and deoxy a and
P
isolated subunits indicating that no difference exists in the electronic structure of iron atom, between the low affinity tetra- mer and the high affinity isolated subunits.In agreement with Huynh et a1 [16], we conclude from our data that even if the small differences observed in the Mossbauer parameters for Hb and des(His- 146
B,
Tyr 145P)
Hb on one side and NES-des-Arg- 141 a-Hb and IHP+
(des-Arg-141 a-Hb on the other side reflect distinct differences in the electronic structure of the ferrous ion in the various derivatives, they cannot be correlated with the distinct functional properties observed in solution, at room temperature, for molecules in R and T state.Referc
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VELOCITY (mrnJs )
FIG. 1. - Mossbauer absorption spectra of : a) deoxyge- nated Hb ; b) HbCPA ; c) NES-des-Arg-Hb ; d ) IHP 4- NES-
des-Arg-Hb.
[5] PERUTZ, M. F., LADNER, J. E., SIMON, S. R. and Ho, C., Biochemistry 13 (1974) 2163.
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