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DIFFERENT PROPERTIES OF FERROUS IRON IN
DEOXYGENATED HEMOGLOBIN CHAINS
A. Trautwein, Y. Alpert, Y. Maeda, H. Marcolin
To cite this version:
JOURNAL DE PHYSIQUE Colloque C6, supplément au n° 12, Tome 37, Décembre 1976, page C6-191
DIFFERENT PROPERTIES OF FERROUS IRON
IN DEOXYGENATED HEMOGLOBIN CHAINS (t)
A. TRAUTWEIN, Y. ALPERT (*), Y. MAEDA (**) and H. E. MARCOLIN Fachbereich Angewandte Physik, Universitat des Saarlandes
6600 Saarbriicken 11, W-Germany
(*) Institut de Biologie Physico-Chimique, 13, rue P.-et-M.-Curie, 75005 Paris, France (**) Research Reactor Institute, Kyoto University, Osaka, Japan
Résumé. — On a étudié par spectrométrie Môssbauer, entre 4,2 K et 200 K, de l'hémoglobine
déoxygénée, enrichie au 5 7Fe, d'un homme adulte (HbA), ses chaînes isolées as H et /?SH et de
l'hémo-globine totale a2(57Fe) /?2(56Fe) partiellement enrichie en 5 7Fe. Compte tenu de la précision
expé-rimentale ( ± 0,006 mm s- 1) l'effet quadrupolaire des HbA des chaînes asir et /3SH varie entre 100 K
et 4,2 K ; de plus l'effet quadrupolaire de HbA n'est pas la moyenne des valeurs trouvées pour les chaînes isolées. L'effet quadrupolaire de a2(57Fe) /?2(56Fe) est pratiquement le même que celui de
HbA. Les mêmes tendances sont retrouvées sur les valeurs du déplacement isomérique. Les largeurs de raies ont des valeurs très voisines pour tous les composés.
Abstract. — Iron 57-enriched, deoxygenated human (adult) hemoglobin (HbA), its isolated asH- and /?sn-chains, and the partially iron 57-enriched, fully functional hemoglobin a2(Fe57)
j82(Fe56) were investigated by Mossbauer spectroscopy in the temperature range 4.2 K-200 K.
According to our experimental accuracy ( ± 0.006 mm s_1) the temperature dependent quadrupole
splittings &EQ(T) of deoxygenated HbA, <XSH- and ^sH-chains are not identical from about 100K down to 4.2 K, and A£Q of HbA is not the average value of the AEVvalues of the isolated chains.
The quadrupole splitting of a2(Fe57) /?2(Fe5S) turns out to be practically the same as that of HbA.
These findings are also reflected by the isomershifts. Line widths are comparable for all compounds.
1. Introduction. — All mammalian hemoglobins
possess four subunits and hence four oxygenation sites (ferrous iron ions). Cooperative interactions between these sites play an important role upon oxygen uptake : The oxygen affinity is low in the fully deoxygenated molecule and increases towards the so called « high-affinity-state ». Associated with this the molecular conformation of these sites switches between two corresponding structures, namely the initial
out-of-plane iron ions move towards the center of the
porphy-rin plane and change their electronic properties [1, 2]. The specific oxygenation characteristic of hemoglobin is attributed to the presence of two different types of subunits, the a- and /?-chains, the aminoacid sequences of which slightly differ in their total number of residues and in several local substitutions. When isolated, a- and /J-chains bind molecular oxygen without any conformational change ; this is interesting especially under the aspect that /J-chains spontaneously aggre-gate to four tetramers [3].
From low - temperature magnetic susceptibility measurements [4] there is evidence, that inter-subunit interactions result in a change of the iron environment when going from isolated to associated chains ;
(f) Supported by Stiftung Volkswagenwerk. One of us (Y. A.) acknowledges the financial support by EMBO during the course of this work.
(such influences are undetectable by crystallographic methods at the present level of resolution). Mossbauer-spectroscopic investigation was expected to corrobo-rate these results, however, recently Huynh et al. [5] reported that hemoglobin and its isolated chains are undistinguishable from their Mossbauer spectra.
2. Experimental. — Isomershift, 8, and quadrupole splitting, AEQ, has been measured between 4.2 K and
200 K for several preparations of deoxygenated Fe5 7 -enriched human (adult) hemoglobin, HbA, and its native and isolated chains with free SH-groups, aSH and /?SH. The sample preparation is described in detail in ref. [6]. All the samples used in our study were found to possess normal electrophoretic behavior, normal optical spectra, and normal cooperative oxyge-nation behavior for reconstituted HbA (Hill-coefficient of about 2.7 is comparable to 2.7-2.9 for native HbA). Another type of sample has been measured, in which two Fe57-enriched a-chains are associated to two unenriched ^-chains (mass hybrids). The Mossbauer spectrum of this a2(Fe57) /?2(Fe56) sample, of course, reveals the properties of iron in a a-chain, which is part of a normal and fully functional HbA molecule. Thus the investigation of mass-hybrids with Moss-bauer spectroscopy opens the possibility to correlate electronic properties of iron in the different subunits with their suggested functional inequivalence [1].
C6-192 A. TRAUTWEIN, Y. ALPERT, Y . MAEDA AND H. E. MARCOLIN
3. Results and discussion. - The experimental qua- drupole splittings, isomershifts and line widths of deoxygenated HbA, a,,, /I,,,and ~ t , ( F e ~ ~ ) j?2(Fe56) are listed in table I. The absolute values of 6(T) are
Experimental quadrupole splittings, AEQ(T), isomevshifts, 6(T), and line widths, T(T), for various Fe57-enriched human hemoglobin compounds
Deoxy hemoglobin Deoxygenated a-chains Deoxygenated /?-chains T(K) AEQ(mm s- l) 6(mm s- I) - - - 4.2 2.492
+
0.008 0.882+
0.010 9.0 2.486+
-
0.884 _+ 0.011 14.0 2.477+
-
0.882+
0.010 20.0 2.472+
- 0.881+
0.01 1 27.0 2.465+
- 0.887+
- 35.0 2.456+
-
0.885+
- 45.0 2.445 f 0.010 0.885 _+ 0.013 55.0 2.435 f 0.008 0.884+
0.011 63.0 2.410+
0.011 0.879 f 0.014 77.0 2.404+
0.008 0.8755
0.011 2.390 _+ 0.007 0.876 _t 0.010 100.0 2.358+
0.009 0.866 0.012 150.0 2.188+
0.010 0.862+
0.013 200.0 1.997+
0.011 0.835 _t 0.014 Deoxy hemoglobin(a-chains (") enriched, /?-chains unenriched)
T(K) AEQ(mm s- I) 6(mm s- I) T(mm s- l)
-
-
-
-
4.2 2.381
+
0.005 0.938+
0.010 0.286+
0.006 77.0 2.319 3- - 0.930+
- 0.304 _+ -120.0 2.239 4 - 0.923
+
- 0.322 _+ -(a) Enrichment with Fe57 is > 90 %.
comparable to these found by other authors [7-111 for deoxygenated hemoglobin or myoglobin. They confirm that iron in these proteins is in the ferrous high-spin state.
3.1 ISOLATED as,- AND fiSH-c~A1~s. - Figure 1 illustrates that the quadrupole splittings of the deoxy-
Psi,
= a St1
-
H b AFIG. 1. - Temperature dependence of experimental quadrupole splittings, AEQ(T) of Fe57-enriched and deoxygenated human
hemoglobin and its chains.
genated a,,- and P,,-chains are definitely not identical according to the experimental accuracy
(1
0.006 mm s-I). This result is in contradiction to the measurement reported by Huynh et al. [5], and we try to find possible reasons for this discrepancy :DIFFERENT PROPERTIES O F PERROUS IRON I N DEOXYGENATED HEMOGLOBIN CHAINS C6-193
valence of our AEQ(T)-values of different chains is emphasized at lower temperatures.
(iii) Huynh et al. also reported values of the iso- mershifts (relative to metallic iron) which show a systematic deviation of about 0.1 mm s - b i t h respect to values reported by other authors [7-101 for deoxy- genated hemoglobins and myoglobins.
(iv) Finally their published spectra reveal the pre- sence of about 20
%
of impurity species, which they consider as unavoidable consequence of the prepara- tional procedure. In contradiction to this we succeeded in preparing samples free of such impurity species (Fig. 2) ; the nature and formation of such impurity species is discussed elsewhere [4, 61.3.2. HbA. - An interesting result of our study is the fact that the quadrupole splitting of deoxygenated HbA is not only different from that of any of the two chains in the temperature range of 4.2 K-100 K, but that it is also different from the average value
4[AEQ(asd
+
AEQ(PsH)].This finding is in agreement with magnetic suscepti- bility measurements [4] and confirms that the elec- tronic properties of iron do measurably change upon chain association.
The absolute AEQ(T)-values we found for HbA are comparable with those of Huynh et al. [5] within the range of errors, however, deviate from those of Eicher et al. [lo] systematically by about 0.05 mm s-l, and from those of Spartalian et al. I l l ] by 0.02 mm s-l. Here again we find it worth to notice that our spectra are free from impurity components in our HbA samples.
FIG. 2.
-
Mossbauer spectra of human deoxy hemoglobin (enriched in vitro with Fe57 by more than 90 %) and its chains at 4.2 K : a ) HbA = az(Fe57) Bz(Fe57) ; b) BSR ;c) az(Fe57) B~(Fe56).
3.3 MASS HYBRIDS.
-
The Mossbauer investigation of the mass hybrid u , ( F ~ ~ ~ ) f i z ( ~ e 5 6 ) allows the ins- pection of the a-chains alone in the fully functional hemoglobin. Its quadrupole splitting is almost the same as that observed for the completely enriched HbA : ~ t , ( F e ~ ~ ) /?,(Fe57). From this, and from the discussion above (3.2) we conclude that the a-/?- association is accompanied by a measurable ligand field modification of the heme iron. More extensive discussion of these last data will appear elsewhere.References
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