Biochimica et Biophysica Acta, 490 (1977) 171-177
© Elsevier/North-Holland Biomedical Press BBA 37510 F U N C T I O N A L PROPERTIES OF P A R T I A L L Y OXIDIZED T R O U T HEMOGLOBINS
G. FALCIONI, G. FORTUNA, B. GIARDINA, M. BRUNORI and J. WYMAN Laboratory of Molecular Biology, University of Camerino, C.N.R. Centre of Molecular Biology, Institutes of Chemistry and Biochemistry, Faculty of Medicine, University of Rome, and Regina Elena Institute (ItalyJ
(Received April 21st, 1976)
SUMMARY This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The 02 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the p H dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic o f the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.
INTRODUCTION The chance of understanding the molecular mechanisms operative in hemoglobin would be greatly increased if it were possible to isolate and characterize frozen intermediates in which one or more of the sites is blocked in the ligand bound state, the others being free to react [1]. It has recently become possible to isolate, in the case of one of the hemoglobin components of trout blood (Hb trout I), all five species which should theoretically exist upon partial oxidation of the tetrameric molecule [2]. This results from the fact that in trout hemoglobins the fast dimer exchange which occurs in the ligand bound form of human hemoglobin [3] is greatly reduced. This note reports, as a preliminary step, a study of the reaction with 02 and CO of the partially oxidized forms of both Hb trout I and Hb trout IV, without, for the time being, attempting to isolate the various intermediates. In these mixtures all the possible forms will be present in proportions determined by the percentage of oxidation. In the analysis given below it is assumed, on the basis of their method o f
172 preparation, that these proportions correspond to a random oxidation, as if the sites were identical and independent. This seems reasonable since the mixtures were prepared starting from the oxygenated derivative at alkaline pH. MATERIALS AND MEYHODS Preparation of trout hemoglobin and purification of the different components was carried out as previously described [4]. The various mixtures were prepared by treating the oxygenated derivative with the desired stoichiometric amount (in terms of heme concentration) of K3[Fe(CN)6] at alkaline pH values ( ~ 8). The amount of Met-Hb in each solution was determined by the ratio of the absorption at 405 nm to that at 419 nm. In flash experiments an estimate of the percent oxidation in each sample was obtained by comparing the total absorption change before and after addition of dithionite to each individual sample. Oxygen dissociation curves were determined spectrophotometrically [5], generally at a protein concentration of 3-5 mg/ml. Flash photolysis measurements were performed with an apparatus similar to that previously described [6]. Spectrophotometric measurements were made with a Beckman D B G T spectrophotometer. Carbon monoxide was obtained from S.I.O. (Rome). All other reagents were analytical grade products. RESULTS
Oxygen equilibrium It may be recalled that the functional behavior of H b trout I is characterized by the presence of positive interactions between the oxygen binding sites (n ~ 2.3) as well as the absence of any significant Bohr effect. In contrast, strong heterotropic interactions, involving both protons and organic phosphates, are present in Hb trout IV: at acid p H (6-6.5) the protein is only partially saturated even under O2 at I a t m and the cooperativity, evident at more alkaline pH values, is completely absent (n ~ 1) (Root effect). o/ The oxygen dissociation curves of mixtures of ferric (from 25 ~ to 75/o) and ferrous form of Hb trout 1 in presence of azide in saturating amount are reported in Fig. 1. The results show a marked decrease of the homotropic interactions in going from 0 ~ oxidation (where n = 2.3) to 75 ~ oxidation (where n = 1.2). Such a drop in cooperativity parallels a great increase ( ~ 7 times) in oxygen affinity. These changes support a progressive shift of the conformational equilibrium towards the highly reactive (R) form of the molecule; in other words, by increasing the number of the oxidized subunit~, we induce a progressive stabilization of a "high affinity" conformational state of the molecule. The results obtained, under the ~,ame experimental conditions for Hb trout IV are summarized in Fig. 2 where the pH dependence of log p½ and n½ is reported. The general effect of increasing oxidation is similar, at each p H value, to that observed for Hb trout I. However, it should be remarked that the decrease in cooperativity, brought about by the increase of oxidation, is more dramatic at low levels of oxidation.
173 1.0
::'.5
r( ~,0
tlolO~
'
0.5
i 0.5
0
0.5
to
log po a
1.5
Fig. 1. Oxygen equilibrium curves of Hb trout I in presence of 2 mM azide as a function of percent oxidation : ( O ) 0 ~ ; (A) 25 ~ ; (~I
© Elsevier/North-Holland Biomedical Press BBA 37510 F U N C T I O N A L PROPERTIES OF P A R T I A L L Y OXIDIZED T R O U T HEMOGLOBINS
G. FALCIONI, G. FORTUNA, B. GIARDINA, M. BRUNORI and J. WYMAN Laboratory of Molecular Biology, University of Camerino, C.N.R. Centre of Molecular Biology, Institutes of Chemistry and Biochemistry, Faculty of Medicine, University of Rome, and Regina Elena Institute (ItalyJ
(Received April 21st, 1976)
SUMMARY This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The 02 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the p H dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic o f the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.
INTRODUCTION The chance of understanding the molecular mechanisms operative in hemoglobin would be greatly increased if it were possible to isolate and characterize frozen intermediates in which one or more of the sites is blocked in the ligand bound state, the others being free to react [1]. It has recently become possible to isolate, in the case of one of the hemoglobin components of trout blood (Hb trout I), all five species which should theoretically exist upon partial oxidation of the tetrameric molecule [2]. This results from the fact that in trout hemoglobins the fast dimer exchange which occurs in the ligand bound form of human hemoglobin [3] is greatly reduced. This note reports, as a preliminary step, a study of the reaction with 02 and CO of the partially oxidized forms of both Hb trout I and Hb trout IV, without, for the time being, attempting to isolate the various intermediates. In these mixtures all the possible forms will be present in proportions determined by the percentage of oxidation. In the analysis given below it is assumed, on the basis of their method o f
172 preparation, that these proportions correspond to a random oxidation, as if the sites were identical and independent. This seems reasonable since the mixtures were prepared starting from the oxygenated derivative at alkaline pH. MATERIALS AND MEYHODS Preparation of trout hemoglobin and purification of the different components was carried out as previously described [4]. The various mixtures were prepared by treating the oxygenated derivative with the desired stoichiometric amount (in terms of heme concentration) of K3[Fe(CN)6] at alkaline pH values ( ~ 8). The amount of Met-Hb in each solution was determined by the ratio of the absorption at 405 nm to that at 419 nm. In flash experiments an estimate of the percent oxidation in each sample was obtained by comparing the total absorption change before and after addition of dithionite to each individual sample. Oxygen dissociation curves were determined spectrophotometrically [5], generally at a protein concentration of 3-5 mg/ml. Flash photolysis measurements were performed with an apparatus similar to that previously described [6]. Spectrophotometric measurements were made with a Beckman D B G T spectrophotometer. Carbon monoxide was obtained from S.I.O. (Rome). All other reagents were analytical grade products. RESULTS
Oxygen equilibrium It may be recalled that the functional behavior of H b trout I is characterized by the presence of positive interactions between the oxygen binding sites (n ~ 2.3) as well as the absence of any significant Bohr effect. In contrast, strong heterotropic interactions, involving both protons and organic phosphates, are present in Hb trout IV: at acid p H (6-6.5) the protein is only partially saturated even under O2 at I a t m and the cooperativity, evident at more alkaline pH values, is completely absent (n ~ 1) (Root effect). o/ The oxygen dissociation curves of mixtures of ferric (from 25 ~ to 75/o) and ferrous form of Hb trout 1 in presence of azide in saturating amount are reported in Fig. 1. The results show a marked decrease of the homotropic interactions in going from 0 ~ oxidation (where n = 2.3) to 75 ~ oxidation (where n = 1.2). Such a drop in cooperativity parallels a great increase ( ~ 7 times) in oxygen affinity. These changes support a progressive shift of the conformational equilibrium towards the highly reactive (R) form of the molecule; in other words, by increasing the number of the oxidized subunit~, we induce a progressive stabilization of a "high affinity" conformational state of the molecule. The results obtained, under the ~,ame experimental conditions for Hb trout IV are summarized in Fig. 2 where the pH dependence of log p½ and n½ is reported. The general effect of increasing oxidation is similar, at each p H value, to that observed for Hb trout I. However, it should be remarked that the decrease in cooperativity, brought about by the increase of oxidation, is more dramatic at low levels of oxidation.
173 1.0
::'.5
r( ~,0
tlolO~
'
0.5
i 0.5
0
0.5
to
log po a
1.5
Fig. 1. Oxygen equilibrium curves of Hb trout I in presence of 2 mM azide as a function of percent oxidation : ( O ) 0 ~ ; (A) 25 ~ ; (~I
