Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.     

Properties of chemically modified Ni(II)-Fe(II) hybrid hemoglobins. Ni(II) protoporphyrin IX as a model for a permanent deoxy-heme

Chemical modifications, NES-Cys(beta 93), des-Arg(alpha 141), and both modifications on the same molecule, were made to Ni-Fe hybrid hemoglobins, and their effect on individual subunits was investigated by measuring oxygen equilibrium curves, the Fe(II)-N epsilon (His F8) stretching Raman lines, and light-absorption spectra. The oxygen equilibrium properties indicated that modified Ni-Fe hybrid hemoglobins remain good models for the corresponding deoxy ferrous hemoglobins, although K1, the dissociation equilibrium constant for the first oxygen to bind to hemoglobin, was decreased by the chemical modifications. Resonance Raman spectra of deoxy alpha 2 (Fe) beta 2 (Ni) and light-absorption spectra of deoxy alpha 2 (Ni) beta 2 (Fe), revealed that the state of alpha hemes in both hybrid hemoglobins underwent a transition from a deoxy-like state to an oxy-like state caused by these chemical modifications when K1 was about 3 mm Hg (1 mm Hg approximately 133.3 Pa). On the other hand, the state of beta hemes in hybrid hemoglobins was little affected, when K1 was larger than 1 mm Hg. Modified alpha 2 (Fe) beta 2 (Ni) gave a Hill coefficient greater than unity with a maximum of 1.4 when K1 was about 4 mm Hg. The two-state model predicts that the K1 value at the maximum Hill coefficient should be much larger than this value. For oxygen binding to unmodified alpha 2 (Ni) beta 2 (Fe), oxygen equilibrium data suggested no structural change, while the spectral data showed a structural change around Ni(II) protoporphyrin IX in the alpha subunits. A similar situation was encountered with modified alpha 2 (Ni) beta 2 (Fe), although K1 was decreased as a result of the structural changes induced by the modifications.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.     

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.     

Nonequivalence in the electronic structure of the prosthetic groups between two alpha-subunits within deoxycobalthemoglobin as determined by single-crystal EPR spectroscopy

An artificial hybrid hemoglobin, alpha(Co)2 beta(Fe)2, the alpha- and beta-subunits of which contain cobaltous and ferrous protoporphyrins IX, respectively, and its complementary hybrid, alpha(Fe)2 beta(Co)2, were prepared from human hemoglobin, crystallized in the deoxy state, and examined by electron paramagnetic resonance (EPR) spectroscopy. The orientations of the porphyrin normals in these deoxy Fe-Co hybrid hemoglobins in terms of the g parallel signals, were closely coincident with those of the heme normals of deoxyhemoglobin determined by x-ray crystallography. Two sets of axially symmetric EPR signals were found in the alpha(Co)-subunits, whereas only one set was observed in the beta(Co)-subunits. Nonequivalence in the electronic structures of the prosthetic groups between the two alpha(Co)-subunits, designated alpha I and alpha II, within deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin was correlated to these two distinct EPR signals. The interaction between the epsilon-nitrogen of the imidazole ring of the proximal histidine and the cobaltous ion in deoxy-alpha I(Co)-subunit is different from that in the deoxy-alpha II(Co)-subunit. The absence of a strict molecular dyad axis in the deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin suggests that the affinity state of the alpha(Co)-subunits may be partially switched to the R-state having a higher affinity for oxygen. Upon partial ligation of carbon monoxide to the beta(Fe)-subunits, the line width of the g parallel and perpendicular signals of the alpha II(Co)-subunit was found to become somewhat narrower without disruption of the crystal structure. This suggests that there may be very close contacts between the alpha- and beta-subunits of different hemoglobin molecules which appear to be responsible for stabilizing the deoxy crystal structure after partial ligation in the crystalline state.     

Polymerization and solubility of Ni(II)-Fe(II) hybrid Hb S

Polymerization of half-liganded Hb S was investigated using Ni(II)-Fe(II) hybrid Hb S, in which heme in either alpha or beta s subunits is replaced by Ni (II) protoporphyrin IX. Studies on the polymerization of these hybrid hemoglobins were carried out under aerobic conditions. Both alpha 2 (Ni) beta 2s (Fe-CO) and alpha 2 (Fe-CO) beta 2s (Ni) polymerized with a distinct delay time as do native deoxy-Hb S and Ni(II) Hb S. However, the critical concentration for polymerization of half-liganded Hb S, alpha 2 (Ni) beta 2s (Fe-CO) and alpha 2 (Fe-CO) beta 2s (Ni), was 4- and 8-times higher, respectively, than that of Ni(II)-Hb S. Kinetics of polymerization of both deoxygenated hybrid hemoglobins with CO completely removed were the same, although the critical concentrations for polymerization were intermediate between those for deoxy-Hb S and Ni(II)-Hb S. These results suggest that the small tertiary conformational change associated with the doubly liganded state may be much less favorable to polymerization than the completely unliganded state of Hb S. The conformational change depends on whether alpha or beta chain is liganded. The ease of polymerization and low solubility of sickle hemoglobin is dependent not only on quaternary, but on tertiary structural changes, as well as on the substitution of Val for Glu at the beta 6 position.     

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.     

Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)-iron(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, alpha(Fe)2 beta(Ni)2 and alpha(Ni)2 beta(Fe)2 have been characterized by proton nuclear magnetic resonance with Ni(II) protoporphyrin IX (Ni-PP) incorporated in apoprotein, which serves as a permanent deoxyheme. alpha(Fe)2 beta(Ni)2, alpha(Ni)2 beta(Fe)2, and NiHb commonly show exchangeable proton resonances at 11 and 14 ppm, due to hydrogen-bonded protons in a deoxy-like structure. Upon binding of carbon monoxide (CO) to alpha(Fe)2 beta(Ni)2, these resonances disappear at pH 6.5 to pH 8.5. On the other hand, the complementary hybrid alpha(Ni)2 beta(Fe-CO)2 showed the 11 and 14 ppm resonances at low pH. Upon raising pH, the intensities of both resonances are reduced, although these changes are not synchronized. Electronic absorption spectra and hyperfine-shifted proton resonances indicate that the ligation of CO in the beta(Fe) subunits induced changes in the coordination and spin states of Ni-PP in the alpha subunits. In a deoxy-like structure, the coordination of Ni-PP in the alpha subunits is predominantly in a low-spin (S = 0) four-coordination state, whereas in an oxy-like structure the contribution of a high-spin (S = 1) five-coordination state markedly increased. Ni-PP in the beta subunits always takes a high-spin five-coordination state regardless of solution conditions and the state of ligation in the partner alpha(Fe) subunits. In the beta(Ni) subunits, a significant downfield shift of the proximal histidyl N delta H resonance and a change in the absorption spectrum of Ni-PP were detected, upon changing the quaternary structure of the hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordination geometry of Cu-porphyrin in Cu(II)-Fe(II) hybrid hemoglobins studied by Q-band EPR and resonance Raman spectroscopies

Cu(II)-Fe(II) hybrid hemoglobins were investigated by UV-vis, Q-band (35 GHz) EPR and resonance Raman spectroscopies. EPR results indicated that Cu-porphyrin in alpha-subunit within hybrid hemoglobin had either 5- or 4-coordination geometry depending on the pH conditions, while Cu-porphyrin in beta-subunit had only 5-coordination geometry at high and low pH values. These results were consistent with UV-vis absorption results. A new resonance Raman band appeared around 190 cm(-1), which was present whenever 5-coordinated Cu-porphyrin existed in Cu(II)-Fe(II) hybrid hemoglobins irrespective of the coordination number in Fe(II) subunit. This Raman band might be assigned to Cu-N(epsilon) (His) stretching mode. These results are direct demonstration of the existence of coordination changes of Cu-porphyrin in alpha-subunit within hybrid hemoglobin by shifting the molecular conformation from fully unliganded state to intermediately liganded state.     

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

On the oxygen-linked anion-binding sites in human hemoglobin. Functional properties of human hemoglobin reacted with 4-isothiocyanatobenzenesulphonic acid and its hybrids

Human hemoglobin, reacted at the four amino termini with 4-isothiocyanatobenzenesulphonic acid (Hb-ICBS), was separated into its constituent chains. Recombination of the ICBS-reacted chains with the unmodified mate chains produced the hybrid tetramers modified at either the beta or the alpha chains: alpha 2 beta 2ICBS and alpha 2ICBS beta 2. All of the modified tetramers show a reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and alpha 2 beta 2ICBS is unaffected by 2,3-bisphosphoglycerate while the oxygen affinity of alpha 2ICBS beta 2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and alpha 2ICBS beta 2 is independent of chloride concentration, while the alpha 2 beta 2ICBS hybrid shows a reduced response to this anion. The tetramers alpha 2ICBS beta 2 and alpha 2ICBS beta 2ICBS show a decreased alkaline Bohr effect, which can be rationalized as being due to disruption of the oxygen-linked chloride-binding sites; in the case of alpha 2 beta 2ICBS the Bohr effect is instead (partially) maintained. The functional properties of artificial tetramers have been studied also from a kinetic point of view by CO combination and the results obtained compare satisfactorily with equilibrium data. The possibility of obtaining selectively modified hemoglobins promises to provide further insight into the properties of the oxygen-linked anion-binding sites in hemoglobin.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Extended X-ray absorption fine structure studies of Zn2Fe2 hybrid hemoglobins: absence of heme bond length changes in half-ligated species

Metal hybrid hemoglobins, in which Zn(II) replaces Fe(II), have been structurally characterized by extended X-ray absorption structure (EXAFS) studies. Since Zn and Fe have very different K absorption edge energies, the structures of the ligated (Fe) and unligated (Zn) sites could be examined independently within a single molecule that mimics an intermediate ligation state. The observed EXAFS spectra and associated structural parameters are compared among the ligand free (alpha Zn)2(beta Zn)2, half-ligated (alpha FeCO)2(beta Zn)2 and (alpha Zn)2(beta FeCO)2, and fully ligated (alpha FeCO)2(beta FeCO)2 systems.     

Resonance Raman spectroscopic studies in copper reconstituted and hybrid hemoglobins: probe into subunit heterogeneity

Copper reconstituted hemoglobin (CuHb), copper containing T-state hybrid hemoglobins like alpha2(Ni)beta2(Cu), and alpha2(Cu)beta2(Ni), and intermediate R-state hybrids like alpha2(CO-Fe)beta2(Cu) and alpha2(Cu)beta2(Fe-CO) are studied using resonance Raman (RR) spectroscopy at two different excitation wavelengths. The high frequency RR region in CuHb indicates the presence of both 4- and 5-coordinate forms of Cu(II). In hybrid Hbs, the presence of two distinct metal ion environments within one particular subunit is evident. This is also consistent with previous findings using EPR spectroscopy and sulfydryl reactivity studies on these hybrid Hbs. The low frequency RR region on these copper derivatives of HbA further suggests the existence of two different heme moieties within the subunit.     

Evidence for significant quantities of creatine kinase MM isoenzyme in human brain

Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.     

The structure of hemoglobin Creteil (beta 89 Ser replaced by Asn) is similar to that of abnormal human hemoglobins having sequence changes at Tyr 145 beta

In normal deoxyhemoglobin A, the beta chain COOH-terminal peptide adopts a well ordered structure which is needed for the full expression of allosteric action. Our crystallographic studies of deoxyhemoglobin Creteil (beta 89 Ser replaced by Asn), a variant hemoglobin characterized by high oxygen affinity and a very low level of allosteric function, show that replacement of Ser 89 beta by asparagine causes severe disordering of the beta chain COOH-terminal tetrapeptide. This results, as shown by our spectroscopic studies, in the destabilization of the quaternary structure of deoxyhemoglobin Creteil. We find, furthermore, that the changes in tertiary structure observed in deoxyhemoglobin Creteil are common to other variant hemoglobins having similar functional abnormalities but very different changes in primary structure. In particular, direct comparison of the difference electron density map of deoxyhemoglobin Creteil with that of deoxyhemoglobin Nancy (beta 145 Tyr replaced by Asp) suggests that these two abnormal hemoglobins may have the same mechanism of dysfunction despite the very different nature of their respective sequence changes.     

Effect of removal of a salt-bridge on the oxygen binding properties and the electronic structure of heme in cobalt-iron hybrid hemoglobin.

In order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined. The EPR spectra of deoxy alpha(Co)2 beta(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g = 2.39 and the other at g = 2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure. The cooperativity of oxygen binding of alpha(Co)2 beta(Fe)2 exhibited a maximum at g = 2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg alpha(Co)2 beta(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg alpha(Co)2 beta(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g = 2.39 was not restored upon conversion to the T structure by addition of IHP. It is therefore concluded that the EPR spectrum of the deoxy alpha(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.     

Interaction of copper(II) with hemoglobins in the unliganded conformation

The interaction of exogenous Cu(II) with stable T-state Ni(II)- and Cu(II)-reconstituted hemoglobins has been studied. The relative binding affinities for the two human hemoglobin Cu(II) binding sites are found to be reversed in these hemoglobins relative to native iron(II) hemoglobin A. Nickel hemoglobin, modified by N-ethylmaleimide (NEM), iodoacetamide, and carboxypeptidase A, is used to establish that the observed differences can be attributed to the protein quaternary conformation and not to the metal substitution. Magnetic interactions between the Cu(II) responsible for oxidation and the metal-heme center suggest that the Cu(II) is closer to the heme in T-state hemoglobin than R-state hemoglobin. This finding suggests a pathway for T-state heme oxidation which does not require the beta-93 sulfhydryl group, consistent with rapid Cu(II) oxidation for NEM-reacted deoxyhemoglobin.