Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

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.     

Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.     

Resonance raman studies of heme structural differences in subunits of deoxy hemoglobin

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d(4)), and the hybrids bearing the deuterated heme in only one type of subunit, which are [alpha(d4)beta(h4)](2) and [alpha(h4)beta(d4)](2). Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the gamma(7) mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.     

Interaction of fully liganded valency hybrid hemoglobin with inositol hexaphosphate. Implication of the IHP-induced T state of human adult methemoglobin in the low-spin state

To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Kinetic studies on methemoglobin reduction by human red cell NADH cytochrome b5 reductase.

The intermediate hemoglobins which were produced by the partial reduction of methemoglobin with human red cell NADH cytochrome b5 reductase were fractionated by the preparative isoelectric focusing. These were found to be composed of alpha3+beta2+ and alpha2+beta3+ valency hybrids by the studies of absorption spectra and inositol hexaphosphate-induced difference spectra. Furthermore, the changes in these intermediate hemoglobins during reduction of methemoglobin by the enzyme were studied in the presence or absence of inositol hexaphosphate using the isoelectric focusing fractionation on Ampholine plate gel...     

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.     

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(Fe); M=Mg(II), Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha2(Fe)beta2(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effectors, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution.We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins.     

Transient kinetics of oxygen dissociation from ferrous subunits of iron-cobalt hybrid hemoglobins. The principal reaction controlling the co-operativity

The oxygen dissociation constants from Fe subunits in the half-ligated intermediate states of Fe-Co hybrid hemoglobins, alpha(Fe-O2)2 beta(Co)2 and alpha(Co)2 beta(Fe-O2)2, have been determined as functions of pH, temperature and inositol hexaphosphate. The oxygen dissociation rates from alpha(Fe-O2)2 beta(Co)2 are estimated to be more than 1300 s-1 for the deoxy quaternary state (T-state) and less than 3 s-1 for the oxy quaternary state (R-state) at 15 degrees C in 50 mM-Tris or Bis-Tris buffer containing 0.1 M-Cl-, while those of alpha(Co)2 beta(Fe-O2)2 are more than 180 s-1 and less than 5 s-1 for the T and R-states, respectively. The pH dependence of the oxygen dissociation rate from Fe subunits is large enough to be accounted for by the R-T transition, and implies that those half-ligated intermediate hybrids mainly exist in the R-state at pH 8.8, and in the T-state at pH 6.6, while other studies indicated that the half-ligated hybrids are essentially in the R-state at pH 7. Large activation energies of the oxygen dissociation process of 19 to 31 kcal/mol determined from the temperature dependence suggest that the process is entropy-driven.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: NMR studies of their tertiary and quaternary structures

Diruthenium-substituted Ru-Fe hybrid hemoglobins (Hb) were synthesized by heme substitution from protoheme to ruthenium (II) carbonyldeuteroporphyrin in the alpha or beta subunits. As the carbon monoxide coordinated to ruthenium (II) is not released under physiological conditions, deoxygenated Ru-Fe hybrid derivatives [alpha(Fe)2 beta(Ru-CO)2 and alpha(Ru-CO)2 beta(Fe)2] can serve as models for half-liganded Hbs. On the basis of proton NMR spectra of hyperfine-shifted proton resonances, these Ru-Fe hybrid Hbs have only small structural changes in the heme environment of the partner subunits at low pH. The proton NMR spectra of the intersubunit hydrogen-bonded protons also showed that the quaternary structures of the two complementary hybrids both remain in the "T-like state" at low pH, suggesting that the T to R structural conversion is induced by ligation of the third ligand molecule. Marked conformational changes in the heme vicinity are observed at high pH only for alpha(Ru-CO)2 beta(Fe)2, and its quaternary structure is converted into the "R state"; the alpha(Fe)2 beta(Ru-CO)2 hybrid does not undergo this change. This implies that the free-energy difference between the two quaternary states is smaller in the alpha-liganded hybrid than in the beta-liganded one.     

Electron transfer kinetics between hemoglobin subunits

The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, alpha or beta, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25 degrees C, the Hb solution (0.1 mm heme) approaches a form with about 60% of beta chains reduced, indicating a faster transfer rate in the direction alpha to beta. There was no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized cross-linked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte.     

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.     

A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions.

An allosteric model proposed previously for structure-function relations in hemoglobin is applied to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model.