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.     

The influence of organic phosphates on the Bohr effect of human hemoglobin valency hybrids.

The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species.     

Structural nonequivalence of the alpha- and beta- heme-pockets in human methemoglobin. A proton magnetic relaxation study in solution.

Solvent-proton longitudinal magnetic relaxation rates as dependent on temperature were measured for human (H)/canine (C) valency hybrids of the type {α^H(III)β^C(II)}₂ and {α^C(II)β^H(III)}₂. The two metheme irons in the human methemoglobin chains induce quite different proton magnetic relaxation (pmr) rates reflecting a tighter β-heme-pocket compared to the α subunit. Both heme-pockets appear to be loosened in the presence of inositol hexaphosphate (IHP) although this allosteric effector binds only to the β chains, the binding assumed to be the same for canine as for human hemoglobin. The subunit nonequivalence is retained also in the T-quaternary state induced by IHP. In the species hybrids the pmr rates due to the metheme iron are sensitive to the valency (ligand) state, which was either CO or H₂O in the partner half of the hybrid. All results show very clearly the interrelationship of the tertiary (protomer) structure with the quaternary (oligomer) structure in hemoglobin.     

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

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.     

Spectral, conformational and chemical properties of opossum methemoglobin

Opossum methemoglobin differs from methemoglobin A in spectral, spin state, conformational and chemical properties. The primary structural alterations in opossum hemoglobin, including the critical substitution at alpha 58 (E7) His leads to Gln result in the following properties. (a) Major contribution of the spectral transitions due to inositol hexakisphosphate binding arises from the alpha chains. (b) The aquomet to hydroxymet (high-spin to low-spin) transition as a function of pH is slightly retarded resulting in considerable high spin at alkaline pH. (c) The tertiary conformation (t) around the beta hemes, upon transition to a T quaternary state, differs from the known hemoglobin t tertiary structure. (d) Both alpha and beta hemes are susceptible to rapid reduction by ascorbic acid (the reduction rate being tenfold faster than that of methemoglobin A). These properties suggest that the heme environments in both the alpha and beta subunits of opossum hemoglobin are different from those of human hemoglobin A.     

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.     

Assignment of proximal histidyl imidazole exchangeable proton NMR resonances to individual subunits in hemoglobins A,Boston, Iwate and Milwaukee

The proton nmr spectra of the synthetic valency hybrids, α₂(β⁺CN)₂, (α⁺CN)₂β₂ of hemoglobin A and the natural valency hybrids of the mutant hemoglobins Boston, Iwate and Milwaukee have led to the unambiguous assignment of the two proximal histidyl imidazole exchangeable proton signals at 64 and 76 ppm to individual α and β subunits, respectively. New single non-exchangeable proton resonances detected in the extreme downfield region of the spectra of Hbs Boston and Iwate are tentatively assigned to the coordinated tyrosine of the mutated α chains.     

Direct spectrophotometric analysis of hemoglobin in isoelectric focusing tube gels

Methods are described for the direct spectrophotometric analysis of human oxyhemoglobin, deoxyhemoglobin, and methemoglobin focused in polyacrylamide tube gels. Visible absorption spectra (350-650 nm) of electrofocused hemoglobin bands were recorded using a diode array rapid-scan spectrophotometer. Direct optical sampling of gels allowed the identification of focused hemoglobin valency hybrids which contain two oxidized monomers per tetramer.     

Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

Heme reactivity of hemoglobins. Azide and fluoride binding equilibria of free and mercuriated ferri-gamma chains.

The free gamma chains, isolated from human foetal hemoglobin, are stable when oxidized and thus suitable for ligand binding and subunit equilibrium studies. The metaquo-ferri chains, with cysteine-F9 in the free state II ag gamma SH) possess several properties which are different from those of their p-mercuribenzoate derivative (III aq gammaSHgR); these are: stronger binding of a high-field ligand (N3- minus), altered spin equilibrium and an altered subunit equilibrium. A quantitative assessment of the free energy changes associated with all individual steps involved in changing the metaquo chains to their azide derivatives has been made. The results show that the higher apparent reactivity of III ag gammaSH (compared to IIIaq gammaSHgR) for the azide ion is not solely due to compensatory effects arising from differences of subunit dissociation or of spin equilibrium: other process(es) occurring in the ligand binding site have to be considered.     

Molecular oxygen binding with α and β subunits within the R quaternary state of human hemoglobin in solutions and porous sol–gel matrices

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O₂) rebinding to α and β subunits within oxygenated human adult hemoglobin in solutions and porous wet sol–gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O₂ binding to the α and β hemes within the R quaternary structure is studied. The individual kinetic properties of the α and β subunits within the hemoglobin encapsulated in sol–gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol–gel-free mediums. Interestingly, the α and β subunits within the encapsulated hemoglobin possess the O₂ rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O₂ rebinding to the α and β subunits within the oxygenated R-state hemoglobin in both solutions and wet sol–gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O₂ rebinding properties, are discussed.     

The solubility of hemoglobin beta 4 S, the mutant subunits of sickle cell hemoglobin

The single subunit hemoglobin β₄^S was found to have a solubility comparable to that of oxygenated rather than deoxygenated Hb S, although it contains twice as many mutant chains as the parent hemoglobin and probably has a quarternary structure similar to deoxyhemoglobin A. This finding supports the assumption that receptor sites in the α chains of sickle hemoglobin are essential for sickling.     

Analysis of the interaction of organic phosphates with hemoglobin.

The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified.     

Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.     

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.     

State of methaemoglobin in normal adults and in patients with hereditary methaemoglobinaemia.

The predominance of valency hybrids over fully oxidized methaemoglobin in the haemolysate from a methaemoglobinaemia patient can be explained by random oxidation of the haems, followed by re-assortment and co-migration of equally charged alpha beta-dimers in the electric field of the isoelectric-focusing gel.     

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.     

Temperature-jump studies on formate binding to methemoglobin and metmyoglobin.

The binding of formate ion to sperm whale metmyoglobin after a temperature-jump is monophasic and not affected by organic phosphate; the Hill coefficient obtained from equilibrium measurements is unity, and there is internal consistency between equilibrium and kinetic results. Formate binding to stripped human methemoglobin, on the other hand, is biphasic. The two relaxation phases can be attributed, on the basis of their equal relaxation amplitudes, to the different kinetic properties of both types of chains. Equilibrium measurements yield a single binding constant. Thus, formate belongs to the class of high-spin ligands which show no binding specificity but strong kinetic heterogeneity for α- and β-chains. There is, however, a lack of consistency between equilibrium and kinetic results, indicating that a reaction scheme which considers only ligand binding to α- and β-chains appears not to be fully adequate. Organic phosphates exert a drastic influence on the kinetics but not on the thermodynamics of ligand binding. In the presence of inositol hexaphosphate the relaxation spectrum is characterized by more than two relaxation processes: A very fast phase—about an order of magnitude faster than the fast process in stripped methemoglobin—appears with high amplitude. The slow relaxation process, however, is only slightly affected. The binding constant of formate obtained from equilibrium measurements is only little changed and the Hill coefficient is 0.97 both in the presence and absence of the phosphate. The phosphate-induced kinetic changes indicate that functionally significant structural changes are introduced in the tertiary structure of one type of chains, presumably the β-chains, to which inositol hexaphosphate is bound.