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.     

Direct measurement of carbon monoxide bound to different subunits of hemoglobin A in solution and in red cells by infrared spectroscopy

Infrared spectra for carbon monoxide bound to alpha and beta subunits of human hemoglobin A have subunit differences near 1950 cm-1 and indicate that 92% of the alpha subunits exist in one conformer and 5% in a second conformer under conditions where 99% of the beta subunit is in only one conformation. The sum of the separated subunit spectra is equivalent to the alpha 2 beta 2 tetramer spectrum. CO infrared spectra indicate that CO displaces O2 from HbO2 in red cells or in solution preferentially at the beta subunits. The measurement of C-O stretch bands provides a direct method for characterization of ligand binding sites within intact cells.     

Crystal structure of horse carbonmonoxyhemoglobin-bezafibrate complex at 1.55-A resolution. A novel allosteric binding site in R-state hemoglobin

Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug. The high resolution crystal structure of horse carbonmonoxyhemoglobin in complex with bezafibrate reveals that the bezafibrate molecule lies near the surface of the E-helix of each alpha subunit and the complex maintains the quaternary structure of the R-state. Binding is caused by the close fit of bezafibrate into the binding pocket, which is composed of some hydrophobic residues and the heme edge, suggesting the importance of hydrophobic interactions. Upon binding of bezafibrate, the distance between Fe and the N epsilon(2) of distal His E7(alpha 58) is shortened by 0.22 A in the alpha subunit, whereas no significant structural changes are transmitted to the beta subunit. Oxygen equilibrium studies of R-state-locked hemoglobin with bezafibrate in a wet porous sol-gel indicate that bezafibrate selectively lowers the oxygen affinity of one type of subunit within the R-state, consistent with the structural data. These results disclose a new allosteric mechanism of bezafibrate and offer the first demonstration of how the allosteric effector interacts with R-state hemoglobin.     

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.     

The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10-->Tyr and HisE7-->Gln on alpha and beta chains probed by infrared spectroscopy

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.     

Molecular dynamics of human methemoglobin: the transmission of conformational information between subunits in an alpha beta dimer

Spectroscopic studies indicate an interaction of the distal histidine with the heme iron as well as the transmission of distal heme perturbations across the alpha1beta1 interface. Molecular dynamics simulations have been used to explain the molecular basis for these processes. Using a human methemoglobin alpha beta dimer, it has been shown that at 235 K after 61 ps, a rearrangement occurs in the alpha-chain corresponding to the formation of a bond with the distal histidine. This transition does not take place in the beta-chain during a 100-ps simulation and is reversed at 300 K. The absence of the distal histidine transition in the isolated chains and with the interface frozen indicate the involvement of the alphabeta interface. A detailed analysis of the simulation has been performed in terms of RMS fluctuations, domain cross-correlation maps, the disruption of helix hydrogen bonds, as well changes in electrostatic interactions and dihedral angles. This analysis shows that the rearrangements in the alpha-chain necessary to bring the histidine closer to the iron involve alterations primarily in the CD loop and at the interface. Communication to the beta-chain distal pocket is propagated by increased interactions of the alpha-chain B helix with the beta-chain G-GH-H segment and the flexibility in the EF loop. The G helices shown to be involved in propagation of perturbation across the alpha1beta1 interface extend into the alpha1beta2 interfaces, providing a mechansim whereby distal interactions can modulate the T<==>R transition in hemoglobin.     

Quantitative determination of carbamino adducts of alpha and beta chains in human adult hemoglobin in presence and absence of carbon monoxide and 2,3-diphosphoglycerate.

The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.     

Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.     

Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state

When methionine beta(55)D6 in human hemoglobin is oxidized to its sulfoxide derivative, the modified protein appears to maintain most of the chemical and structural properties typical of the native protein. On the contrary, the functional behavior is drastically changed, being characterized (like that of the isolated chains) by high oxygen affinity (p50 = 0.47 torr in 0.1 M Tris (pH 7.3) + 0.1 M NaCl at 25 degrees C), absence of cooperativity (n = 1), and lack of Bohr effect. The complete destabilization of the T-state as a result of this modification is related to a perturbation of the alpha 1 beta 1 subunit interface, which in native hemoglobin remains static during the quaternary ligand-linked transition. Results also suggest that methionyl sulfoxide-containing hemoglobin, obtained under different conditions, assumes functionally different R-states, none of which is exactly comparable with that typical of the native protein.     

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.     

Nanosecond optical spectra of iron-cobalt hybrid hemoglobins: geminate recombination, conformational changes, and intersubunit communication

Hybrid hemoglobins were prepared in which cobalt was substituted for the heme iron in either the alpha or beta subunits. Transient optical absorption spectra were measured at room temperature for these hybrids at time intervals between 0 and 50 ms following photodissociation of the carbon monoxide complex with 10-ns laser pulses. The cobalt porphyrins do not bind carbon monoxide, making it possible to investigate the time-resolved response of the cobalt-containing subunits to photodissociation of carbon monoxide in the iron-containing subunits. At the same time the response of the iron-containing subunits to the photolysis event can be studied, permitting an independent determination of the kinetics of ligand rebinding and conformational changes in the alpha and beta subunits of an intact tetramer. The data were analyzed by using singular-value decomposition to obtain the kinetic progress curve for ligand rebinding, the deoxyheme and cobalt porphyrin spectral changes, and the time course of these spectral changes. The geminate rebinding kinetics following photodissociation of alpha(Co)2 beta(Fe-CO)2 were very similar to those found unsubstituted hemoglobin, alpha(Fe-CO)2 beta(Fe-CO)2, indicating equivalence of the geminate kinetics for alpha and beta subunits within the R-state tetramer. The results for alpha(Fe-CO)2 beta(Co)2 were consistent with this conclusion, even though the analysis was complicated by the presence of comparable populations of R- and T-state species. Comparison of the deoxyheme spectral changes and relaxation times among the three molecules indicated that both alpha and beta subunits contribute to the deoxyheme spectral changes that signal tertiary and quaternary conformational changes in the unsubstituted tetramer. The response of the cobalt porphyrins to photodissociation was similar in the two hybrids. No structural changes were detected in the cobalt-containing subunits until the second tertiary conformational change in the iron-containing subunits observed at 1-2 microseconds. Much larger structural changes, as judged by the amplitude of the spectral changes, occurred in the cobalt-containing subunits concomitant with the R----T quaternary change at about 20 microseconds.     

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.     

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.     

Mutational effects at the subunit interfaces of human hemoglobin: evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the alpha 1 beta 2 interface

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of beta Y145, the residues with the most significant effects on these kinetics are found in the hinge region of the alpha 1 beta 2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both alpha or both beta subunits, were prepared for beta W37E, beta W37A, alpha Y140G, and alpha Y140A, hinge region variants causing large kinetic changes, and for beta Y145G. Such hybrids permit measurements of the kinetics of CO binding to only the heme-containing alpha or beta subunits within the unliganded hemoglobin tetramer. Mutations at beta 37 and alpha 140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of beta Y145 has a large effect on the beta subunits in the deoxygenated T state, but very little effect on the alpha subunits. Oxygen equilibria measurements on the crystalline T state of beta W37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the alpha 1 beta 2 interface.     

Unusual CO bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.     

Cooperative ligand binding of crosslinked hemoglobins at very high temperatures

Human hemoglobin was reacted with the bifunctional reagent bis(3,5-dibromosalicyl) fumarate to yield a derivative (Hb alpha alpha) crosslinked between the two alpha-chains; when the reaction was carried out with HbA already crosslinked between the two beta-chains by 2-nor-2-formylpyridoxal 5'-phosphate, a doubly crosslinked derivative (Hb alpha alpha beta beta) was obtained. We have observed that both modified hemoglobins are extremely stable up to temperatures of at least 85 degrees C. The carbon monoxide binding kinetics of both crosslinked hemoglobins, studied at temperatures between 15 and 85 degrees C, by means of stopped flow and flash photolysis techniques, show that the ligand-linked allosteric transition is maintained even at the highest temperatures. These results are also relevant to the mechanism of thermal unfolding of human hemoglobin, since they show that dissociation into alpha beta dimers (and exposure of the relatively hydrophobic dimer-dimer interfaces) is an obligatory step in the irreversible denaturation of deoxy and carbon monoxy hemoglobin.     

The intermediate compounds between human hemoglobin and carbon monoxide at equilibrium and during approach to equilibrium

The procedure of Perrella et al. (Perrella, M., Benazzi, L., Cremonesi, L., Vesely, S., Viggiano, G., and Rossi-Bernardi, L. (1983) J. Biol. Chem. 258, 4511-4517) for trapping the intermediate compounds between human hemoglobin and carbon monoxide was validated by quantitatively determining during the approach to equilibrium all the species present in a solution containing large amounts of intermediates. An accurate estimate of the intermediate compounds at 50% carbon monoxide saturation in 0.1 M KCl, pH 7, at 22 degrees C, allowed the calculation, according to Adair's scheme, of the four equilibrium constants. At 50% ligand saturation, the pool of intermediate species was about 12% of the total. A slightly greater concentration of tri-liganded than mono-liganded species was found. Carbon monoxide bound to beta chains in slightly greater excess with respect to alpha chains in both the mono- and tri-liganded species. The symmetrical bi-liganded intermediates, alpha 2 beta CO2 and alpha 2CO beta 2, were absent. The nature of the bi-liganded intermediate found to be present in detectable amounts by our technique has yet to be clarified: it could be either the asymmetrical species (alpha beta) (alpha CO beta CO) and (alpha beta CO) (alpha CO beta) or both of them. Such a finding on the functional heterogeneity among the four possible bi-liganded intermediates is consistent with hypotheses of the existence of more than two quaternary structures in the course of ligand binding to hemoglobin.     

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.     

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.