Role of the tertiary and quaternary structure in the formation of bis-histidyl adducts in cold-adapted hemoglobins

All tetrameric hemoglobins from Antarctic fish, including that from Trematomus bernacchii, HbTb form in the ferric state, promptly and distinctively from all the other tetrameric hemoglobins, a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the β subunits. The role of the tertiary and quaternary structure in the hemichrome formation is unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the β-chain of HbTb (β-HbTb). Similarly to the human β-chains, β-HbTb self-assembles to form the homotetramer β(4)-HbTb; however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (β(4)-HbA). A molecular dynamics study of the isolated β subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of β-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. On the bases of these experimental and computational results on the isolated chain, the influence of the quaternary structure on the stability of the endogenous ferrous and ferric hexa-coordination is also discussed.     

Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

The bis-histidyl complex in hemoproteins: A detailed conformational analysis of database protein structures and the case of Antarctic fish hemoglobins

The properties of hemoproteins strictly depend on the type and orientation of axial ligands. Here, the orientations of axially coordinated His in bis-His complexes and the heme geometry in protein data bank have been analyzed. The effect of the bis-histidyl formation on the heme cavity of Antarctic fish hemoglobins has been also evaluated. The results show that protein matrix exerts a major effect on the conformation of axially ligated histidines: the imidazoles in bis-His complexes occupy a preferred relative orientation in globins and in model systems, whereas they adopt a variety of relative orientations in other hemoproteins. The bis-histidyl adducts affect the heme geometry inducing larger distortions from planarity with respect to other ligands. These deviations are larger in bis-His multiheme cytochromes than in globins. In Antarctic fish hemoglobins the bis-histidyl adduct adopts preferentially a distorted coordination and the formation of the bis-His complex induces a slight but significant modification in the shape, area and volume of the heme cavity.     

Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.     

Crystal structures of Parasponia and Trema hemoglobins: differential heme coordination is linked to quaternary structure

Hemoglobins from the plants Parasponia andersonii (ParaHb) and Trema tomentosa (TremaHb) are 93% identical in primary structure but differ in oxygen binding constants in accordance with their distinct physiological functions. Additionally, these proteins are dimeric, and ParaHb exhibits the unusual property of having different heme redox potentials for each subunit. To investigate how these hemoglobins could differ in function despite their shared sequence identity and to determine the cause of subunit heterogeneity in ParaHb, we have measured their crystal structures in the ferric oxidation state. Furthermore, we have made a monomeric ParaHb mutant protein (I43N) and measured its ferrous/ferric heme redox potential to test the hypothesized link between quaternary structure and heme heterogeneity in wild-type ParaHb. Our results demonstrate that TremaHb is a symmetric dimeric hemoglobin similar to other class 1 nonsymbiotic plant hemoglobins but that ParaHb has structurally distinct heme coordination in each of its two subunits that is absent in the monomeric I43N mutant protein. A mechanism for achieving structural heterogeneity in ParaHb in which the Ile(101(F4)) side chain contacts the proximal His(105(F8)) in one subunit but not the other is proposed. These results are discussed in the context of the evolution of plant oxygen transport hemoglobins, and other potential functions of plant hemoglobins.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman,crystallography, and DFT calculation

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or β subunit contains a ferric heme in the α₂β₂ tetramer. Though the ferric subunit cannot bind O₂, it regulates O₂ affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm^-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe³⁺-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.     

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

Structural basis for the heterotropic and homotropic interactions of invertebrate giant hemoglobin

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 A resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.     

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: The oxidation reaction

The oxidation by ferricyanide of the dimeric (HbI) and tetrameric (HbII) hemoglobins from the bivalve mollusc Scapharca inaequivalvis has been studied in static and kinetic experiments. Both hemoglobins give rise to hemichromes as stable oxidation products.Oxidation of deoxyHbI yields a hemichrome by a simple bimolecular process. No intermediate Met form can be detected during the reaction even in rapid mixing experiments. The HbI hemichrome undergoes a reversible pH-dependent dissociation into monomers. A simple model has been proposed to account for the linkage between proton binding and subunit dissociation.In the case of tetrameric HbII, oxidation yields an intermediate Met form. Thus, the kinetics of the oxidation reaction are always biphasic; the fast reaction is a bimolecular process and yields the Met derivative. The slow reaction is a monomolecular process and corresponds to the conversion of the Met form into the hemichrome: its rate is independent of the state of ligation of the ferrous protein and decreases with increase of pH. The HbII hemichrome is tetrameric when newly formed: it tends to dissociate into lower molecular weight species with the same optical properties. The rate of dissociation is relatively fast at neutral pH ($\text{t}\text{1}\text{2}$ ≈ 12 min) and markedly less at alkaline pH values.The HbI and HbII hemichromes are reduced by dithionite yielding the spectra of the native deoxygenated proteins: in the case of HbII, the tetrameric structure of the native protein is re-acquired.     

Protonation of histidine 55 affects the oxygen access to heme in the alpha chain of the hemoglobin from the Antarctic fish Trematomus bernacchii

The Root effect describes the drastic drop of oxygen affinity and loss of cooperativity at acidic pH expressed in the hemoglobins (Hb) of certain fish. The comparison between the deoxy structures of the Root effect Hb from the Antarctic fish Trematomus bernacchii (HbTb) at different pHs (pH = 6.2 and pH = 8.4) shows that the most significant differences are localized at the CDα region, where a salt bridge between Asp48 and His55 breaks during the low-to-high pH transition. In order to shed light on the relationship between pH, CDα loop structure and dynamics, and oxygen access to the active site in the alpha chain of HbTb, different computer simulation techniques were performed. Our results highlight the importance of the protonation of His55 in regulating oxygen access, underscoring its pivotal role in the structural and functional properties of HbTb. These data provide further support to the hypothesis that this residue might contribute to the release of Root protons in HbTb and underline the fact that an efficient transport of molecular oxygen in Hbs relies on a subtle balance of tertiary structure and protein conformational flexibility.     

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.     

Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.     

An Order-Disorder Transition Plays a Role in Switching Off the Root Effect in Fish Hemoglobins

The Root effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α₁β₂ and α₂β₁ interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

Crystal structure of Trematomus newnesi haemoglobin re-opens the root effect question

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 A R-state structure of Trematomus newnesi Hb 1 is presented. The structure is similar to that of Root effect fish Hbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T. bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha-->Ile, Ala97 (G3)alpha-->Ser and His41 (C7)beta-->Tyr at the alpha1beta2-interface is discussed.