Cooperative Oxygen Binding Research Articles

Cooperative Protein Dynamics of Heterotetrameric Hemoglobin from Scapharca inaequivalvis.

Hemoglobins achieve cooperative oxygen binding by diverse strategies based on different assemblies of globin subunits. Heterotetrameric hemoglobin from Scapharca inaequivalvis (HbII) consists of two AB-dimers, whose structure closely resembles that of homodimeric hemoglobin from the same organism (HbI). Herein, we investigated the structural dynamics of HbII following carbon monoxide (CO) dissociation using time-resolved resonance Raman (RR) spectroscopy. The observed spectra showed that the heme structure of the transient dissociated form of HbII was similar to that of HbI; however, the transition from the transient dissociated form to the equilibrium unligated form was faster for HbII than for HbI. Furthermore, the dependence of the time-resolved spectra on the yield of CO dissociation revealed that the transition became faster as the number of dissociated ligands increased from one to four. The positive correlation between the rate constants and number of dissociated ligands indicates that the structural transition of HbII following CO dissociation is cooperative.

Cooperative oxygen binding in beta-semihemoglobins caused by a chemical modification in the alpha1beta1 interface

A beta-semihemoglobin is an alpha-beta dimer of hemoglobin (Hb) in which the beta-subunit carries heme, while the alpha-subunit is heme-less, in apo form. It is characterised by displaying a high affinity for oxygen, and absence of cooperative binding of oxygen. We have modified chemically the residue beta112Cys (G14), located adjacent to the alpha1beta1 interface, and studied the impact of such a modification on the oligomeric state and oxygenation properties of the derivatives. We also studied the impact of modifying beta93Cys (F9) since its modification was unavoidable. For this, we used N-Ethyl maleimide and iodoacetamide. For the alkylation of beta112Cys (G14) in isolated subunits, we used N-Ethyl maleimide, iodoacetamide, or additionally, 4,4’-Dithiopyridine. Seven native and chemically modified beta-subunit derivatives were prepared and analysed. Only those derivatives treated with iodoacetamide showed oxygenation properties that were indistinguishable from those of native beta-subunits. These derivatives were then converted into their respective semihemoglobin forms, and four additional derivatives were prepared and analysed .in terms of ligation-linked oligomeric state, and oxygenation function, and contrasted against native Hb and unmodified beta-subunits. Strikingly, beta-semiHbs with modifications in beta112Cys showed indications of cooperative oxygen binding in various degrees, which suggested the possibility of assembly of two beta-semiHbs. The derivative modified with 4-Thiopyridine in beta112Cys showed a highly cooperative binding of oxygen (nmax = 1.67). A plausible allosteric scheme that could explain allostery in beta-semiHb system is suggested.

Photocatalytic Synthesis of a Polydopamine-Coated Acellular Mega-Hemoglobin as a Potential Oxygen Therapeutic with Antioxidant Properties.

Hemoglobin-based oxygen carriers (HBOCs) are being developed to overcome limitations associated with transfusion of donated red blood cells (RBCs) such as potential transmission of blood-borne pathogens and limited ex vivo storage shelf-life. Annelid erythrocruorin (Ec) derived from the worm Lumbricus terrestris (Lt) is an acellular mega-hemoglobin that has shown promise as a potential HBOC due to the large size of its oligomeric structure, thus overcoming limitations of unmodified circulating cell-free hemoglobin (Hb). With a large molecular weight of 3.6 MDa compared to 64.5 kDa for human Hb (hHb) and 144 oxygen-binding globin subunits compared to the 4 globin subunits of hHb, LtEc does not extravasate from the circulation to the same extent as hHb. LtEc is stable in the circulation without RBC membrane encapsulation and has a lower rate of auto-oxidation compared to acellular hHb, which allows the protein to remain functional for longer periods of time in the circulation compared to HBOCs derived from mammalian Hbs. Surface coatings, such as poly(ethylene glycol) (PEG) and oxidized dextran (Odex), have been investigated to potentially reduce the immune response and improve the circulation time of LtEc in vivo. Polydopamine (PDA) is a hydrophilic, biocompatible, bioinspired polymer coating used for biomedical nanoparticle assemblies and coatings and has previously been investigated for the surface coating of hHb. PDA is typically synthesized via the self-polymerization of dopamine (DA) under alkaline (pH > 8.0) conditions. However, at pH > 8.0, the oligomeric structure of LtEc begins to dissociate. Therefore, in this study, we investigated a photocatalytic method of PDA polymerization on the surface of LtEc using 9-mesityl-10-methylacridinium tetrafluoroborate (Acr-Mes) to drive PDA polymerization under physiological conditions (pH 7.4, 25 °C) over 2, 5, and 16 h in order to preserve the size and structure of LtEc. The resulting structural, biophysical, and antioxidant properties of PDA surface-coated LtEc (PDA-LtEc) was characterized using various techniques. PDA-LtEc showed an increase in measured particle size, molecular weight, and surface ζ-potential with increasing reaction time from t = 2 to 16 h compared to unmodified LtEc. PDA-LtEc reacted for 16 h was found to have reduced oxygen-binding cooperativity and slower deoxygenation kinetics compared to PDA-LtEc with lower levels of polymerization (t = 2 h), but there was no statistically significant difference in oxygen affinity. The thickness of the PDA coating can be controlled and in turn the biophysical properties can be tuned by changing various reaction conditions. PDA-LtEc was shown to demonstrate an increased level of antioxidant capacity (ferric iron reduction and free-radical scavenging) when synthesized at a reaction time of t = 16 h compared to LtEc. These antioxidant properties may prove beneficial for oxidative protection of PDA-LtEc during its time in the circulation. Hence, we believe that PDA-LtEc is a promising oxygen therapeutic for potential use in transfusion medicine applications.

ZIF-8 metal organic framework nanoparticle loaded with tense quaternary state polymerized bovine hemoglobin: potential red blood cell substitute with antioxidant properties.

Due to several limitations associated with blood transfusion, such as the relatively short shelf life of stored blood, low risk of developing acute immune hemolytic reactions and graft-versus-host disease, many strategies have been developed to synthesize hemoglobin-based oxygen carriers (HBOCs) as universal red blood cell (RBC) substitutes. Recently, zeolite imidazole framework-8 (ZIF-8), a metal-organic framework, has attracted considerable attention as a protective scaffold for encapsulation of hemoglobin (Hb). Despite the exceptional thermal and chemical stability of ZIF-8, the major impediments to implementing ZIF-8 for Hb encapsulation are the structural distortions associated with loading large quantities of Hb in the scaffold as the Hb molecule has a larger hydrodynamic diameter than the pore size of ZIF-8. Therefore to reduce the structural distortion caused by Hb encapsulation, we established and optimized a continuous-injection method to synthesize nanoparticle (NP) encapsulated polymerized bovine Hb (PolybHb) using ZIF-8 precursors (ZIF-8P-PolybHb NPs). The synthesis method was further modified by adding EDTA as a chelating agent, which reduced the ZIF-8P-PolybHb NP size to <300 nm. ZIF-8P-PolybHb NPs exhibited lower oxygen affinity (36.4 ± 3.2 mm Hg) compared to unmodified bovine Hb, but was similar in magnitude to unencapsulated PolybHb. The use of the chemical cross-linker glutaraldehyde to polymerize bovine Hb resulted in the low Hill coefficient of PolybHb, indicating loss of Hb's oxygen binding cooperativity, which could be a limitation when using PolybHb as an oxygen carrier for encapsulation inside the ZIF-8 matrix. ZIF-8P-PolybHb NPs exhibited slower oxygen offloading kinetics compared to unencapsulated PolybHb, demonstrating successful encapsulation of PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant properties when exposed to H2O2. Incorporation of PolybHb into the ZIF-8 scaffold resulted in reduced cytotoxicity towards human umbilical vein endothelial cells compared to unloaded ZIF-8 NPs and ZIF-8 NPs loaded with bovine Hb. We envisage that such a monodisperse and biocompatible HBOC with low oxygen affinity and antioxidant properties may broaden its use as an RBC substitute.

A retrospective on statistical mechanical models for hemoglobin allostery.

Understanding allosteric interactions in proteins has become one of the major research areas in protein science. The original aim of the famous theoretical model of Monod, Wyman, and Changeux (MWC) was to explain the regulation of enzymatic activity in biochemical pathways. However, its first successful quantitative application was to explain cooperative oxygen binding by hemoglobin, often called the "hydrogen molecule of biology." The combination of its original application and the enormous amount of research on hemoglobin has made it the paradigm for studies of allostery, especially for multi-subunit proteins, and for the development of statistical mechanical models to describe how structure determines function. This article is a historical account of the development of statistical mechanical models for hemoglobin to explain both the cooperative binding of oxygen (called homotropic effects by MWC) and how oxygen binding is affected by ligands that bind distant from the heme oxygen binding site (called heterotropic allosteric effects by MWC). This account makes clear the many remaining challenges for describing the relationship of structure to function for hemoglobin in terms of a satisfactory statistical mechanical model.

Cooperative Oxygen Binding with Hemoglobin as a General Model in Molecular Biophysics

Cooperative Oxygen Binding with Hemoglobin as a General Model in Molecular Biophysics

An Analysis of Models of Cooperative Oxygen Binding by Hemoglobin

An Analysis of Models of Cooperative Oxygen Binding by Hemoglobin

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Evolutionary and functional insights into the mechanism underlying body-size-related adaptation of mammalian hemoglobin.

Hemoglobin (Hb) represents a model protein to study molecular adaptation in vertebrates. Although both affinity and cooperativity of oxygen binding to Hb affect tissue oxygen delivery, only the former was thought to determine molecular adaptations of Hb. Here, we suggest that Hb affinity and cooperativity reflect evolutionary and physiological adaptions that optimized tissue oxygen delivery. To test this hypothesis, we derived the relationship between the Hill coefficient and the relative affinity and conformational changes parameters of the Monod-Wymann-Changeux allosteric model and graphed the 'biophysical Hill landscape' describing this relation. We found that mammalian Hb cooperativity values all reside on a ridge of maximum cooperativity along this landscape that allows for both gross- and fine-tuning of tissue oxygen unloading to meet the distinct metabolic requirements of mammalian tissues for oxygen. Our findings reveal the mechanism underlying body size-related adaptation of mammalian Hb. The generality and implications of our findings are discussed.

Conjugation of para-benzoquinone of Cigarette Smoke with Human Hemoglobin Leads to Unstable Tetramer and Reduced Cooperative Oxygen Binding.

Besides multiple life-threatening diseases like lung cancer and cardiovascular disease, cigarette smoking is known to produce hypoxia, a state of inadequate oxygen supply to tissues. Hypoxia plays a pivotal role in the development of chronic obstructive pulmonary disease. Smoking during pregnancy imposes risk for the unborn child. In addition to carbon monoxide, conjugation of para-benzoquinone (pBQ), derived from cigarette smoke, with human hemoglobin (HbA) was also reported to contribute in hypoxia. In fact, conjugation of pBQ is more alarming than carbon monoxide as it is an irreversible covalent modification. In the present study, the functional assay of Hb-pBQ, performed through oxygen equilibrium curve, showed a significant decrease in both P50 and cooperativity. However, the structural changes associated with the observed functional perturbation of the hemoglobin conjugate (Hb-pBQ) are unknown to date. Enhanced sensitivity and high resolution of nano-ESI mass spectrometry platform have enabled to investigate the native structure of oligomers of hemoglobin in a single scan. The structural integrity of Hb-pBQ measured through the dissociation equilibrium constants (Kd) indicated that compared to HbA, Kd of tetramer-dimer and dimer-monomer equilibria were increased by 4.98- and 64.3-folds, respectively. Using isotope exchange mass spectrometry, we observed perturbations in the inter-subunit interactions of deoxy and oxy states of Hb-pBQ. However, the three-dimensional architecture of Hb-pBQ, monitored through collision cross-sectional area, did not show any change. We propose that the significant destabilization of the functionally active structure of hemoglobin upon conjugation with pBQ results in tighter oxygen binding that leads to hypoxia. Graphical Abstract ᅟ.

Manipulating hemoglobin oxygenation using silica nanoparticles: a novel prospect for artificial oxygen carriers

AbstractRecently, nanoparticles have attracted much attention as new scaffolds for hemoglobin-based oxygen carriers (HBOCs). Indeed, the development of bionanotechnology paves the way for the rational design of blood substitutes, providing that the interaction between the nanoparticles and hemoglobin at a molecular scale and its effect on the oxygenation properties of hemoglobin are finely controlled. Here, we show that human hemoglobin has a high affinity for silica nanoparticles, leading to the adsorption of hemoglobin tetramers on the surface. The adsorption process results in a remarkable retaining of the oxygenation properties of human adult hemoglobin and sickle cell hemoglobin, associated with an increase of the oxygen affinity. The cooperative oxygen binding exhibited by adsorbed hemoglobin and the comparison with the oxygenation properties of diaspirin cross-linked hemoglobin confirmed the preservation of the tetrameric structure of hemoglobin loaded on silica nanoparticles. Our results show that silica nanoparticles can act as an effector for human native and mutant hemoglobin. Manipulating hemoglobin oxygenation using nanoparticles opens the way to the design of novel HBOCs.

1 - Interactions of zebrafish Cytoglobins with oxygen and nitric oxide

Cytoglobin (Cygb) is a ubiquitously expressed heme protein that has been identified in the genome of most higher vertebrate clades including fish, birds and mammals. Since its discovery, extensive study has revealed many structural and functional characteristics of the human Cygb protein, although its physiologic functions remain unknown. Fish have two Cygb genes (Cygb1 and Cygb2); these proteins show small but significant differences, with Cygb2 being more similar to mammalian Cygbs. To gain insights about Cygb function, we compared the properties of zebrafish cytoglobins to the mammalian proteins. Human Cygb exhibits high-affinity, cooperative oxygen binding (P50 of roughly 1 torr, Hill coefficient up to 1.6), as well as nitric oxide (NO) scavenging through NO dioxygenation and NO production via nitrite reduction. These reactions may modulate NO signaling in vivo. Reduced cytochrome b5 (CYB5) has been shown to rapidly reduce Cygb. We have reported that human CYB5 efficiently reduces both human and zebrafish Cygbs, suggesting a conserved role for the CYB5 reduction pathway. In this work, we characterize the two zebrafish Cygbs, measuring key parameters and comparing these results to human Cygb. Specifically, we examine oxygen binding for affinity and cooperativity, NO dioxygenation activity, and the ability of zebrafish CYB5 and CYB5R to reduce zebrafish Cygbs during NO dioxygenation. Oxygen affinity measurements suggest high oxygen affinity, with P50 values of

Tertiary and quaternary structural basis of oxygen affinity in human hemoglobin as revealed by multiscale simulations

Human hemoglobin (Hb) is a benchmark protein of structural biology that shaped our view of allosterism over 60 years ago, with the introduction of the MWC model based on Perutz structures of the oxy(R) and deoxy(T) states and the more recent Tertiary Two-State model that proposed the existence of individual subunit states -“r” and “t”-, whose structure is yet unknown. Cooperative oxygen binding is essential for Hb function, and despite decades of research there are still open questions related to how tertiary and quaternary changes regulate oxygen affinity. In the present work, we have determined the free energy profiles of oxygen migration and for HisE7 gate opening, with QM/MM calculations of the oxygen binding energy in order to address the influence of tertiary differences in the control of oxygen affinity. Our results show that in the α subunit the low to high affinity transition is achieved by a proximal effect that mostly affects oxygen dissociation and is the driving force of the allosteric transition, while in the β subunit the affinity change results from a complex interplay of proximal and distal effects, including an increase in the HE7 gate opening, that as shown by free energy profiles promotes oxygen uptake.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy.

Using the MWC model to describe heterotropic interactions in hemoglobin.

Hemoglobin is a classical model allosteric protein. Research on hemoglobin parallels the development of key cooperativity and allostery concepts, such as the ‘all-or-none’ Hill formalism, the stepwise Adair binding formulation and the concerted Monod-Wymann-Changuex (MWC) allosteric model. While it is clear that the MWC model adequately describes the cooperative binding of oxygen to hemoglobin, rationalizing the effects of H+, CO2 or organophosphate ligands on hemoglobin-oxygen saturation using the same model remains controversial. According to the MWC model, allosteric ligands exert their effect on protein function by modulating the quaternary conformational transition of the protein. However, data fitting analysis of hemoglobin oxygen saturation curves in the presence or absence of inhibitory ligands persistently revealed effects on both relative oxygen affinity (c) and conformational changes (L), elementary MWC parameters. The recent realization that data fitting analysis using the traditional MWC model equation may not provide reliable estimates for L and c thus calls for a re-examination of previous data using alternative fitting strategies. In the current manuscript, we present two simple strategies for obtaining reliable estimates for MWC mechanistic parameters of hemoglobin steady-state saturation curves in cases of both evolutionary and physiological variations. Our results suggest that the simple MWC model provides a reasonable description that can also account for heterotropic interactions in hemoglobin. The results, moreover, offer a general roadmap for successful data fitting analysis using the MWC model.

Can the hemoglobin characteristics of vesicomyid clam species influence their distribution in deep-sea sulfide-rich sediments? A case study in the Angola Basin

Vesicomyids live in endosymbiosis with sulfur-oxidizing bacteria and therefore need hydrogen sulfide to survive. They can nevertheless live in a wide range of sulfide and oxygen levels and depths, which may explain the exceptional diversity of this clam family in deep-sea habitats. In the Gulf of Guinea, nine species of vesicomyid clams are known to live in cold-seep areas with pockmarks from 600 to 3200m deep, as well as in the organic-rich sediments of the Congo deep-sea fan at 5000m deep. Our previous study showed that two species living in a giant pockmark have different oxygen carriers, suggesting different adaptations to hypoxia. Here, we studied the hemoglobin structure and oxygen affinity in three other species, Calyptogena valdiviae, Elenaconcha guiness and Abyssogena southwardae to determine whether the characteristics of their oxygen carriers contribute to their distribution in sulfide-rich sediments at a regional scale. Documenting pairwise species associations in various proportions, we give a semi-quantitative account of their local distribution and oxygen and sulfide measurements at seven sites. Mass spectrometry showed that each vesicomyid species has four intracellular monomeric hemoglobin molecules of 15–16kDa, all differing in their molecular mass. As expected, the monomers showed no cooperativity in oxygen binding. Their oxygen affinities were very high (below 1Torr), but differed significantly. C. valdiviae had the highest affinity and was dominant in the Harp pockmark, the site with the lowest oxygen content (half the value of fully oxygenated water). A. southwardae dominated in the Congo Lobe area, the site with the deepest sulfides. We discuss how hemoglobin may favor an active, vertical distribution of vesicomyids in sulfide-rich sediments.

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer.

Human hemoglobin (Hb), which is an α2β2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by ‘cooperativity’. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the β subunit simply enhances the O2-affinity of α subunit.

Structural basis for cooperative oxygen binding and bracelet-assisted assembly of Lumbricus terrestris hemoglobin.

The iron-containing hemoglobins (Hbs) are essential proteins to serve as oxygen transporters in the blood. Among various kinds of Hbs, the earthworm Hbs are the champions in carrying oxygen due to not only their large size but also the unusually high cooperativity of ligand binding. However, the cooperative oxygen binding mechanisms are still mostly unknown. Here we report the cryo-electron microscopy structure of Lumbricus terrestris Hb in its native, oxygenated state at 9.1 Å resolution, showing remarkable differences from the carbon monoxide-binding X-ray structure. Our structural analysis first indicates that the cooperative ligand binding of L. terrestris Hb requires tertiary and quaternary transitions in the heme pocket and a global subunit movement facilitated by intra-ring and inter-ring contacts. Moreover, the additional sinusoidal bracelet provides the confirmation for the long-standing debate about the additional electron densities absent in the X-ray crystal structure.

New look at hemoglobin allostery.

New look at hemoglobin allostery.