Vertebrate Hemoglobins Research Articles

Probes and Point Mutations on the Web

Dorit Rotenberg (University of Wisconsin-Madison, USA) was fascinated to see crystalline structures within the gut of rhabditids in culture when crystal violet was used to inhibit bacterial contamination. Have others encountered the phenomenon? Indeed they have! David Bird (North Carolina State University at Raleigh, USA) submitted A.F.Bird et al. Observations on crystalloid bodies in the pseudocoelom of Eutobrilus heptapapillatus. J. Nematol. 23, 39–47, 1991 [function unknown]. Michal Brzeski (Instytut Warzywnictwa, Poland) had seen crystals large enough to fill the entire gut lumen in most specimens of Enchodelis feeding on green moss. (M.W. Brzeski, Enchodelus repis new species and comments on Enchodelis laevisThorne, 1939 and Enchodelis microdoroides Baqri & Jairajpuri, 1974 (Nematoda:Dorylaimoidea) from Korea. Fundam Appl Nematol 15, 319–326, 1992.) He did not think they represented waste material as the worms have a functional anus. However, Orville G. Marti, Jr (University of Georgia, USA) thought those in the intestine of Noctuidonema, an ectoparasite of moths which has an imperforate anus, might be ‘an alternative to the usual method of waste elimination’. Jay Burr (Simon Fraser University, Canada) described crystalline haemoglobin found in the eye of mature adult female Mermis nigrescens ‘the only example of naturally crystalline hemoglobin [but with] the same dichroic spectrum as artificial crystals of vertebrate hemoglobins ... its unique function is to cast a shadow on a photoreceptor and thus provide mature females with the ability to discriminate the direction of light during phototaxis ...[it] appears to have been selected ... because of its strong light-absorbing property, rather than its oxygen-bearing ability.’ (A.H.J. Burr and C.P.F. Babinszki. Scanning motion, ocellar morphology and orientation mechanisms in the phototaxis of the nematode M. nigrescens. J. Compar. Physiol. A. 167, 257–268, 1990.) Sarah Rosloski (University of Guelph, Canada) had read that nematodes use extracellular freezing to protect themselves from freezing. She wondered if crystals might be caused by the release of nucleating substances into the lumen of the intestine, but David Wharton (University of Otago, New Zealand) thought this unlikely. ‘Most nematodes seem to rely on inoculative freezing, ie. ice from the surrounding medium penetrating through a body opening and initiating the freezing of the body contents.’ The antarctic nematode Panagrolaimus davidi can survive extensive intracellular freezing, and he suspects the ability to survive intracellular ice formation is common among nematodes which survive freezing. Jihua Wu (Chinese Academy of Sciences, Wuhan, Hubei), while not sure if they were the same thing as crystals, said ‘crystalloids’ occur in some freshwater genera (Monhystera, Ironus, Tobrilus and Tripyla), perhaps acting as a detoxification system by preventing harmful accumulation of metal sulphides (L.J. Jacobs and J. Heyns, An ecological strategy in the genus Monhystera – an hypothesis. Rev. Nematol. 13, 109–111, 1990). Michal Brzeski agreed with the distinction: crystalloids occur in the body cavity, large crystals in the gut.

ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha1beta2 interface and favors the dissociation into dimers. The ATP-dependent dimer left arrow over right arrow tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals' metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.

Regulation of the Salmonella typhimuriumFlavohemoglobin Gene: A NEW PATHWAY FOR BACTERIAL GENE EXPRESSION IN RESPONSE TO NITRIC OXIDE

Flavohemoglobins, a family of two-domain proteins with homology to vertebrate hemoglobins, are found in a variety of prokaryotic and eukaryotic microorganisms. Recent studies suggest a role for these proteins in nitrogen oxide metabolism. We now show that nitric oxide donors positively regulate a chromosomal flavohemoglobin (hmp)/lacZ operon fusion in Salmonella typhimurium. hmp gene expression in the presence of NO. is independent of the SoxS, OxyR, and FNR transcription factors and instead relies on inactivation of the iron-dependent Fur repressor. Other Fur-repressed promoters in S. typhimurium are also activated by an NO. donor. In contrast to the wild-type strain, an hmp- mutant requires markedly lower concentrations of NO to induce the hmp/lacZ fusion, whereas its response to iron chelation is equivalent to wild type. These data unveil a new pathway for NO-dependent gene expression in S. typhimurium.

Evolution of cooperativity in hemoglobins: What can invertebrate hemoglobins tell us?

While vertebrate hemoglobins typically are tetrameric and show highly regulated and cooperative ligand binding, little is known of the evolution of these properties. We are studying the structural and functional properties of the hemoglobins from Caudina arenicola, an echinoderm. The echinoderms are in the lineage most closely related to the vertebrates to express hemoglobin. C. arenicola has three sets of red cells, in the water vascular system, the coelomic cavity, and in an intestinal vein. Each of these expresses a distinct array of globins. The hemoglobins are cooperative and exhibit unusual ligand-linked associative properties, being dimeric when oxygenated and forming tetramers and higher aggregates on deoxygenation. The major coelomic hemoglobins have been subjected to a detailed examination by a combination of ligand binding analyses and protein and DNA sequencing, as well as X-ray crystallography. Two typical globin introns were identified, along with a unique intron that bisects an N-terminal extension of the globin from the remainder of the gene. X-ray crystallographic analysis shows that the subunit interfaces of C. arenicola hemoglobins differ radically from those of vertebrate hemoglobins and indeed from some other invertebrate hemoglobins, but closely resemble the packing arrangements found in a clam hemoglobin (Scapharca). However, the residues implicated in cooperativity in these two types of hemoglobins differ substantially.

Role for the Salmonella Flavohemoglobin in Protection from Nitric Oxide

Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms. Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus. Determination of a function for these flavohemoglobins has been elusive. A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide. The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function. These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.

Self-association, cooperativity and supercooperativity of oxygen binding by hemoglobins.

Cooperative ligand binding by tetrameric vertebrate hemoglobins (Hbs) makes possible the delivery of oxygen at higher pressures than would otherwise occur. This cooperativity depends on changes in dimer-dimer interactions within the tetramer and is reflected in a 50 000-fold increase in the tetramer-dimer dissociation constant in human Hb upon oxygenation at pH 7.4, from approximately 2x10(-11)mol l-1 to approximately 10(-6)mol l-1. Hbs that undergo such ligand-dependent changes in association are widespread in non-vertebrates, where the mechanisms are very different from those in vertebrates. Oligomeric Hbs have been identified in organisms in five phyla (molluscs, echinoderms, annelids, phoronids and chordates) that dissociate to subunits upon oxidation of the heme iron and reassociate with the binding of ferric iron ligands such as CN-, N3- or NO2-. Thus, the valence and ligand state of the heme iron control the stability of a critical subunit interface. The broad distribution of this phenomenon suggests a common mechanism of communication between heme and interface that may be almost universal among non-vertebrate Hbs. This interaction may be similar to that known for the homodimeric Hb of the mollusc Scapharca inaequivalvis. Although muscle tissue Hbs or myoglobins (Mbs) are usually monomeric, with non-cooperative O2 binding, the radular muscles of gastropod molluscs and chitons have homodimeric Mbs that bind O2 cooperatively. Cooperative non-muscle tissue Hbs have also been identified. These include the neural Hb of the nemertean worm Cerebratulus lacteus and the Hb of the diving beetle Anisops assimilis, which exhibit deoxygenation-dependent self-association of monomers that is associated with high Hill coefficients. Calculations suggest that the 2-3 mmol l-1 concentration of Hb on a heme basis in the brain of Cerebratulus should substantially extend the time as an active predator in an anaerobic or hypoxic environment. Oxygen from the Hb of Anisops is delivered to a gas bubble and thereby controls the buoyant density. Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2. This phenomenon results in steeper O2-binding curves than exhibited by tetramers alone. The increased cooperativity should result in an increase in the amount of O2 delivered to the tissues and should be especially valuable for avian flight muscles.

Adaptation of the Oxygen Transport System to Hypoxia in the Blue Crab,Callinectes sapidus

Among the oxygen carrying proteins, two groups are known to adapt to environmental challenges in the adult stage. Both vertebrate hemoglobins and arthropod hemocyanins adapt to chronic hypoxia by responding to the actions of allosteric co-factors. The design strategy, however, differs fundamentally in the two groups. Even within the arthropods, chelicerate and crustacean hemocyanins respond to co-factors very differently. Only in the crustaceans does the oxygen carrier adapt by shifts in intrinsic molecular properties. In hypoxic blue crabs, increases in the ratio of the primitive 1 × 6-meric oligomer bring about a higher oxygen affinity relative to that in normoxic animals, in which greater proportions of the derived 2 × 6-meric oligomer are responsible for a lower oxygen affinity

Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin alpha and beta chains.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1; 15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21-30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of "two globin strains in annelid hemoglobin", which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin alpha and beta chains.

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibilty tensor, and the major axis found tilted from the normal in a manner similar to that found for the FeCO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

The Crystal Structure of Horse Deoxyhaemoglobin Trapped in the High-affinity (R) State

Co-operative oxygen binding by the vertebrate haemoglobins arises from an equilibrium between a quaternary structure with low affinity (T), favoured in the absence of ligand, and a high affinity form (R) adopted by the fully ligated protein. While R state haemoglobin has an oxygen affinity close to that of isolated subunits, the affinity of the T state is roughly 300-fold lower. The mechanism by which the T state restrains ligand binding, and the pathway of the quaternary transition, have been largely revealed by detailed crystallographic analyses of a number of haemoglobin molecules in the equilibrium states, as well as intermediate forms of the T state including partially ligated species. The ligation intermediates of the R state, however, have not been as well characterized structurally. We report here the crystal structure of one such intermediate species, namely, horse deoxyhaemoglobin in the R state, at 1.8 Å resolution. While ligand binding in the T state may result in unfavourable stereochemistry in and around the haem – ligand complex, the more plastic R structure appears to accommodate equally well both liganded and ligand-free haem. Loss of ligand at the R state haem results in movements of the haem and shifts of the FG corners, which form characteristic intersubunit contacts that distinguish the quaternary states. The shifts are comparable in magnitude to the corresponding movements associated with de-ligation in the T state, although they differ in direction. These and other differences illustrate how the structural changes in the haem pocket are communicated to the subunit interfaces and how the movements that can occur in the R state may be impeded in the T state.

8] Preparation and characterization of invertebrate globin complexes

The hemoglobins of vertebrates are uniformly intracellular and, with the exception of agnathan hemoglobins, have a heterotetrameric subunit structure. The hemoglobins of the agnathans (jawless cyclostomes) that consist of hagfish and lampreys occur as homopolymers and exhibit oxygenation-linked monomerization. Moreover, hemoglobin is ubiquitous in the vertebrates, being absent only in subantarctic fish of the family Chaenichtydae and in larvae of the European eel Anguilla anguilla. Hemoglobins from invertebrates can be intra- or extracellular and their occurrence is episodic; thus, closely related species living in identical environments may have no hemoglobin, have an intracellular hemoglobin, or have an extracellular hemoglobin. In some cases, intra- and extracellular hemoglobins coexist in the same species. Whenever intracellular hemoglobins are present, they occur in nucleated erythrocytes. Moreover, invertebrate hemoglobins vary widely in molecular size, ranging from single-chain, and myoglobin-like molecules of ∼17 kDa to giant heteromultimeric complexes of up to 10,000 kDa. The majority of intracellular invertebrate hemoglobins are monomeric, many instances of oligomerization to dimers and tetramers and of the formation of heterodimers and heterotetramers are also known.

Absence of ligand binding-induced tertiary changes in the multimeric earthworm Lumbricus terrestris hemoglobin. A resonance Raman study.

In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding or changes in quaternary state are manifested at the heme as reflected in resonance Raman spectral changes involving the iron-proximal histidine stretching mode. No such changes are observed for Lumbricus terrestris hemoglobin. The iron-histidine stretching mode and the porphyrin breathing motion in the deoxy-, oxy-, or CO-photodissociated forms of Lumbricus hemoglobin and human hemoglobin A (pH 7.0 and 9.2, the latter to effect Lumbricus hemoglobin subunit dissociation) were studied using pulsed (10 ns) light at 435 nm. In contrast to that observed for hemoglobin A, a comparison of the spectra of the deoxy and photoproduct forms of Lumbricus hemoglobin reveal minimal differences in the region of the iron-histidine and the pi electron distribution in the heme moiety. The spectral frequencies are similar to that observed in R-state vertebrate hemoglobins. Such average behavior of the approximately 192 hemes present in Lumbricus hemoglobin is more analogous to the Raman spectral properties observed in myoglobin.

Metastable CO binding sites in the photoproduct of a novel cooperative dimeric hemoglobin

The infrared absorption spectrum of the CO-photoproduct from Scapharca inaequivalvis hemoglobin (Hbl) at 10 K yields only a single line in the "B" state region at 2132 cm-1. This is the same frequency as the B1 line observed in photodissociated vertebrate HbCO and MbCO. No evidence was found for the B2 line detected in vertebrate hemoglobins and myoglobin in the 2118-2120 cm-1 region. These data demonstrate that the protein does not have the same conformationally accessible ligand-binding sites as do vertebrate hemoglobins and myoglobins. The absence of the B2 line indicates that only a single weak site is accessible to the photolyzed CO molecule. These results are in accord with geminate rebinding experiments and ligand escape pathway calculations which have shown that the distal properties of Hbl are distinct from those of tetrameric hemoglobins and vertebrate myoglobins.

Cooperative homodimeric hemoglobin from Scapharca inaequivalvis: cDNA cloning and expression of the fully functional protein in E. coli

The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc. Scapharca inaequivalvis, has been accomplished in E. coli from its cDNA. The latter was isolated by PCR amplification of total RNA and sequenced. The cDNA-derived sequence differed by a single amino acid when compared to that previously obtained from purified protein. Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins. The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.

Hemoglobin Structure and Function in the Rat-Tailed Sea Cucumber, Paracaudina chilensis.

The rat-tailed sea cucumber, Paracaudina chilensis, has abundant hemoglobin-filled hemocytes in its perivisceral coelom, water vascular system, and hemal system. The perivisceral oxyhemoglobin consisted of 34 kDa dimers and molecules with an apparent molecular weight of ca. 50 kDa. The perivisceral hemoglobin had a high oxygen affinity with a P50 of 1.5 mm Hg at 15°C. It exhibited cooperative oxygen binding with a Hill coefficient of 1.26 to 1.86. Oxygen affinity appeared to be pH dependent, but the effect was not significant. The heat of oxygenation was -11.2 kcal mol-1. At high hemoglobin concentrations, the perivisceral hemoglobin oxygen affinity was lower and the apparent pH effect and cooperativity were increased. Perivisceral and water vascular hemoglobins had spectral characteristics similar to those of other invertebrate and vertebrate hemoglobins. The perivisceral hemoglobin appeared to be electrophoretically heterogeneous and was structurally distinguishable from water vascular hemoglobin. The oxygen affinity of water vascular hemoglobin was not different from that of the perivisceral hemoglobin in spite of the difference in structure and location in the animal. The exceptionally high oxygen affinity hemoglobin of P. chilensis, a burrowing sea cucumber, may be adaptive to this animal's oxygen-limited habitat.

Heme-heme interactions in a homodimeric cooperative hemoglobin. Evidence from transient Raman scattering.

The comparison of the resonance Raman spectrum of the deoxy dimeric hemoglobin (HbI) from the Arcid clam, Scapharca inaequivalvis, to its CO photoproduct at 10 ns reveals several significant differences in the low frequency vibrational modes including those involving motions of the peripheral substituents on the heme such as the propionates. This finding reflects the involvement of the propionates in a hydrogen bonding network which connects the two heme groups across the subunit interface and is sensitive to ligand binding. A frequency difference in the iron-histidine stretching mode of 6 cm-1 in this invertebrate hemoglobin is substantially smaller than that detected in tetrameric vertebrate hemoglobins under similar conditions. Time evolution studies of the vibrational modes from the photoproduct demonstrate that the transient form relaxes to the deoxy structure concertedly with a half-life of 1 microseconds, the time scale in which tertiary relaxations of tetrameric hemoglobins occur. These data establish that the tertiary and quaternary structural changes take place on the same time scale and confirm a mechanism of cooperativity involving direct interaction between the two heme groups through the peripheral substituents. This direct structural communication provides a very tight linkage between the heme groups prohibiting modulation of the oxygen affinity by factors in the physiological milieu.

The differential effects of carbon monoxide and oxygen on the pressure dissociation of Lumbricus terrestris hemoglobin

We have explored the subunit affinities of Lumbricus terrestris hemoglobin (LtHb) under a variety of conditions using high-pressure spectroscopy. While only small changes were observed for LtHb-oxy below 1.0 kbar, higher pressures resulted in a 1000 cm −1 red shift and 2-fold increase in fluorescence intensity with a concomitant 12-fold decrease in scattering intensity, all of which reached completion by approx. 2.2 kbar. In the presence of 1 M MgCl 2 or at acidic pH (4.2), the curves shifted by 400 and 1000 bar corresponding to significant destabilization. At pH 9.1, the initial spectral parameters were almost equal to the final endpoints and were unaffected by pressure. While the pressure curve of the CO form was similar to the oxy form at pH 7.2, the midpoints of the other samples were shifted to higher pressures relative to their oxy counterpart, indicating tighter subunit contacts. This stabilization was unexpected based upon the sequence homology to vertebrate hemoglobins, and the minimal structural differences between these two liganded forms of human hemoglobin. These data indicate that the differences are the result of the additive nature of the interactions involved in subunit packing whose effects become significant in larger aggregates.

Primary structure of chain I of the heterodimeric hemoglobin from the blood clam Barbatia virescens.

The blood clam Barbatia virescens has a heterodimeric hemoglobin in erythrocytes. Interestingly, the congeneric clams B. reeveana and B. lima contain quite different hemoglobins: tetramer and polymeric hemoglobin consisting of unusual didomain chain. The complete amino acid sequence of chain I of B. virescens has been determined. The sequence was mainly determined from CNBr peptides and their subpeptides, and the alignment of the peptides was confirmed by sequencing of PCR-amplified cDNA for B. virescens chain I. The cDNA-derived amino acid sequence matched completely with the sequence proposed from protein sequencing. B. virescens chain I is composed of 156 amino acid residues, and the molecular mass was calculated to be 18,387 D, including a heme group. The sequence of B. virescens chain I showed 35-42% sequence identity with those of the related clam Anadara trapezia and the congeneric clam B. reeveana. An evolutionary tree for Anadara and Barbatia chains clearly indicates that all of the chains are evolved from one ancestral globin gene, and that the divergence of chains has occurred in each clam after the speciation. The evolutionary rate for clam hemoglobins was estimated to be about four times faster than that of vertebrate hemoglobin. We suggest that blood clam hemoglobin is a physiologically less important molecule when compared with vertebrate hemoglobins, and so it evolved rapidly and resulted in a remarkable diversity in quaternary and subunit structure within a relatively short period.

Molecular adaptation of hemoglobin function in mammals

Vertebrate hemoglobins are tetramers made of two pairs of α and β subunits each containing a hydrophobic pocket where a heme molecule binds tightly and allows for the reversible binding of oxygen. Both tertiary and quaternary structures are ideally suited for the loading and unloading of oxygen necessary for the metabolic requirements of the organisms. Starting from a single ancestor hemoglobin subunit, evolutionary processes have led to heterologous tetramers exhibiting a moderate oxygen affinity due to heme-heme interaction (allosteric mechanism) which may be futher modulated through electrostatic interactions with chloride and/or organophosphate anions present in the red cells. These effectors, which bind preferentially to the deoxyHb tetramers at a distance from the heme groups, play a major role in the adaptation of the respiratory properties of hemoglobin to either allometric-dependent oxygen needs or to various hypoxic environments such as altitude, burrowing, or foetal life. In most cases the existence or the strength of the effector-Hb complexes, hence the changes in the allosteric equilibrium, may be ascribed to one or a few mutations of residues at the effector binding sites. Typical examples of these mechanisms are described.

Hemoglobins: Current Opinion in Structural Biology 1991, 1:915–921

Recent work on human hemoglobin has provided new insight into the energetics of ligand binding and the requirements for the shift to the quaternary R state. Comprehensive studies of myoglobin by site-directed mutagenesis have delineated the importance of polar interactions with water in the heme pocket for controlling ligand entry. Structural studies of invertebrate hemoglobins show that their subunit interactions and cooperative mechanisms are wholly different from those of vertebrate hemoglobins.