Binding modes of L35 to alpha- and beta-semihemoglobins: structural insights into the inequivalence of alpha- and beta-subunits of hemoglobin

It has been thought for several years that the greatly lowered oxygen affinity, high cooperativity, and heterotropic modulation displayed by tetrameric human hemoglobin (Hb) was an exclusive result of the assembly of high affinity alpha(1)beta(1) dimers into alpha(2)beta(2) tetramers. However, in recent times, it has been shown that alpha- and beta-semihemoglobins, namely alpha(heme)beta(apo) and alpha(apo)beta(heme), which are dimers of Hb characterized by a high affinity for oxygen and lack of cooperativity do respond to effectors such as 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35), a bezafibrate (BZF) related compound, by decreasing the ligand affinity to a considerable extent (between 60- and 130-fold). In order to shed some light on the structural basis of this phenomenon, we have developed a binding mode of L35 to semihemoglobins through docking analysis using the program GRID. Molecular modelling studies did identify sites on semihemoglobins where favourable interactions with L35 can occur. We found that the effector binds differently to the two semihemoglobins exhibiting high affinity only for the alpha chain heme pocket. The proposed binding models are consistent with the experimental findings and may be rationalized in terms of different hydrophobic and hydrophilic characteristics between alpha- and beta-heme pockets of Hb.     

Covalent fixation of polymer-linked benzene hexacarboxylate onto human haemoglobin.

The reaction of human deoxy and oxyhaemoglobin with a macromolecular effector, monomethoxypolyoxyethylene-linked benzene hexacarboxylate, in the presence of a water soluble carbodiimide, produces under defined conditions, the same conjugates preferentially acylated at the two valines beta 1. The oxygen affinity of both these conjugates is decreased by approximately 5-fold compared with that of native Hb (at pH 7.2, in 0.05 M Tris buffer, 25 degrees C, P50: 20.1 and 20.7 Torr versus about 4 Torr for Hb). This difference appears to be due to an overstabilization of the T state probably together with a decrease of the oxygen affinity of the R state. Addition of IHP to the conjugate solutions does not influence the P50 but addition of IHP to the reaction mixtures before the coupling limits the substitution of Hb by the macromolecular effector, to 20% (instead of 100% in absence of IHP). The cooperativity curve is shifted to the right with an Nmax of 3 at about 90% oxygen saturation, which corresponds to a potential release of 48% of oxygen at pH 7.2, 25 degrees C, between 100 and 40 Torr, compared with 40% for blood. Such kinds of conjugates especially those obtained from oxyhaemoglobin which are easily prepared, could be of a great interest as non-diffusing oxygen carriers in transfusional and perfusional fluids.     

Description of hemoglobin oxygenation under universal solution conditions by a global allostery model with a single adjustable parameter

The Monod-Wyman-Changeux allosteric model parameters evaluated from accurate oxygen equilibrium curves (OECs) of hemoglobin that were measured in an extremely wide range of structural constraints, imposed by allosteric effectors, yielded a closed circle when log K(T) and log K(R) were plotted against log L(0) and log L(4), respectively, showing novel phenomena that L(0) and L(4) have a maximal value and a minimal value, respectively, and K(T) and K(R) vary by more than three orders of magnitude. These phenomena were successfully described by a global allostery model, which mathematically keeps the frame work of the MWC model, but allows that K(T) under a set of solution conditions becomes larger than K(R) under another set of solution conditions and postulates that a representative allosteric effector binds to both the T and R states with a lower affinity but with a larger stoichiometry for the R state than for the T state. Thus, this global model can describe any given OEC measured under universal solution conditions with the single adjustable parameter, the concentration of the representative effector.     

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.     

Liganded hemoglobin structural perturbations by the allosteric effector L35

Effector binding to liganded hemoglobin (Hb) provides a new understanding of structural determinants of Hb function. L35, a bezafibrate-related compound, is one of the more potent synthetic regulators of Hb oxygen (O(2)) affinity. In the presence of inositol hexaphosphate and bezafibrate (or derivatives), liganded Hb at low pH (pH approximately 6.5) exhibits extremely low O(2) affinity and very low cooperativity. In this study, the nature of L35 binding to COHbA at pH 6.35, an altered R-state, is presented. Solution-active site-specific spectroscopic probings by front-face fluorescence and circular dichroism reveal that L35 induces a global heterogeneous conformation in COHbA at pH 6.35 that includes: a T-like structural feature at the alpha1beta2 interface; an R-like structural feature within the heme environment; and an intermediate-like state at the central cavity. These long-range structural perturbations appear to stem from L35 binding to two classes of binding sites: the central cavity (primarily at the alphaalpha cleft) and the surface. These results indicate that L35 induces an allosteric transition species, characterized by domain-specific tertiary and quaternary-like conformation within a global R-quaternary structure.     

The binding of hemoglobin to red cell membrane lowers its oxygen affinity

The mode of interaction of human hemoglobin (Hb) with the red cell membrane was investigated with special reference to the effect on oxygen binding properties and Hb-membrane binding constants. Compared to free native Hb, the membrane-bound native Hb showed a strikingly lowered oxygen affinity and smaller response to organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate. Similar effects of membrane binding were also observed for intermediately cooperative Hbs such as N-ethylmaleimide-treated Hb (NES-Hb) and iodoacetamide-treated Hb (AA-Hb), but very small effects were observed for non-cooperative Hb, i.e., carboxypeptidase A-treated Hb (des-His-Tyr Hb). The magnitude of the affinity lowering was in the order: NES-Hb greater than native Hb greater than AA-Hb much greater than des-His-Tyr Hb. In the presence of inositol hexaphosphate, the three chemically modified Hbs showed an increased oxygen affinity when bound to the red cell membrane, probably due to partial replacement of bound inositol hexaphosphate by membrane. The binding to membrane caused a slight decrease in cooperativity for native Hb, but no distinct change in cooperativity was observed for the three modified Hbs. These results imply: a) the red cell membrane binds to deoxyHb more strongly than to oxyHb; b) the difference in membrane binding affinity between oxyHb and deoxyHb is closely related to the quaternary structure change in the Hb molecule occurring upon oxygenation. The higher affinity of the membrane for deoxyHb than for oxyHb apparently disagrees with the conclusion drawn by earlier investigators. However, the present binding experiments by means of ultrafiltration proved that the red cell membrane actually binds to deoxyHb much more strongly than to oxyHb, validating the present conclusion based on oxygenation experiments. Our results are consistent with those obtained recently by other investigators using a synthetic peptide or the cytoplasmic fragment of red cell membrane band 3.     

Allosteric kinetics and equilibria of triligated, cross-linked hemoglobin.

Using modulated excitation, we have measured the forward and reverse rates of the allosteric transition between relaxed (R) and tense (T) quaternary structures for triply ligated hemoglobin (Hb), cross-linked between the alpha chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris buffers at neutral pH. Since the cross-link prohibits disproportionation, triply ligated aquomet Hb species with ferrous beta chains were specifically isolated by isoelectric focusing. Modulated excitation provides rate pairs and therefore gives equilibrium constants between quaternary structures. To coordinate with that information, oxygen binding curves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is determined by modulated excitation to be of order unity for O2 or CO (1.1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subunits it is much greater (> or = 23). R-->T conversion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from HbXL O2 were used to obtain a unique allosteric decomposition of the ferrous O2 binding curve in terms of KT, KR, and L3. From these values and the O2 binding curve of tri-aquomet HbXL, L3 was calculated to be 2.7 for the tri-aquomet derivative. Consistency in L3 values between equilibrium and modulated excitation data for tri-aquomet-HbXL can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than that for binding to the beta chains in the R state, while the cooperative properties remain homogeneous. The results are in quantitative agreement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the alpha subunits with almost no change in the affinity of the beta subunits, leaving the allosteric parameters L and c unchanged.     

Conformational states of human rat sarcoma (Ras) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.     

Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features. These findings provide a new global view for allosteric interactions and invoke for a modern interpretation of the role of allosteric effectors and a reformulation of the Monod-Wyman-Changeaux model for control of allosteric systems, and other complementary models as well.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.     

Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.     

Molecular modelling of Trematomus newnesi Hb 1: insights for a lowered oxygen affinity and lack of root effect

Three-dimensional structural models of the hemoglobin (Hb 1) of the Antarctic fish Trematomus newnesi were built by homology modelling, using as template the X-ray crystallographic structures of Trematomus (previously named Pagothenia) bernacchii Hb 1, both in R and T state. The Hbs of these two fishes, although showing remarkably different oxygen binding properties, differ only by 4 residues in the alpha chain (142 aa) and 10 residues in the beta chain (146 aa). T. newnesi Hb1 R-state model, essentially performed as a quality control of the adopted modelling procedure, showed a good correspondence with the crystallographic one. Modelling of T. newnesi Hb1 in the T state was performed taking into account that the proton uptake by aspartate residues, proposed to be responsible for half of the Root effect in T. bernacchii Hb 1 (showing sharp pH dependent oxygen affinity and T-state overstabilization at low pH, i. e. Bohr and Root effect), does not occur in T. newnesi Hb1 (having nearly pH-independent lower oxygen affinity). Comparison with the template structure (submitted to the same minimization procedure) indicates that, in T. newnesi Hb1 T-state model, the substitution of Ile for Thr in 41 C6, in central position of the switch region, induces at the alpha(1)beta(2) interface structural modifications able to hamper the protonation. Similar modifications are also found in T. bernacchii Hb 1 modelled in the T state with the single substitution Thr-->Ile in 41alpha. These models also suggest that the lower oxygen affinity observed in T. newnesi Hb1 is related to structural differences at the alpha(1)beta(2) interface leading to a more stable low-affinity T state. Proteins 2000;39:155-165.     

Thermodynamics of oxygen binding to arctic hemoglobins. The case of reindeer

The most surprising characteristic of reindeer hemoglobin (Hb) concerns its response to changes in temperature. Thus, the shape of the oxygen-binding curve is strongly temperature dependent due to the difference in the enthalpy of oxygenation between the T and R state of the molecule. In fact, delta H of oxygen binding to the T state is strongly exothermic whereas that of the R state is very close to zero or possibly positive after correction for the heat of oxygen solubilization. Moreover, the allosteric transition T0----R0 has been found to display a negative delta H and a contemporaneous decrease in entropy, a behavior which is precisely the opposite of what has been reported for other hemoglobins. As a whole, reindeer Hb represents a beautiful example of the significance that comparative studies may have in assessing the general validity of the main properties of the hemoglobin molecule.     

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.     

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O(2) binding.

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O(2) affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the "osmotic stress" method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl(-) and of DPG concentration. We find that only approximately 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with approximately 72 that bind in the presence of more than 50 mM Cl(-) or more than 15 microM DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl(-), DPG, and H(2)O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.     

Macromolecular isoforms of Daphnia magna haemoglobin

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4-4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7-5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.     

Allosteric interactions in sipunculid and brachiopod hemerythrins

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.