Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy-entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.     

A cooperative hemoglobin with directly communicating hemes. The Scapharca inaequivalvis homodimer

The unique functional properties of the homodimeric hemoglobin (HbI) extracted from the Arcid blood clam Scapharca inaequivalvis are discussed in the light of the unusual assembly of this protein. At variance with vertebrate hemoglobins, in S. inaequivalvis HbI, the heme-carrying E and F helices form the subunit interface and bring the heme groups almost into direct contact. This creates a new pathway for transferring information about the ligation state of the heme from one subunit to the other which allows cooperativity in the binding of heme ligands to be displayed by a homodimer. The tight coupling between the two subunits and the two heme groups also manifests itself in other reactions that are cooperative in S. inaequivalvis HbI, but not in human hemoglobin, namely, the cleavage of the proximal histidine-heme iron bond and the modification of specific residues located at the subunit interface.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

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.     

The Bohr effect of hemoglobin intermediates and the role of salt bridges in the tertiary/quaternary transitions

Understanding mechanisms in cooperative proteins requires the analysis of the intermediate ligation states. The release of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the protein tertiary/quaternary transitions, useful for testing models of cooperativity. The Bohr effects due to ligation of one subunit of a dimer and two subunits across the dimer interface are not additive. The reductions of the Bohr effect due to the chemical modification of a Bohr group of one and two alpha or beta subunits are additive. The Bohr effects of monoliganded chemically modified hemoglobins indicate the additivity of the effects of ligation and chemical modification with the possible exception of ligation and chemical modification of the alpha subunits. These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition.     

Inhibition of Ice Nucleus Growth in Water by Alanine Dipeptide

A molecular dynamics simulation has been carried out for the mixture of an ice nucleus, supercooled water and a molecule of alanine dipeptide (AD). The dipeptide molecule has been allocated near the nucleus surface which corresponds to the prism plane of ice crystal. The molecule is found to approach the ice surface so that the two hydrophilic sites on one side of the molecule (Oc2 and Hn1) are closest to the surface. The hydrogen bond between Hn1 site and the oxygen atom on the prism plane of the ice nucleus is expected. The perturbations of two hydrophilic sites (Oc1 and Hn2), which are surrounded by hydrophobic sites and are pointing away from the surface, attenuate the approach of water molecules to these sites. Thus, these water molecules diffuse. The hydrogen bond between the oxygen atoms on the prism plane and the hydrogen atoms of water molecules is attenuated by the diffusion.     

Structures of Invertebrate Hemoglobins

Hemoglobin is widely distributed among the invertebrates. Intracellularhemoglobins consist of relatively small molecules with mol wtsof 15–17,000 or dimeric, tetrameric or octameric aggregatesof 15–17,000 mol wt subunits. Sequence homology is presentbut not extensive in those pigments which have been studiedand the characteristic myoglobin fold of vertebrate hemoglobinoccurs in at least two invertebrate hemoglobins. The wide arrayof aggregation states among invertebrate hemoglobins providessome simple models for understanding homotropic functional propertiesexhibited by many of these pigments. Polymeric extracellularhemoglobins are present in annelids molluscs crustacean arthropodsand nematodes. Annelid extracellular hemoglobins and chlorocruorinsconsist of 3 x 10⁶ mol wt two-tiered hexagonal arrays of submultipleswhich in turn are based on polypeptide chain subunits of molwt 14–16 000. Molluscan extracellular hemoglobins areconstructed from a different subunit arrangement. In the planorbidsnail and clam extracellular hemoglobins the subunits appearto be 175 000 and 300 000 mol wt linear series of 15–17000 dalton oxygen binding domains respectively. Planorbid snailnative hemoglobin presents circular structures 200 Å indiameter in the electron microscope with 10-fold symmetry inat least one view, and clam extracellular hemoglobins are huge345 by 1200 Å rodlike structures. Crustacean extracellularhemoglobins are also polymeric pigments and at least in a fewspecies appear to have subunits which are tandemly linked oxygenbinding domains. The polymeric hemoglobins of nematodes havemolecular weights of about 330 000. The subunit molecular weightand heme content suggest a value of 40,000 daltons which setthe nematode pigments apart from all other hemoglobins so farstudied. An overview of invertebrate hemoglobin structures andsome of the questions they pose are presented in this paper.     

A novel mechanism of heme-heme interaction in the homodimeric hemoglobin from Scapharca inaequivalvis as manifested upon cleavage of the proximal Fe-N epsilon bond at low pH

The CO-binding kinetics and the optical spectra of the NO derivative of the homodimeric hemoglobin from Scapharca inaequivalvis have been investigated over the range between pH 7.0 and 2.0. In the deoxygenated derivative, protonation of the proximal imidazole at very low pH values and the consequent cleavage of the Fe-N epsilon bond result in a approximately 50-fold enhancement of the rate constant for CO binding, as found in other hemoproteins. However, in the case of the hemoglobin from S. inaequivalvis, the pH profile displays a cooperative behavior (n = 1.8 +/- 0.1), a unique feature that differentiates this protein from any other hemoprotein investigated thus far. Cleavage of the proximal bond in the NO derivative of S. inaequivalvis hemoglobin likewise displays a very steep pH transition. The mode of assembly of the homodimer, in which the heme-carrying E and F helices provide the subunit interface and bring the hemes at a much shorter distance (18.4 A) than in vertebrate hemoglobins, is likely to provide the structural basis for this unique behavior.     

Do water molecules mediate protein-DNA recognition?

A comprehensive analysis of interfacial water molecules in the structures of 109 unique protein-DNA complexes is presented together with a new view on their role in protein-DNA recognition. Location of interfacial water molecules as reported in the crystal structures and as emerging from a series of molecular dynamics studies on protein-DNA complexes with explicit solvent and counterions, was analyzed based on their acceptor, donor hydrogen bond relationships with the atoms and residues of the macromolecules, electrostatic field calculations and packing density considerations. Water molecules for the purpose of this study have been categorized into four classes: viz. (I) those that contact both the protein and the DNA simultaneously and thus mediate recognition directly; (II) those that contact either the protein or the DNA exclusively via hydrogen bonds solvating each solute separately; (III) those that contact the hydrophobic groups in either the protein or the DNA; and, lastly (IV) those that contact another water molecule. Of the 17,963 crystallographic water molecules under examination, about 6% belong to class I and 76% belong to class II. About three-fourths of class I and class II water molecules are exclusively associated with hydrogen bond acceptor atoms of both protein and DNA. Noting that DNA is polyanionic, it is significant that a majority of the crystallographically observed water molecules as well as those from molecular dynamics simulations should be involved in facilitating binding by screening unfavorable electrostatics. Less than 2% of the reported water molecules occur between hydrogen bond donor atoms of protein and acceptor atoms of DNA. These represent cases where protein atoms cannot reach out to DNA to make favorable hydrogen bond interactions due to packing/structural restrictions and interfacial water molecules provide an extension to side-chains to accomplish hydrogen bonding.     

Refined atomic model of glutamine synthetase at 3.5 A resolution

An atomic model of 43,692 non-hydrogen atoms has been determined for the 12-subunit enzyme glutamine synthetase from Salmonella typhimurium, by methods of x-ray diffraction including restrained least-squares atomic refinement against 65,223 unique reflections. At 3.5 A resolution the crystallographic R-factor (on 2 sigma data) is 25.8%. As reported earlier for the unrefined structure, the 12 subunits are arranged in two layers of six; at the interface of pairs of subunits within each layer, cylindrical active sites are formed by six anti-parallel beta strands contributed by one subunit and two strands by the neighboring subunit. This interpretation of the electron density map has now been supported by comparison with glutamine synthetase from Escherichia coli by the Fourier difference method. Each active site cylinder holds two Mn2+ ions, with each ion having as ligands three protein side chains and two water molecules (one water shared by both metals), as well as a histidyl side chain just beyond liganding distance. The protein ligands to Mn2+ 469 are Glu-131, Glu-212, and Glu-220; those to Mn2+ 470 are Glu-129, His-269, and Glu-357. The two layers of subunits are held together largely by the apolar COOH terminus, a helical thong, which inserts into a hydrophobic pocket formed by two neighboring subunits on the opposite ring. Also between layers, there is a hydrogen-bonded beta sheet interaction, as there is between subunits within a ring, but hydrophobic interactions account for most of the intersubunit stability. The central loop, which extends into the central aqueous channel, is subject to attack by at least five enzymes and is discussed as an enzyme "passive site."     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

Hemoglobin of the Antarctic fishes Trematomus bernacchii and Trematomus newnesi: structural basis for the increased stability of the liganded tetramer relative to human hemoglobin

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.     

Cooperativity in Scapharca dimeric hemoglobin: simulation of binding intermediates and elucidation of the role of interfacial water

Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin of Scapharca (HbI), which is composed of subunits with the same fold as in HbA, is also highly cooperative but the structural changes on ligand binding are small. A re-orientation of Phe97 in the binding pocket and changes in the number of interfacial water molecules have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated models of partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel, which can be open or closed, is made of hydrophilic residues. The implication of the present results for the cooperative mechanism of HbI is discussed.     

Interdomain interactions in aspartic proteases of higher organisms and their analogs in retroviral enzymes

A continuous chain of hydrogen bonded groups, which forms cross-hands interaction between domains in molecules of pepsin-like enzymes, has been revealed. The chain contains a pair of 6 symmetrically related hydrogen bonds between main chain atoms and the two conserved water molecules. The peptide groups forming hydrogen bond with the inner oxygens of the active carboxyls are important elements of the chain. The so-called "fireman grip" hydrogen bonding, consisting of a pair of the two symmetrically related bonds, is an integral part of this system of interactions. One of the water molecules in this system has a zero accessibility and forms a very short hydrogen bond with the active site interacting peptide group. This chain connects tightly the two regions of domains which have a high correlation in conformational mobility. The retroviral enzymes have an abortive chain of the interdomain interaction in this region which is reduced to the "fireman grip" net.     

Variable Subunit Contact and Cooperativity of Hemoglobins

Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact. Received: 12 January 2001 / Accepted: 3 April 2001     

Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

Conservation and diversity of ancient hemoglobins in Bacteria

A group of single-domain proteins in Bacteria similar to thermoglobin, an oxygen-avid hemoglobin representative of the ancestral form, reveals the primordial structure, function, and evolvability of the family. Conserved residues at specific positions function to bind ligand or participate in hydrophobic packing of the protein core during protein folding. A potential hydrogen bond network consisting of a tyrosine and glutamine residue in the distal ligand-binding site of most hemoglobins suggests that the ancestral protein bound oxygen avidly. Two divergent hemoglobins with mutations at generally conserved positions contain non-canonical ligand-binding sites, illustrating plasticity of the fold. One binds heme in a manner similar to cytochromes and may represent an evolutionary link to the precursor of the hemoglobin fold. Conservation suggests specific biochemical properties of the ancestral protein; diversity suggests an evolvability of this group of hemoglobins tolerant of mutations that perturb conserved biochemical properties for adaptation to novel functions.     

Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes.

The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.