Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.     

Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum.

1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arginyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.     

Solution studies on heme proteins. Circular dichroism and optical rotation of Glycera dibranchiata hemoglobins

Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of several liganded derivatives of the monomer and polymer hemoglobin components of the marine annelid, Glycera dibranchiata were measured over the wavelength range 650--195 nm. The differences observed between the monomer and polymer components for the heme dichroic bands in the visible, Soret and ultraviolet wavelength regions seem to result from changes in the heme environment, geometry and coordination state of the central heme iron in these proteins. Within the Soret region, the liganded derivatives of the monomer hemoglobin exhibit predominantly negative circular dichroic bands. The heme band at 260 nm is also absent for the monomer hemoglobin. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region suggest also differences in the alpha-helix content of the monomer and polymer hemoglobins. The values for the single-chain G. dibranchiata hemoglobin are in the expected range (about 70% alpha-helix) as predicted by the X-ray structure of this protein. The lower estimates of the alpha-helix content for the polymer hemoglobin (approx. 50%), may reflect the differences in amino acid composition, primary structure and polypeptide chain foldings. Changes in oxidation state and ligand binding appears to have no pronounced effect on the helicity of either the monomer or polymer hemoglobins. The removal of the heme moiety from the monomer hemoglobin did result in a major decrease in its helix content similar to the loss of heme from myoglobin.     

High and low oxygen affinity conformations of T state hemoglobin

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.     

Orally administrated cerium chloride induces the conformational changes of rat hemoglobin, the hydrolysis of 2,3-DPG and the oxidation of heme-Fe(II), leading to changes of oxygen affinity

The structure and oxygen affinity of hemoglobin from erythrocytes of CeCl(3) fed Wistar rats in the dose range of 0.2-20.0 mg/kg body weight/day were investigated by means of various spectroscopic methods. The changes in oxygen saturation curves of hemoglobin are dependent upon both feeding dose and feeding time. After 40 days feeding with 20 mg CeCl(3)/kg body weight/day, the curve changed to a double sigmoid shape and the oxygen affinity in low oxygen pressure increases. It regained the sigmoid form after 80 days feeding, but the degree of oxygen saturation in higher oxygen pressure became higher than that in the control. These results indicate that CeCl(3) can increase the oxygen affinity of hemoglobin of rat erythrocytes. This effect is further demonstrated by the analysis of M?ssbauer spectra of erythrocytes. Increase of hemoglobin content in erythrocytes was found in rats fed with CeCl(3). It might be the offset response to the poor oxygen-releasing capability of the hemoglobin. CD and FT-IR deconvoluted spectra indicate that secondary structures of hemoglobin have remarkable changes, characterized by a gradual decrease of alpha-helix content, in a dose- and feeding time-dependent fashion. Meanwhile, the 31P NMR spectra demonstrate that the level of 2,3-diphosphoglyceric acid (2,3-DPG) in erythrocytes, an allosteric regulator of oxygen release from hemoglobin, decreases due to its hydrolysis. In addition, the M?ssbauer and ESR spectra show clearly that a fraction of the heme-iron changes from Fe (II) to Fe (III) in CeCl(3) fed rats. The results indicate that the oral administration of CeCl(3) leads to a microenvironment changes of heme in intracellular hemoglobin. Oxygen affinity changes might be attributed to a series of events triggered by the binding of Ce (III) to hemoglobin and 2,3-DPG, including conformational changes of hemoglobin and 2,3-DPG hydrolysis, respectively and also the partial transformation from heme-Fe (II) to heme-Fe (III).     

The hemoglobins of a fresh-water teleost, Cichlasoma cyanoguttatum (Baird and Girard). II. Subunit structure and oxygen equilibria of the isolated components

Three major components constitute at least 80% of the total hemoglobin in hemolysates of the Rio Grande cichlid, Cichlasoma cyanoguttatum. All three of these appear to share a common β chain. Two components have unique α chains, and the other component has both of these unique α chains.Two of the major components have identical oxygen equilibria. The effects of pH (Bohr effect) and of adenosine triphosphate are the same for each of the three components. Although one of the components has a slightly higher oxygen affinity than the other two the effects of pH and of adenosine triphosphate appear to be indistinguishable in the different components. The Hill coefficient, n, is pH-dependent for all components. The data indicate that the ion exchange chromatography was without effect on the oxygen-binding properties.The oxygen equilibria of the components cannot be interpreted in terms of dimers and appear to require a tetrameric structure of the hemoglobin in the concentration range studied.     

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.     

The hemoglobin of the common sting-ray, Dasyatis sabina: structural and functional properties.

1. The hemoglobin of the sting-ray, Dasyatis sabina, is both polymorphic and heterogeneous; three components predominate. 2. One major component has two kinds of polypeptide chain, of which one, presumably an alpha-chain, has a blocked NH2-terminus and an arginyl COOH-terminus, whereas carboxypeptidases A and B release tyrosine and histidine from the COOH-terminus of the beta-chain. 3. The amino acid sequence of the beginning NH2-terminal segment of the beta-chain of the major component has been determined. 4. The hemoglobin of the sting-ray, Dasyatis sabina, is highly resistant to urea and does not dissociate readily into subunits. 5. Oxygen binding by the hemoglobin is not affected by organic phosphates or high concentrations of either NaCl or urea. 6. The hemoglobin does not polymerize beyond tetramers. 7. Cooperativity, as monitored by n in the Hill equation, is pH-dependent and maximal between pH 8.5 and 9.0. 8. The hemoglobin has a large Bohr effect; the oxygen affinity is 16 times higher at pH 10 than at pH 6.5.     

Structure of Annelid High Molecular Weight Hemoglobins (Erythrocruorins)

The extracellular vascular hemoglobins (erythrocruorins) ofannelids are polymeric oxygen carriers with molecular weightsof approximately 3 x 10⁶ or about 46 times the molecular weightof a vertebrate hemoglobin tetramer. The molecule appears asa dodecamer of 12 large submultiples arranged at the verticesof two regular hexagons one on top of the other in electronmicrographs. The dimensions are about 250 Å across theface of the hexagon and about 170 Å in height. The molecularweight of a one-twelfth submultiple is approximately 250 000.Biochemical studies suggest that each submultiple contains 16to 18 subunits and that the intact hemoglobin molecule containsapproximately 200 subunits. Unlike vertebrate hemoglobin whichcontains one heme moiety for each polypeptide chain the annelidhemoglobins apparently contain one heme per 15 to 20 chains.The reasons for this lack of a 11 heme chain stoichiometry arenot known at present. One possibility may be that it is theresult of insufficient purification of the hemoglobin. Alternatively,more than one globin chain might share a heme certain globinchains might lack the heme moiety and have a non hemoglobinfunction, or certain globin chains may lose their heme duringpurification of the hemoglobin. We are presently determiningthe amino acid sequence of one globin chain of Lumbricus terrestrishemoglobin. This information should be helpful in understandingthe structure of these interesting polymers.     

Cobalt hemoglobin: pH dependence of Adair constants and the Bohr effects.

Precise oxygen equilibrium curves have been obtained for cobalt hemoglobin at pH values from 5.5 to 8.2. The Hill plots are symmetric having asymptotes with slopes of unity. At pH 7.0, cobalt hemoglobin has p0.5 = 116 toor (15.45 kPa), pm = 117 torr (15.58 kPa) and a Hill coefficient of n = 1.72. The values of n decrease slightly with either decrease or increase of pH; the protein is almost non-cooperative at pH greater than 8.2. The Adair constants have been calculated with a non-linear least-squares program. From deltalnpm/deltapH a maximum of 2.5 Bohr protons was calculated at physiological pH values. The majority of alkaline Bohr protons are released after binding of the first and the third oxygen with maxima at pH 7.6 and 7.3, respectively. The acid Bohr effect was also observed with the majority of the protons taken up following the first and third oxygen bound. Smaller alkaline Bohr effects were obtained by differential titration and at higher pH than that calculated from oxygen equilibria. The discrepancy can be largely attributed to the binding of salt components to cobalt hemoglobin.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Restricting the ligand-linked heme movement in Scapharca dimeric hemoglobin reveals tight coupling between distal and proximal contributions to cooperativity.

Cooperative ligand binding in the dimeric hemoglobin from the blood clam Scapharca inaequivalvis results primarily from tertiary, rather than quaternary, structural changes. Ligand binding is coupled with conformational changes of key residues, including Phe 97, which is extruded from the proximal heme pocket, and the heme group, which moves deeper into the heme pocket. We have tested the role of the heme movement in cooperative function by mutating Ile 114, at the base of the heme pocket. Replacement of this residue with a Met did not disturb the hemoglobin structure or significantly alter equilibrium ligand binding properties. In contrast, substitution with a Phe at position 114 inhibits the ligand-linked movement of the heme group, and substantially reduces oxygen affinity and cooperativity. As the extent of heme movement to the normal position of the ligated state is diminished, Phe 97 is inhibited from its movement into the interface upon ligand binding. These results indicate a tight coupling between these two key cooperative transitions and suggest that the heme movement may be an obligatory trigger for expulsion of Phe 97 from the heme pocket.     

Amino Acid Sequence of Kalinowaski's Tinamou (Nothoprocta Kalinowskii) Hemoglobin and the Rate of Evolution of Bird αD-Globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou α^D-, α^A-, and β-globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the α^D-globin chain. This has not been reported in the literature, except in pigeon embryonic α^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, α^D = α^A > β, was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.     

Kinetic properties of sodium transport pathways in the river lamprey Lampetra fluviatilis erythrocytes

To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 to 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9-10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6-1.0 microM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration of Na+: for the semimaximal stimulation of H+ outcome amounted to 19 mM. The obtained results indicate the presence in lamprey erythrocytes of only one binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+ binding from the extracellular side of the Na+/H+ exchanger. Its efflux from cells in the Na+ -free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equation with n 1.5. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm the concept of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Residue F4 plays a key role in modulating oxygen affinity and cooperativity in Scapharca dimeric hemoglobin

Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.     

The structure of annelid and mollusc hemoglobins.

The polypeptide chain composition and the chemical properties of several annelid hemoglobins and chlorocruorins are presented. In agreement with earlier studies on the hemoglobin from Arenicola cristata (Waxman, L. (1971) J. Biol. Chem. 246, 7318-7327), nearly all of the pigments which have been examined consist of one or more different disulfide-linked polypeptide chains of 13,000 to 16,000 daltons, and the heme-protein stoichiometry suggests that more than one polypeptide is associated with each heme. Except for the prosthetic group, there is no outstanding chemical difference between the chlorocruorins and the hemoglobins, nor is ther any apparent differnce between those hemoglobins which show cooperative oxygen binding properties and those which do not. The results suggest that all these hemoglobins have similar structures. On the other hand, the polypeptide chains of mollusc hemoglobins have molecular weights of greater than 220,000. Each polypeptide binds many heme groups. Thus, annelids use small polypeptide chains while molluscs use giant polypeptides to carry O2.     

Isolation of polypeptide chains with heme from the extracellular hemoglobin of Amphitrite ornata (Polychaeta, Annelida)

From the extracellular hemoglobin of Amphitrite ornata four constituent polypeptide chains containing heme and designated AI, AII, BI and BII according to the elution order were obtained by DE52-cellulose ion-exchange chromatography with dithiothreitol (DTT) as a reducing reagent. The NH2-terminal sequences for the chains are AI, Asp-Ser-Asn-Ala; AII, Glu-Tyr-Thr; BI, Asp-Phe-Asn-Thr; and BII, Asp-Ser-Glu. Each of the isolated chains showed spectra similar to those of vertebrate hemoglobins, and they bound oxygen reversibly. Acid urea polyacrylamide gel electrophoresis separated four bands, corresponding to the isolated chain, from the intact extracellular hemoglobin reduced with DTT. These results and our failure to detect an appreciable amount of non-heme protein suggest that the extracellular hemoglobin of A. ornata is composed of four polypeptide chains, each containing a heme.     

Quaternary structure of rice nonsymbiotic hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.