Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

Polypeptide chain composition diversity of hexagonal-bilayer haemoglobins within a single family of annelids, the alvinellidae.

Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 +/- 14 kDa (A. caudata), 3822 +/- 28 kDa (P. grasslei) and 3750 +/- 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull. 179, 366-373.     

Electrospray ionization mass spectrometric determination of the molecular mass of the approximately 200-kDa globin dodecamer subassemblies in hexagonal bilayer hemoglobins.

Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.     

Hemoglobins from deep-sea hydrothermal vent scaleworms of the genus Branchipolynoe: a new type of quaternary structure

Branchipolynoe symmytilida and B. seepensis are two scaleworms (Polychaeta; Polynoidae) living commensally in the mantle cavity of deep-sea hydrothermal vent and cold-seep mussels. In contrast with littoral members of this family, the two species exhibit a large amount of extracellular hemoglobin (Hb) in their coelomic fluid. Gel filtration revealed the existence of four different Hbs: one minor, high molecular mass (3x10(6) Da) Hb, V1-Hb, reminiscent of a vascular hexagonal bilayer annelid Hb; two major coelomic Hbs, C1-Hb, and C2-Hb, with unusual masses for extracellular annelid Hbs of 153 and 124 kDa respectively; and a minor probably coelomic Hb of 23 kDa (C3-Hb). Using electrospray ionization mass spectrometry, SDS-PAGE after subtilisin treatment, and tandem mass spectrometry, we showed that C1-Hb is a trimer of a 57,996 Da chain and C2-Hb is a dimer of a 57,648 Da chain, each chain being a four-domain/four-heme polypeptide. This multimeric, multidomain arrangement is unique among annelid Hbs and appears different from that of other known multidomain Hbs.     

A thermostable protein inhibitor of protein kinase from the swine brain. Isolation of the inhibitor and interaction with the enzyme

A heat-stable protein inhibitor of cAMP-dependent protein kinase has been isolated from pig brain tissue. During gel filtration the protein is eluted in three peaks corresponding to the tetramer, dimer and monomer. The monomer fraction was purified 609-fold. The molecular mass of the monomeric form as determined by gel filtration and electrophoresis is equal to 11,000 Da and 8000 Da, respectively. The inhibition of the phosphotransferase reaction with respect to ATP occurs via a non-competitive mechanism, while that for histone--via a competitive mechanism. A formal kinetic analysis of various modes of the inhibitor binding to different protein kinase forms, e. g., the cAMP-dependent protein kinase catalytic subunit, protein kinase holoenzyme in the presence and absence of cAMP as well as of holoenzyme preparations modified by dimethylsuberimidate and cupric 1,10-phenanthroline, has been carried out. It was demonstrated that 3-4 inhibitor molecules are involved in the interaction with protein kinase.     

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: An insight on the sulfide binding-site

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b–e) and four linker chains (L1–L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a–f) and five for C1 (a–e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a Mr of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vetimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the “strain A” family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs. Proteins 29:562–574, 1997. © 1997 Wiley-Liss, Inc.     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Functional differentiation in trematode hemoglobin isoforms.

The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis. The Hbs are monomeric and resemble the so-called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO. O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half-saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H-bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7-Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs. The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform. The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990-998.] Intermediate state(s) due to slow reorientation of E7-Tyr may account for this difference. Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs. In marked contrast to vertebrate Hbs, CO does not seem to compete effectively with O2 in trematode Hbs, as evident from partition coefficient values (M) below 1.     

The ontogeny of multiple hemoglobins in Chironomus thummi (Diptera): The effects of a compound with juvenile hormone activity

The multiple hemoglobins (Hbs) of Chironomus thummi show distinct and significant ontogenetic changes during development from the third instar through the fourth instar and metamorphosis into the pupa. A total of nine Hbs are resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hbs 2 and 3, which are stage specific for the fourth instar, are first detected on the fourth day of this stage by electrophoresis and immunoprecipitation. Hb 4 is the predominant Hb species in the early and middle fourth instar, but during the late fourth instar and prepupa, Hb 1 predominates. The concentrations of Hbs 5–9 remain relatively constant in middle instars and decrease during later development. The Hb content of larval hemolymph exhibits changes that coincide with developmental stages; molting is characterized by low Hb content, whereas, the hemolymph of intermolt animals contains relatively high levels of Hbs. Treatment of fourth instars with a juvenile hormone analog, Altosid, prolongs this stage and inhibits the progress of normal development resulting in the formation of larval-pupal intermediates. Altosid also appears specifically to inhibit the accumulation of soluble hemolymph proteins related to pupation and metamorphosis, without affecting the concentration of Hb. Most significantly, it induces the precocious appearance of Hbs 2 and 3, which remain elevated above control levels in the late larval and prepupal stages. The present results strongly suggest that Altosid stimulates the appearance and accumulation of larval-specific proteins in vivo, while it inhibits the appearance of pupation-related proteins.     

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

Quadrupole time-of-flight mass spectrometry of the native hemocyanin of the deep-sea crab Bythograea thermydron

Since electrospray ionization mass spectrometry (ESI-MS) has demonstrated capabilities for observing intact, weak interactions, there has been increasing interest in studying by this method noncovalently bound complexes. In this communication, we report for the first time the structure obtained by a commercial ESI quadrupole time-of-flight spectrometer on a native hemocyanin of deep-sea crab Bythograea thermydron with a molecular mass of 1.3 MDa. ESI-MS analysis of the native hemocyanin revealed the formation of a 18-mer noncovalent assembly with a measured molecular mass of 1354940 +/- 480 Da. ESI-MS data also revealed that this huge structure is an equilibrium with several assemblages, dodecamer (measured molecular weight = 902570 +/- 110 Da), hexamer (measured molecular weight = 450310 +/- 260 Da), and monomeric structures (measured molecular weight = 74999 +/- 85 Da).     

Hemoglobin M: effect of the proximal or distal histidine replacement on circular dichroism in the visible region

To examine the effects of a replacement of the proximal or the distal histidine on the structure of hemoglobin (Hb), absorption and circular dichroic (CD) spectra of five species of Hbs M in the visible region were measured. Four Hbs M had a characteristic but a similar absorption spectrum upon amino acid substitution, however, the proximal histidine replaced Hbs M (Hb M Iwate and Hb M Hyde Park) showed considerably different CD spectra from those of the distal histidine replaced ones (Hb M Boston and Hb M Saskatoon). The former exhibited large positive CD but the latter gave a complex CD spectrum with positive and negative extrema. On the other hand, absorption and CD spectra of Hb M Milwaukee did not changed very much from those of Hb A.     

Analyses of In Vitro Nonenzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.     

Variant subunit specificity in the quaternary structure of Artemia hemoglobin

The brine shrimp Artemia has three extracellular hemoglobins (Hbs) that are developmentally expressed and exhibit distinct oxygen-binding characteristics (Heip, Moens, and Kondo 1978; Heip et al. 1978 ). These Hbs are composed of two polymers, each of which comprises nine covalently linked globin domains. Although the cDNA sequences of two nine-domain globins from Artemia have been published, there is evidence for the existence of further expressed globin genes (Manning, Trotman, and Tate 1990 ). In the present study extensive analysis at the cDNA and genomic levels was performed in order to determine the globin gene copy number in Artemia. Sequence and Southern analysis suggest that four Hb polymers (T1, T2, C1, and C2) are expressed in Artemia. In addition, there is also at least one globin pseudogene. Protein sequencing of the native Hbs revealed that there are limitations on which two polymers can associate. The composition of the Hbs has been determined to be: Hb I, C1C2; Hb II, C1T2; and Hb III, T1T2. These pairings allow the levels of the three Artemia Hbs to be regulated independently by polymer expression alone, therefore explaining the previously inconsistent developmental and hypoxia-induced expression patterns.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

Surprising differences in the respiratory protein of insects: A spectroscopic study of haemoglobin from the European honeybee and the malaria mosquito

Only recently it was discovered that haemoglobin (Hb) belongs to the standard gene repertoire of insects, although their tracheal system is used for respiration. A classical oxygen-carrying function of Hb is only obvious for hexapods living in hypoxic environments. In other insect species, including the common fruit fly Drosophila melanogaster, the physiological role of Hb is yet unclear. Here, we study recombinant haemoglobin from the European honeybee Apis mellifera (Ame) and the malaria mosquito Anopheles gambiae (Aga). Spectroscopic evidence shows that both proteins can be classified as hexacoordinate Hbs with a strong affinity for the distal histidine. AgaHb1 is proposed to play a role in oxygen transport or sensing based on its multimeric state, slow autoxidation, and small but significant amount of five-coordinated haem in the deoxy ferrous form. AmeHb appears to behave more like vertebrate neuroglobin with a complex function given its diversified distribution in the genome.     

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.