Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

A myoglobin evolved from indoleamine 2,3-dioxygenase.

Hemoglobins and myoglobins are some of the best studied proteins. They are distributed in animals, plants and bacteria, and the characteristic two intron-three exon structure is widely conserved in animal globin genes (Jhiang et al., 1988). To date, all of the hemoglobins and myoglobins are believed to have a common origin, and so they are considered to be homologous. We have isolated a completely new type of myoglobin from the red muscle of the abalone Sulculus diversicolor aquatilis. The myoglobin consists of an unusual 41 kDa polypeptide chain, contains one heme per chain and forms a homodimer under physiological conditions. The cDNA-derived amino acid sequence of Sulculus myoglobin showed no significant homology with any other globins, but, surprisingly, showed high homology (35% identity) with human indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. This clearly indicates that Sulculus myoglobin evolved from a gene for indoleamine dioxygenase, but not from a globin gene. Sulculus myoglobin lacks the enzyme activity of indoleamine dioxygenase. However, in the presence of tryptophan, the autoxidation rate of oxymyoglobin was greatly accelerated, suggesting that a tryptophan binding site remains near or in the heme cavity as a relic of the molecular evolution.     

Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

Isolation and cDNA-derived amino acid sequences of hemoglobin and myoglobin from the deep-sea clam Calyptogena kaikoi

The heterodont clam Calyptogena kaikoi, living in the cold-seep area at a depth of 3761 m of the Nankai Trough, Japan, has abundant hemoglobins and myoglobins in erythrocytes and adductor muscle, respectively. Two types of hemoglobins (Hb I and Hb II) were isolated, and the complete amino acid sequences of Hb I (145 residues) and Hb II (137 residues) were obtained with combination of cDNA and protein sequencing. The amino acid sequences of C. kaikoi Hbs I and II differed from homologous chains of the congeneric clam Calyptogena soyoae in eight and five positions, respectively. The distal (E7) His, one of the functionally important residues in hemoglobin and myoglobin, was replaced by Gln in hemoglobins of C. kaikoi. A phylogenetic analysis of clam hemoglobins indicates that the evolutionary rate of Calyptogena hemoglobins is rather faster than those of other clams, suggesting that the mutation rate might be accelerated in the deep-sea animals around the areas of cold seeps or hydrothermal vents. On the other hand, it was found unexpectedly that two myoglobins Mbs I and II, isolated from the red adductor muscle, are identical in amino acid sequence Hbs I and II, respectively. Thus it was assumed that genes for Hbs I and II are also expressed in the muscle of C. kaikoi in substitution for myoglobin gene. This suggests that the major physiological role of globins in C. kaikoi is storage of oxygen under the low oxygen conditions, rather than circulating of oxygen.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Amino acid sequence of the dimeric hemoglobin (Hb I) from the deep-sea cold-seep clam Calyptogena soyoae and the phylogenetic relationship with other molluscan globins

The deep-sea cold-seep clam Calyptogena soyoae has two homodimeric hemoglobins (Hbs I and II) in erythrocytes. The complete amino acid sequence of Hb I has been determined. It is composed of 144 amino acid residues, has a high content of hydrophobic residues, and a calculated molecular weight of 16,350 including a heme group. The sequence of Calyptogena Hb I showed high homology (42% identity) with that of Calyptogena Hb II (Suzuki, T., Takagi T. and Ohta, S. (1989) Biochem. J. 260, 177-182), although it has a long insertion of seven residues in the C-terminal region compared with Hb II. On the other hand, it showed low homology (12-20% identity) with other molluscan globins. As well as Hb II, Calyptogena Hb I lacked the N-terminal extension of 7-9 residues characteristic of molluscan intracellular hemoglobins, and the distal (E7) histidine was replaced by glutamine. A phylogenetic tree was constructed from 13 molluscan globins belonging to the five families Aplysiidae, Galeodidae, Potamididae, Arcidae and Vesicomyidae. The globin sequences of Calyptogena (Vesicomyidae) were found to be rather distant from other globin sequences, suggesting that they might conserve a primitive form of molluscan globins.     

Concentration-dependent effects of anions on the anaerobic oxidation of hemoglobin and myoglobin

The redox potentials of hemoglobin and myoglobin and the shapes of their anaerobic oxidation curves are sensitive indicators of globin alterations surrounding the active site. This report documents concentration-dependent effects of anions on the ease of anaerobic oxidation of representative hemoglobins and myoglobins. Hemoglobin (Hb) oxidation curves reflect the cooperative transition from the T state of deoxyHb to the more readily oxidized R-like conformation of metHb. Shifts in the oxidation curves for Hb A(0) as Cl(-) concentrations are increased to 0.2 m at pH 7.1 indicate preferential anion binding to the T state and destabilization of the R-like state of metHb, leading to reduced cooperativity in the oxidation process. A dramatic reversal of trend occurs above 0.2 m Cl(-) as anions bind to lower affinity sites and shift the conformational equilibrium toward the R state. This pattern has been observed for various hemoglobins with a variety of small anions. Steric rather than electronic effects are invoked to explain the fact that no comparable reversal of oxygen affinity is observed under identical conditions. Evidence is presented to show that increases in hydrophilicity in the distal heme pocket can decrease oxygen affinity via steric hindrance effects while increasing the ease of anaerobic oxidation.     

Modulation of the oxygen affinity of cobalt-porphyrin by globin

We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.     

The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Modification of isolated α and β chains of human hemoglobin by the metal tetrasulfonated phthalocyanines. Studies on hybrid phthalocyanine-heme intermediates

α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands

Three hemoglobins have been isolated from the symbiont-harboring gill of the bivalve mollusc Lucina pectinata. Oxyhemoglobin I (Hb I), which may be called sulfide-reactive hemoglobin, reacts with hydrogen sulfide to form ferric hemoglobin sulfide in a reaction that may proceed by nucleophilic displacement of bound superoxide anion by hydrosulfide anion. Hemoglobins II and II, called oxygen-reactive hemoglobins, remain oxygenated in the presence of hydrogen sulfide. Hemoglobin I is monomeric; Hb II and Hb III self-associate in a concentration-dependent manner and form a tetramer when mixed. Oxygen binding is not cooperative. Oxygen affinities are all nearly the same, P50 = 0.1 to 0.2 Torr, and are independent of pH. Combination of Hb I with oxygen is fast; k'on = (estimated) 100-200 x 10(6) M-1 s-1. Combination of Hb II and Hb III with oxygen is slow: k'on = 0.4 and 0.3 x 10(6) M-1 s-1, respectively. Dissociation of oxygen from Hb I is fast relative to myoglobin: koff = 61 s-1. Dissociation from Hb II and Hb III is slow: koff = 0.11 and 0.08 s-1, respectively. These large differences in rates of reaction together with differences in the reactions of carbon monoxide suggest differences in configuration of the distal heme pocket. The fast reactions of Hb I are comparable to those of hemoglobins that lack distal histidine residues. Slow dissociation of oxygen from Hb II and Hb III suggest that a distal residue may interact strongly with the bound ligand. We infer that Hb I may facilitate delivery of hydrogen sulfide to the chemoautotrophic bacterial symbiont and Hb II and Hb III may facilitate delivery of oxygen. The midpoint oxidation-reduction potential of the ferrous/ferric couple of Hb I, 103 +/- 8 mV, was independent of pH. Potentials of Hb II and Hb III were pH-dependent. At neutral pH all three hemoglobins have similar midpoint potentials. The rate constant for combination of ferric Hb I with hydrogen sulfide increases 3000-fold from pH 10.5 to 5.5, with apparent pK 7.0, suggesting that undissociated hydrogen sulfide is the attacking ligand. At the acid limit combination of ferric Hb I with hydrogen sulfide, k'on = 2.3 x 10(5) M-1 s-1, is 40-fold faster than combination with ferric Hb II or myoglobin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.     

Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues

Vertebrates possess multiple respiratory globins that differ in terms of structure, function, and tissue distribution. Three types of globins have been described so far: hemoglobin facilitates the transport of oxygen in the blood, myoglobin serves oxygen transport and storage in the muscle, and neuroglobin has a yet unidentified function in nerve cells. Here we report the identification of a fourth and novel type of globin in mouse, man, and zebrafish. It is expressed in apparently all types of human tissue and therefore has been called cytoglobin (CYGB). Mouse and human CYGBs comprise 190 amino acids; the zebrafish CYGB, 174 amino acids. The human CYGB gene is located on chromosome 17q25. The mammalian genes display a unique exon-intron pattern with an additional exon resulting in a C-terminal extension of the protein, which is absent in the fish CYGB. Phylogenetic analyses suggest that the CYGBs had a common ancestor with vertebrate myoglobins. This indicates that the vertebrate myoglobins are in fact a specialized intracellular globin that evolved in adaptation to the special needs of muscle cells.     

Nitrosyliron(III) hemoglobin: autoreduction and spectroscopy

Nitrosyl complexes of the iron(III) forms of myoglobin, human hemoglobin, Glycera dibranchiata hemoglobins (Hbm and Hbh), and model iron(II) and iron(III) synthetic porphyrins including octaethylporphyrin (OEP) have been prepared. The iron(III) heme proteins are electron spin (paramagnetic) resonance (ESR) silent, while hexacoordinate solution structures are indicated for [Fe(OEP)(NO)2]ClO4 and for Hbm(II)NO, which has an ESR spectrum similar to that of Mb(II)NO and the hexacoordinate iron(II) model complex Fe(OEP)NO(BzIm). The splitting of the alpha- and beta-bands in the optical spectrum of Mb(III)NO and Hbh(III)NO contrasts markedly with the sharp, single bands observed in that of Hbm-(III)NO. The nondegeneracy of the dxz and dyz orbitals in Mb(III)NO and Hbh(III)NO is attributed to the influence of the distal histidine. Circular dichroism spectra were obtained for Hbm(III)NO, Hbm(II)NO, Hbh(III)NO, Hbh(II)NO, Mb(II)NO, and Mb(III)NO. The vicinal chiral center contribution that governs the heme protein CD leads to low Kuhn anisotropies, which have been used to assign certain electronic transitions. The Hb(III)NO spectrum is not stable but transforms into that of Hb(II)NO. This autoredox process follows kinetics that are first order in FeIIINO. The relative rates of autoreduction (25 degrees C, 1 atm NO) are Mb(III)NO less than Hbm(III)NO less than Hb alpha(III)NO less than HbA(III)NO. At high NO partial pressure or after "recycling" of HbA, the rates of reduction decrease. The first step in the reaction of NO with the ferric heme is the reversible formation of the formally iron(III) adduct. This reacts with another molecule of NO, generating the final heme(II)-NO via nitrosylation of NO itself or of an endogenous nucleophile. Kinetic and spectroscopic evidence shows involvement of trans-heme-(NO)2 in the reaction. The activation parameters delta H and delta S were determined. The overall reaction is photoenhanced.