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A group of inherited blood defects is known as Thalassemia is among the world’s most prevalent hemoglobinopathies. Thalassemias are of two types such as Alpha and Beta Thalassemia. The cause of these defects is gene mutations leading to low levels and/or malfunctioning α and β globin proteins, respectively. In some cases, one of these proteins may be completely absent. α and β globin chains form a globin fold or pocket for heme (Fe++) attachment to carry oxygen. Genes for alpha and beta-globin proteins are present in the form of a cluster on chromosome 16 and 11, respectively. Different globin genes are used at different stages in the life course. During embryonic and fetal developmental stages, γ globin proteins partner with α globin and are later replaced by β globin protein. Globin chain imbalances result in hemolysis and impede erythropoiesis. Individuals showing mild symptoms include carriers of alpha thalassemia or the people bearing alpha or beta-thalassemia trait. Alpha thalassemia causes conditions like hemolytic anemia or fatal hydrops fetalis depending upon the severity of the disease. Beta thalassemia major results in hemolytic anemia, growth retardation, and skeletal aberrations in early childhood. Children affected by this disorder need regular blood transfusions throughout their lives. Patients that depend on blood transfusion usually develop iron overload that causes other complications in the body systems like renal or hepatic impairment therefore, thalassemias are now categorized as a syndrome. The only cure for Thalassemias would be a bone marrow transplant, or gene therapy with currently no significant success rate. A thorough understanding of the molecular basis of this syndrome may provide novel insights and ideas for its treatment, as scientists have still been unable to find a permanent cure for this deadly disease after more than 87 years since it is first described in 1925.

AIMS The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. BACKGROUND For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as "Synechocystis hemoglobin (SynHb)". The "three histidines" linkages to heme are novel to this cyanobacterial hemoglobin. OBJECTIVE Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. METHODS Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. RESULTS The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a "molten globule" like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. CONCLUSION Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

Hemoglobins (Hbs) are evolutionarily conserved small globular proteins with characteristic 3-over-3 α-helical sandwich structure that is typically known as “globin fold”. Hbs have been found to be involved in diverse biological functions and the characteristic property of oxygen transportation is relatively a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2, and glob3) and it was previously reported that adequate expression of glob1 is required for various aspects of development, and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of glob1 gene in Drosophila development. We demonstrate a dynamic expression pattern of glob1 in larval tissues which largely concentrate around F-actin rich structures and also co-precipitate. Reduced expression of glob1 leads to developmental abnormalities which appeared to be largely mediated by inappropriately formed F-actin based cytoskeletal structures. Our subsequent analysis in FLP/FRT mediated somatic clones establishes specific role of Drosophila glob1 in maintenance of the integrity of F-actin based cytoskeleton during development. For the first time, we report interaction between Glob1 and actin, and propose a novel role of glob1 in maintenance of F-actin based cytoskeleton in Drosophila.

In this study, the structural motifs that can be represented as combinations of small motifs such as β-hairpins, S- and Z-like β-sheets and βαβ-units, and the П-like module are described and analyzed. The П-module consists of connected elements of the β-strand-loop-β-strand type arranged in space so that its overall fold resembles a clip or the Greek letter П. In proteins, the П-module itself and the structural motifs containing it exhibit unique overall folds and have specific sequence patterns of the key hydrophobic, hydrophilic and glycine residues. All this together enables us to conclude that these structural motifs can fold independently of the remaining part of the molecule and can act as nuclei and/or “ready-made” building blocks in protein folding. This article is protected by copyright. All rights reserved.

Latex clearing proteins (Lcps) are rubber oxygenases that catalyse the extracellular cleavage of poly (cis-1,4-isoprene) by Gram-positive rubber degrading bacteria. Lcp of Streptomyces sp. K30 (LcpK30) is a b-type cytochrome and acts as an endo-type dioxygenase producing C20 and higher oligo-isoprenoids that differ in the number of isoprene units but have the same terminal functions, CHO-CH2– and –CH2-COCH3. Our analysis of the LcpK30 structure revealed a 3/3 globin fold with additional domains at the N- and C-termini and similarities to globin-coupled sensor proteins. The haem group of LcpK30 is ligated to the polypeptide by a proximal histidine (His198) and by a lysine residue (Lys167) as the distal axial ligand. The comparison of LcpK30 structures in a closed and in an open state as well as spectroscopic and biochemical analysis of wild type and LcpK30 muteins provided insights into the action of the enzyme during catalysis.

Globin coupled sensors (GCS) are O2-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O2 dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O2 dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

Background: Hemoglobins (Hbs) are evolutionarily conserved heme-containing metallo-proteins of the “Globin” protein family which harbour the characteristic “globin fold”. Hemoglobins have been functionally diversified during evolution and its usual property of oxygen transport is rather a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2 and glob3) and we have reported earlier that adequate expression of glob1 is required for the various aspects of development and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of Drosophila globin1 in progression of oogenesis. Results: We demonstrate a dynamic expression pattern of glob1 in somatic and germ cell derivatives of developing egg chambers during various stages of oogenesis which largely confines around the F-actin rich cellular components. Reduced expression of glob1 leads to various types of abnormalities during oogenesis which were primarily mediated by the inappropriately formed F-actin based cytoskeleton. Our subsequent analysis in the somatic and germ line clones shows cell autonomous role of glob1 in the maintenance of the integrity of F-actin based cytoskeleton components in the somatic and germ cell derivatives. Conclusions: Our study establishes a novel role of glob1 in maintenance of F-actin based cytoskeleton during progression of oogenesis in Drosophila. This article is protected by copyright. All rights reserved.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.

The Rossmann fold is one of the most common and widely distributed super-secondary structures. It is composed of a series of alternating beta strand (β) and alpha helical (α) segments wherein the β-strands are hydrogen bonded forming a β-sheet. The initial beta-alpha-beta (βαβ) fold is the most conserved segment of Rossmann folds. As this segment is in contact with the ADP portion of dinucleotides such as FAD, NAD, and NADP it is also called as an "ADP-binding βαβ fold". The Proteopedia entry on the Rossmann fold (Available at: http://proteopedia.org/w/Rossmann_fold) was generated to illustrate several structural aspects of super families of FAD and NAD(P) binding proteins: (1) The coenzymes FAD and NAD(P) share the basic adenosine diphosphate (ADP) structure. (2) The βαβ fold motif that is common to both FAD and NAD(P) binding enzymes accommodates the common ADP component of these two coenzymes. (3) In both FAD and NAD(P) binding sites, the tight turn between the first β-strand and the α-helix is in contact with the two phosphate groups of ADP. (4) This hairpin curve includes the first two conserved glycines (Gly-x-Gly) that allow the sharp turn of the polypeptide backbone. (5) The two β-strands of the βαβ fold may constitute the core of a larger β-sheet that may include up to seven β-strands generally in parallel orientation. (6) The structures of segments between additional strands vary greatly and may be composed of a variety of structures such as multiple short helices or coils.

My first experiences with crystallography were as a graduate student at the University of Glasgow determining the structure of aromatic hydrocarbons using only primitive hand calculation techniques. I had my first experience using electronic computers as a post-doc in the lab of William Lipscomb in Minnesota. That gave me a good preparation for the structure determination of haemoglobin with Max Perutz in Cambridge. The discovery of the conserved globin fold had a major impact on my subsequent activities. It also provided the foundation for the development of the molecular replacement method and non-crystallographic symmetry electron density averaging that was essential for the structure determination of simple icosahedral viruses. The structure determination of a common cold virus was also a major stimulus for the study of more complex viruses and the development of the use of cryo-electron microscopy. This provided the basis for the investigation of lipid-enveloped viruses such as Sindbis virus and deng...

Trajectories of diatomic ligands in heme proteins have provided a wealth of information on how such molecules can enter and exit the protein, which must fluctuate in order to permit their passage. The time-resolved X-ray crystallography technique has provided the most detailed images of the transport of the carbon monoxide ligand (CO) to various cavities within proteins (1–5). However, the function of protein cavities is still an open question. Indeed, it is difficult to assign a general function based on the analysis of a single protein, since the functional role of cavities may vary from one protein to another. Thus far, two heme proteins, Sperm Whale myoglobin (SWMb) (2–3) and Scapharca inaequivalvis dimeric hemoglobin (HbI) (4–5), have been the most extensively studied by time-resolved crystallography. Although one is a myoglobin and the other a hemoglobin they both have the reversible binding of O2 as their primary function. Despite the rich set of conformational changes associated with the allosteric cooperative transition in photolyzed HbI*CO, the nature of the CO trajectory in that globin has certain similarities with SWMb*CO (The asterisk indicates a photolyzed carbon monoxide molecule, following photon absorption by the heme that leads to rupture of the Fe-CO bond). Both have a distal docking site B, where the initially photolyzed CO molecule resides. The B-site in both proteins is a distinct ligand docking site in the distal pocket, not observed as the Xe binding site in either proteins. (2, 4) Based on experiments designed to block the Xe-binding cavities in SWMb*CO and HbI*CO, and on time-resolved crystallographic studies, it has been deduced that the cavities in these proteins do not constitute an exit route for diatomic ligands (6–7). Instead, exit occurs near the distal histidine in both SWMb*CO and HbI*CO. Moreover, previous temperature derivative spectroscopy (TDS) studies show the presence of a nearby docking site and one or more further secondary sites in SWMb (8–10) and HbI (11) where photolyzed CO molecules can reside. Why then does CO migrate to a number of Xe-binding cavities in these proteins after it leaves the B-site? This fundamental question has been studied extensively in SWMb using many spectroscopic techniques including TDS and methods such as kinetic hole burning (12), which makes the connection between the conformation-dependent energy of recombination and the spectroscopic energy of a heme charge transfer band. In this study we propose that in order to understand the possible function of the cavities in various heme proteins, it is necessary to compare a protein with a significant difference in function. We conclude that ligand dynamics studied by time-resolved X-ray and the protein cavity in DHP identified by a Xe binding site (13) are consistent with an open architecture in the distal pocket of the bifunctional hemoglobin, DHP, which explains how it can carry out multiple functions. These features distinguish DHP from the more specialized oxygen transport proteins SWMb and HbI. Dehaloperoxidase-hemoglobin (DHP), first isolated from the terebellid polychaete Amphitrite ornata, is a bifunctional protein, which displays significant peroxidase activity under physiological conditions, while also having a globin fold and an associated oxygen transport function (14). As a hemoglobin, DHP can reversibly bind with O2 and as a peroxidase, DHP can oxidize 2,4,6-trihalophenol into the corresponding 2,6-dihaloquinone in the presence of H2O2. However, normal peroxidase function inactivates the ability of DHP to bind oxygen leading to a paradox because of the failure of the DHP hemoglobin function. (15) Recently, this functional paradox of DHP has been resolved by establishing that oxyferrous DHP can serve as the resting state of the peroxidase reaction cycle. (16–17) For a typical peroxidase, e.g. Horseradish Peroxidase and Cytochrome c Peroxidase, the ferric state of the heme Fe is the resting state. Thus, DHP appears to have a new type of peroxidase cycle, a ferrous peroxidase cycle. (15) Although it has high peroxidase activity, the oxyferrous peroxidase cycle and unique internal binding sites possessed by DHP distinguish it from other heme peroxidases. (18–19) The replacement of O2 bound to the heme Fe by H2O2 required by the ferrous peroxidase mechanism suggests DHP must simultaneously accommodate both O2 and H2O2 in the distal pocket in order to function. This requirement is quite different from other well-studied hemoglobins and myoglobins. For example, SWMb, which is by far the best studied protein, cannot simultaneously accommodate both CO and H2O in the distal pocket (20–22). The reasons for this may become clearer in comparison with the bifunctional protein, DHP, which has a different requirement for the function of the distal pocket, namely entry of multiple molecules both for activation and for inhibition of enzymatic function. In order to address the dynamics of diatomic ligands in DHP, we have conducted a time-resolved X-ray crystallography study of the trajectory of photolyzed carbon monoxide in DHP*CO. The time-dependent structures reveal a qualitatively different CO trajectory than those observed previously in SWMb*CO and HbI*CO.

article i nfo Genome of the model dicot flowering plant, Arabidopsis thaliana, a popular tool for understanding molecular biology of plant physiology, encodes all three classes of plant hemoglobins that differ in their sequence, ligand binding and spectral properties. As such these globins are of considerable attention. Crystal structures of few members of plant class I nonsymbiotic hemoglobin have been described earlier. Here we report the crystal structure of Arabidopsis class I hemoglobin (AHb1) to 2.2 Ǻ and compare its key features with the structures of similar nonsymbiotic hemoglobin from other species. Crystal structure of AHb1 is homologous to the related members with similar globin fold and heme pocket architecture. The structure is homodimeric in the asymmetric unit with both distal and proximal histidines coordinating to the heme iron atom. Residues lining the dimeric interface are also conserved in AHb1 with the exception of additional electrostatic interac- tion between H112 and E113 of each subunit and that involving Y119 through two water molecules. In addi- tion, differences in heme pocket non-covalent interactions, a novel Ser residue at F7 position, Xe binding site variability, internal cavity topology differences, CD loop conformation and stability and other such properties might explain kinetic variability in AHb1. Detailed cavity analysis of AHb1 showed the presence of a novel long tunnel connecting the distal pockets of both the monomers. Presence of such tunnel, along with confor- mational heterogeneity observed in the two chains, might suggest cooperative ligand binding and support its role in NO scavenging. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. © 2013 Elsevier B.V. All rights reserved.

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-Delta N20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-Delta N20Z mutant)] have been investigated. In keeping with the MaPgb*-Delta N20 mutant crystal structure, here reported at 2.0 angstrom resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-Delta N20. CO binding to ferrous MaPgb*-Delta N20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of alpha-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. (c) 2013 Elsevier B.V. All rights reserved

This study reports a comparative analysis of the topological properties of inner cavities and the intrinsic dynamics of non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana. The two proteins belong to the 3/3 globin fold and have a sequence identity of about 60%. However, it is widely assumed that they have distinct physiological roles. In order to investigate the structure–function relationships in these proteins, we have examined the bis-histidyl and ligand-bound hexacoordinated states by atomistic simulations using in silico structural models. The results allow us to identify two main pathways to the distal cavity in the bis-histidyl hexacoordinated proteins. Nevertheless, a larger accessibility to small gaseous molecules is found in AHb2. This effect can be attributed to three factors: the mutation Leu35(AHb1) → Phe32(AHb2), the enhanced flexibility of helix B, and the more favorable energetic profile for ligand migration to the distal cavity. The net effect of these factors would be to facilitate the access of ligands, thus compensating the preference for the fully hexacoordination of AHb2, in contrast to the equilibrium between hexa- and pentacoordinated species in AHb1. On the other hand, binding of the exogenous ligand introduces distinct structural changes in the two proteins. A well-defined tunnel is formed in AHb1, which might be relevant to accomplish the proposed NO detoxification reaction. In contrast, no similar tunnel is found in AHb2, which can be ascribed to the reduced flexibility of helix E imposed by the larger number of salt bridges compared to AHb1. This feature would thus support the storage and transport functions proposed for AHb2. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Some carbohydrate-active enzymes display a modular structure in which catalytic modules that target an insoluble substrate are often attached to one or more noncatalytic carbohydrate-binding modules (CBMs) that assist enzymatic activity. CBMs have been classified into more than 60 families based on amino acid sequence similarities. CBM family 13 (CBM13) and family 42 (CBM42) possess a β-trefoil fold and are grouped into CBM fold family 2. The β-trefoil fold contains a sequence of approximately 45 amino acid residues that is repeated 3 times, resulting in three subdomains (α, β and γ) that fold into an overall globular structure. Each subdomain is composed of four β-strands that fold into a Y-shaped β-hairpin structure. CBM13 and CBM42 have multivalent sugar-binding ability. In this review, I describe the sugar-binding mechanisms of the CBM13 and CBM42 domains of a β-xylanase, a β-L-arabinopyranosidase, and an α-L-arabinofuranosidase.

Arc repressor is a homodimeric protein with a ribbon-helix-helix fold. A single polar-to- hydrophobic substitution (N11L) at a solvent-exposed position leads to population of an alternate dimeric fold in which 310 helices replace a b-sheet. Here we find that the variant Q9V/N11L/R13V (S-VLV), with two additional polar-to-hydrophobic surface mutations in the same b-sheet, forms a highly stable, reversibly folded octamer with approximately half thea-helical content of wild-type Arc. At low protein concentration and low ionic strength, S-VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9-14 of S-VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre-amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space.

Globins are heme containing proteins with the capability to bind oxygen reversibly. All globins can be classified as M (myoglobin-like), S (sensor) and T (truncated) globins; among them, M and S globins have a typical 3/3 α-helical globin fold and T globins have a 2/2 α-helical fold. Till date, all the myxobacterial genomes available in NCBI data base harbor one or more genes for globins but none of the globins from this group of bacteria have been characterized as yet. In this study, in-silico analysis and homology modeling approach was exploited to explore the physiochemical properties and 3-D structure of trHbN (group I T-globin) of Myxococcus xanthusDK1622, a fruiting myxobacteria. After generating the 3-D model, assessment of the structure and stereo-chemical analysis was carried using different evaluation and validation tools.As homologs of the trHbN exist in dimeric as well as monomeric form, dimerization studies were performed and it was observed that dimeric form of the protein is more stable as compared to the monomer. Further, heme group was appended to the model structure of trHbN of M. xanthus by Patchdock web server and model-heme complex was further refined by Firedock.The 3-D model structure will enhance the understanding for functional analysis of experimentally derived crystal structure.

Recently the existence of prototype mutants in β globin genes was proved. In this study a further and similar investigation was performed. The investigation concerns the Homo sapiens α globin. The prototype mutants were screened in the α globin DNA sequences. The results proved their existence. A selected α globin fragment contains the prototype mutant was used for searching both of the nucleotide and protein databases. The used fragment consists of nine nucleotides GGC AAG GTT and contain two silent mutants with the following amino acids sequences GKV. Searching the Blast protein database for the existence of correct mutants using the V00493.7 Homo sapiens 27 amino acids fragment was led to find 50 different sequences. Interestingly the protein sequences show similarity with marsupials, even-toed ungulates, odd-toad ungulates, placental, bats, rodents, carnivores, whales and dolphins; primates, insectivores, rabbits and hares. Species responsible for human bit or blood transfer are located and suggest gene recombination between different species occure through blood transfer. The restriction map for the 81 nucleotides for four nucleotide sequences (one prototype mutant, two correct mutants, and one normal) proved change in the restriction enzymes profile of the α globin sequences. That might be a proof for the existence of reduced α+ globin (also in case of β+ globin) macromolecules. The protein modelling in the selected different fragments show differences in the 3D structure and in their hydrophobicity. The unexpected exo-sources for the α globin put another load on the possibility of the haemoglobin illness solving. However, avoiding marriage from relatives, and from the same ethnic groups might be the best choice. Additionally avoiding blood contact with animals have compatible mutants is recommended. Further investigations are in need to prove/disprove the role of the gene recombination between species through blood in the transfer of the α globin mutants or even other ones.

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.

Summary Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions. 2011 IUBMB IUBMB Life, 63(3): 166–174, 2011

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 Å. The overall fold is a classic three-on-three α-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered α-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency νFe-His stretching mode (251 cm−1), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (−134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.

A binding site for iron protoporphyrin IX (heme) was designed and embedded in a photosynthetic non-heme protein, phycocyanin, which forms a globin-like backbone structure, called a globin fold, but lacks sequence similarity to the globin family containing myoglobins and hemoglobins. Based on the structural alignment of the phycocyanin and myoglobin molecules, the proximal and distal His residues were repositioned in the phycocyanin sequence for heme ligation. The heme-binding pocket was created around the His residues by several residue replacements in the phycocyanin core. The synthesized phycocyanin variant, designated as HPY, bound one heme per protein molecule and showed spectroscopic features characteristic of six-coordinated heme proteins. The heme-binding HPY exhibited redox activity with an electrochemical midpoint potential of -130 mV against the standard hydrogen electrode, which was approximately 200 mV lower than the potential of natural myoglobins but 50 mV higher than the typical values of designed heme proteins with four-helix bundle or globin scaffolds. HPY also displayed native-like folding properties, in contrast to these designed heme proteins. However, the bound ferrous heme of HPY was quickly reoxidized by air and did not stably bind O(2), unlike the natural globins. The present results demonstrated that the globin fold of a non-globin protein is suitable for binding heme but is not sufficient for the reversible O(2) binding and myoglobin functions. The comparison of HPY with the natural globins may yield new insights into the essential features for realizing the natural heme protein functions.

Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent α globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human α globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to β globin. We analyzed 6 human α globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and β globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N α globins bind β globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coli and erythroid cells, α globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some α globin variants and support the hypothesis that AHSP promotes α globin chain stability during human erythropoiesis.

The crystal structure of the uncharacterized protein SO2946 from Shewanella oneidensis MR-1 was determined with single-wavelength anomalous diffraction (SAD) and refined to 2.0 Å resolution. The SO2946 protein consists of a short helical N-terminal domain and a large C-terminal domain with the “jelly-roll” topology. The protein assembles into a propeller consisting of three C-terminal blades arranged around a central core formed by the N-terminal domains. The function of SO2946 could not be inferred from the sequence since the protein represents an orphan with no sequence homologs, but the protein’s structure bears a fold similar to that of proteins containing carbohydrate-binding modules. Features such as fold conservation, the presence of a conserved groove and a metal binding region are indicative that SO2946 may be an enzyme and could be involved in binding carbohydrate molecules.

SUMMARY The importance of molybdoenzymes is exemplified both by the debilitating and fatal human diseases caused by their deficiency and by their persistence throughout evolution. Here, we show that the protein fold of the molybdopyranopterin-containing domain of sulfite oxidase (the SUOX fold) can be found in all three domains of life. Analyses of sequence data and protein structure comparisons (secondary structure matching) show that the SUOX fold is found in enzymes that have quite distinct macromolecular architectures comprising one or more domains and sometimes subsidiary subunits. These are summarized as follows: (i) animal SUOXs that contain an N-terminal cytochrome b 5 domain and an SUOX fold fused to a C-terminal dimerization domain; (ii) plant SUOX that contains an SUOX fold fused to a C-terminal dimerization domain; (iii) the YedY protein from Escherichia coli , which comprises only the SUOX fold; (iv) the sulfite dehydrogenase from Starkeya novella that contains the SUOX fold, a dimerization domain, and an additional c -type cytochrome subunit; and (v) the plant-type nitrate reductases, exemplified by that of Pichia angusta , that contain an N-terminal SUOX fold, a dimerization domain, a cytochrome b 5 domain, and a C-terminal NADH binding flavin adenine dinucleotide-containing domain. We used the primary sequences of the proteins containing an SUOX fold to mine 559 sequences of related proteins. A phylogeny of a nonredundant subset of these sequences was generated, and the resultant clades were categorized by sequence motif analyses in the context of the available protein structures. Based on the motif analyses, cladistics, and domain conservations, we are able to postulate a plausible pathway of SUOX fold enzyme evolution.

The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 A using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the alpha/beta-hydrolase fold family. Structural superposition onto representative alpha/beta-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands beta6 and beta7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent Delta G degrees (UN(w))=-3.1+/-0.3 kcal mol(-1); m(UN)=-1.7 kcal mol(-1) M(-1)] and predominant alpha-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (beta(I)=0.9+/-0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.

Neuroglobin (Ngb), a recently discovered ancient heme protein, presents the typical globin fold and is around 20% identical to myoglobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.

Neuroglobin (Ngb), a protein in the globin family, is found in vertebrate brains. It binds oxygen reversibly. Compared with myoglobin (Mb), the amino acid sequence has limited similarity, but key residues around the heme and the classical globin fold are conserved in Ngb. The CO adduct of Ngb displays two CO absorption bands in the IR spectrum, referred to as N 3 (distal histidine in the pocket) and N 0 (distal histidine swung out of the pocket), which have absorption spectra that are almost identical with the Mb mutants L29F and H64V, respectively. The Mb mutants mimic the heme pocket structures of the corresponding Ngb conformers. The equilibrium protein dynamics for the CO adduct of Ngb are investigated by using ultrafast 2D-IR vibrational echo spectroscopy by observing the CO vibration's spectral diffusion (2D-IR spectra time dependence) and comparing the results with those for the Mb mutants. Although the heme pocket structure and the CO FTIR peak positions of Ngb are similar to those of the mutant Mb proteins, the 2D-IR results demonstrate that the fast structural fluctuations of Ngb are significantly slower than those of the mutant Mbs. The results may also provide some insights into the nature of the energy landscape in the vicinity of the folded protein free energy minimum.

The \beta-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, \beta-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the \beta-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a \beta-prism I fold lectin, griffthsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of \beta-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion.It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the \beta-prism II fold, is related to the role of plant lectins in defence.

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His64(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction-modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel beta-sheet that forms a 'half pipe'. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg(2+). These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.

Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions.

To study the sequence determinants governing protein fold evolution, we generated hybrid sequences from two homologous proteins with 40% identity but different folds: Pfl 6 Cro, which has a mixed α + β structure, and Xfaso 1 Cro, which has an all α-helical structure. First, we first examined eight chimeric hybrids in which the more structurally conserved N-terminal half of one protein was fused to the more structurally divergent C-terminal half of the other. None of these chimeras folded, as judged by circular dichroism spectra and thermal melts, suggesting that both halves have strong intrinsic preferences for the native global fold pattern, and/or that the interfaces between the halves are not readily interchangeable. Second, we examined 10 hybrids in which blocks of the structurally divergent C-terminal region were exchanged. These hybrids showed varying levels of thermal stability and suggested that the key residues in the Xfaso 1 C terminus specifying the all-α fold were concentrated near the end of helix 4 in Xfaso 1, which aligns to the end of strand 2 in Pfl 6. Finally, we generated hybrid substitutions for each individual residue in this critical region and measured thermal stabilities. The results suggested that R47 and V48 were the strongest factors that excluded formation of the α + β fold in the C-terminal region of Xfaso 1. In support of this idea, we found that the folding stability of one of the original eight chimeras could be rescued by back-substituting these two residues. Overall, the results show not only that the key factors for Cro fold specificity and evolution are global and multifarious, but also that some all-α Cro proteins have a C-terminal subdomain sequence within a few substitutions of switching to the α + β fold.

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-angstrom resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix. Proteins 2007. © 2006 Wiley-Liss, Inc.

A number of homo- and heterodimeric hemoglobins has been identified in several phyla of invertebrates, lower vertebrates and even in unicellular organisms (bacteria, algae, fungi). These hemoglobins are characterized by different modes of assembly of the typical globin fold and hence represent ideal systems for testing thermodynamic models and mechanistic hypotheses aimed at explaining cooperative phenomena. In turn, the structural diversity of the subunit interfaces implies that in dimeric hemoglobins cooperativity in ligand binding can arise from quite diverse molecular mechanisms. The homodimeric hemoglobin fromScapharca inaequivalvis (HbI) is prototypic of systems where cooperative ligand binding originates within stable dimers and is thus mainly homotropic in nature. The availability of high resolution crystallographic data and the extensive characterization of the dynamic and equilibrium features of the ligand binding reactions allow a detailed description of the events that lead from the unliganded to the liganded form. InScapharca HbI cooperativity results primarily from tertiary rather than quaternary structural rearrangements. It can be ascribed to direct communication between the heme groups that are in close contact across the subunit interface, with the propionate groups providing a direct pathway for heme-heme interaction. The cooperative mechanism entails the ligand-triggered sinking of the heme into its pocket and the movement of key residues,e.g. the extrusion of Phe 97 from the proximal side of the heme into the subunit interface. In turn, the reorientation of the Phe 97 side chain brings about changes in the number of ordered interfacial water molecules. The oxygen binding isotherms of HbI can be described in terms of a simp le two-state MWC model under a symmetry constraint that imposes a constant KR/KT ratio. Kinetically, cooperative ligand binding manifests itself in the rate of oxygen dissociation and in the rate of carbonmonoxide combination. Several independent specroscopic probes indicate that the structural rearrangements accompanying ligand binding take place in a concerted fashion in the μsecond time-regime.

Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.