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Apis mellifera is an important provider of ecosystem services, and during flight and foraging behaviour is exposed to environmental pollutants including airborne particulate matter (PM). While exposure to insecticides, antibiotics, and herbicides may compromise bee health through alterations of the gut microbial community, no data are available on the impacts of PM on the bee microbiota. Here we tested the effects of ultrapure Titanium dioxide (TiO2) submicrometric PM (i.e., PM1, less than 1 µm in diameter) on the gut microbiota of adult bees. TiO2 PM1 is widely used as a filler and whitening agent in a range of manufactured objects, and ultrapure TiO2 PM1 is also a common food additive, even if it has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen in Group 2B. Due to its ubiquitous use, honey bees may be severely exposed to TiO2 ingestion through contaminated honey and pollen. Here, we demonstrated that acute and chronic oral administration of ultrapure TiO2 PM1 to adult bees alters the bee microbial community; therefore, airborne PM may represent a further risk factor for the honey bee health, promoting sublethal effects against the gut microbiota.

This laboratory has reported previously that fragmentation of brain mitochondrial fractions resulted in a 7–25 fold increase of the antimycin A-sensitive NADH oxidase, provided that ionic compounds such as inorganic phosphate were present in the incubation media used for enzymatic determination ( Giuditta and Strecker, 1959 ). The oxidation of NADH and of succinate by the Keilin-Hartree heart muscle preparation has been shown also to be stimulated by ionic compounds and by globin ( Keilin and Hartree, 1949 ; Slater, 1949 ; Bonner, 1954 ). Similar observations have been made now with liver and heart mitochondria. Evidence is presented to indicate that the stimulatory effects obtained with added ionic compounds depends at least partially on interaction with a heat stable non-dialyzable factor, which appears to be present exclusively in the mitochondrial fractions of heart, liver or brain homogenates.

The importance of the Arctic, in contributing to the knowledge of the overall ensemble of adaptive processes influencing the evolution of marine organisms, calls for investigations on molecular adaptations in Arctic fish. Unlike the vast majority of Antarctic Notothenioidei, several Arctic species display high hemoglobin multiplicity. The blood of four species, the spotted wolffish of the family Anarhichadidae and three Gadidae, contains three functionally distinct major components. Similar to many Antarctic notothenioids, Arctic Liparis tunicatus (suborder Cottoidei, family Liparidae) has one major hemoglobin (Hb 1) accompanied by a minor component (Hb 2). This paper reports the structural and functional characterisation of Hb 1 of L. tunicatus. This hemoglobin shows low oxygen affinity, and pronounced Bohr and Root effects. The amino-acid sequence of the β chain displays an unusual substitution in NA2 (β2) at the phosphate-binding site, and the replacement of Val E11 (β67) with Ile. Similar to some Antarctic fish Hbs, electron paramagnetic resonance spectra reveal the formation of a ferric penta-coordinated species even at physiological pH. The amino-acid sequences have also been used to gain insight into the evolutionary history of globins of polar fish. L. tunicatus globins appear close to the notothenioid clades as predicted by teleostean phylogenies. Close phylogenetic relationships between Cottoidei and Notothenioidei, together with their life style, seem to be the main factor driving the globin-sequence evolution.

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.

Since haemoglobins of all animal species have the same haem group, differences in their properties, including oxygen affinity, electrophoretic mobility and pH sensitivity, must result from the interaction of the prosthetic group with specific amino-acid residues in the primary structure. For this reason, fish globins have been the subject of extensive studies in recent years, not only for their structural characteristics, but also because they offer the possibility to investigate the evolutionary history of these ancient molecules in marine and freshwater species living in a great variety of environmental conditions. This review summarizes the current knowledge on the structure, function and phylogeny of haemoglobins of notothenioid fishes. On the basis of crystallographic analysis, the evolution of the Root effect is analysed. Adaptation of the oxygen transport system in notothenioids seems to be based on evolutionary changes, involving levels of biological organization higher than the structure of haemoglobin. These include changes in the rate of haemoglobin synthesis or in regulation by allosteric effectors, which affect the amount of oxygen transported in blood. These factors are thought to be more important for short-term response to environmental challenges than previously believed.

1. Bathydraco marri Norman is a cold-adapted Antarctic teleost (Family: Bathydraconidae), living preferably at depths between 400 and 1200 m. 2. The blood of this species contains a single hemoglobin, in which oxygen binding is pH-regulated (Bohr and Root effects). 3. The complete amino acid sequence of the alpha and beta chains of the hemoglobin of B. marri has been elucidated.

Liganded and unliganded forms of two Antarctic fish haemoglobins, from Trematomus newnesi and T. bernacchii, have been crystallized in low-salt media using polyethylene glycol as precipitant. In particular, crystals of air-exposed T. newnesi carbomonoxy haemoglobin were found to be isomorphous to the crystals grown in high-salt media. Preliminary X-ray analysis of the diffraction data revealed that the beta-haem iron of this haemoglobin is in the haemichrome state, with both the proximal and distal histidyl residues linked to the iron. This is the first crystallization of a haemichrome intermediate of a vertebrate haemoglobin.

In order to study the molecular mechanisms of enzyme cold adaptation, direct amino acid sequence, catalytic features, thermal stability and thermodynamics of the reaction and of heat inactivation of L-glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (suborder Notothenioidei, family Channichthyidae) were investigated. The enzyme shows dual coenzyme specificity, is inhibited by GTP and the forward reaction is activated by ADP and ATP. The complete primary structure of C. aceratus GDH has been established; it is the first amino acid sequence of a fish GDH to be described. In comparison with homologous mesophilic enzymes, the amino acid substitutions suggest a less compact molecular structure with a reduced number of salt bridges. Functional characterisation indicates efficient compensation of Q(10), achieved by increased k(cat) and modulation of S(0.5), which produce a catalytic efficiency at low temperature very similar to that of bovine GDH at its physiological temperature. The structural and functional characteristics are indicative of a high extent of protein flexibility. This property seems to find correspondence in the heat inactivation of Antarctic and bovine enzymes, which are inactivated at very similar temperature, but with different thermodynamics.

In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 Angstrom R-state structure of Trematomus newnesi Ho 1 is presented. The structure is similar to that of Root effect fish Kbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T, bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha --> Ile, Ala97 (G3)alpha --> Ser and His41 (C7)beta --> Tyr at the alpha(1)beta(2)-interface is discussed.

The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae, Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the alpha and beta chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation.

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid alpha-globin genes but have lost the gene that encodes adult beta-globin. Here we demonstrate that loss of expression of the major adult alpha-globin, alpha1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5' end of the notothenioid alpha1-globin gene. The wild-type, functional alpha1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3'-untranslated region of the notothenioid alpha1-globin gene. The residual, nonfunctional alpha-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire beta-globin gene and the 5' end of the linked alpha1-globin gene.

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a side pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effects in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobins of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain of Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His beta 143 that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.

The functional properties of the single haemoglobin (Hb) of Emperor penguin (Aptenodytes forsteri) have been investigated at different temperatures as a function of proton and organic phosphate concentration. The complete amino acid sequence has been established. Comparison with that of human HbA shows 12 substitutions in the contact regions of alpha beta dimers. In addition to overall similarities shared with most of the avian Hbs previously described, this Hb shows significant differences, which could be related to the peculiar behaviour of this penguin. In particular we may consider that: (1) the shape of the Bohr effect curve seems well adapted for gas exchange during very prolonged dives, preserving penguin Hb from a sudden and not controlled stripping of oxygen; (2) the very minor enthalpy change observed at lower pH could be an example of molecular adaptation, through which oxygen delivery becomes essentially insensitive to exposure to the extremely low temperatures of the environment. Moreover, the small alkaline Bohr effect has been found to be only chloride-linked, since the pH dependence of the oxygen affinity is totally abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha and beta-chains.

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.

Inorganic phosphate, a strong activator of glutamate dehydrogenase at pH 8.0–9.0, is an inhibitor at pH 6.0–7.6. The extent of inhibition increases with the decrease of pH. The same effect is shown by other electrolytes, including Tris-hydroxymethyl-aminomethane and NaCl. The combined effect of pH and ionic strength also alters the allosteric characteristics of the enzyme. Lowering the pH minimizes the activation by high concentrations of NAD; phosphate partially restores this activation. The allosteric activation by ADP disappears at pH around neutrality; in the pH range 6.0–7.0, ADP becomes a strong inhibitor, the inhibition being enhanced by the addition of ionic compounds. Similarly, the extent of allosteric inhibition by guanosine 5′-triphosphate (pyro) (GTP), which is maximal at pH 9.0, decreases at lower pH values and a slight activation is observed in the presence of electrolytes at pH 6.0. Glutamate dehydrogenase, selectively desensitized by dinitrophenylation in the presence of ADP, can be activated by ADP at pH 9.0, but is no longer inhibited by the same effector at pH 6.0, high salt concentration. The densensitized enzyme is not inhibited by GTP at pH 9.0, but is activated by this effector at pH 6.0 in the presence of ionic compounds. Conversely, GTP-protected dinitrophenylated glutamate dehydrogenase is desensitized only to the effect of the activating modifier, ADP at pH 9.0, GTP at pH 6.0, high salt concentration. These findings suggest that the conformation of each allosteric site of glutamate dehydrogenase is changed by pH and ionic strength so that it keeps its specificity for the ligand which brings about a given effect, activation or inhibition, independently from its chemical structure.

At pH 9.0 the activity of crystalline glutamate dehydrogenase was increased by the addition to the incubation mixture of various electrolytes including sodium chloride, potassium phosphate, Tris, 2-amino-2-methyl-1, 3-propanediol and methionine. Potassium phosphate was most stimulatory and, at an optimum concentration, increased activity three to four-fold. No stimulation was obtained at pH 7.6. Potassium phosphate and the other electrolytes abolished activation by high concentrations of NAD or NADP and altered the inhibitory effects obtained by compounds which presumably do not interact at the NAD binding site. These electrolytes also overcame the inhibition by GTP, a ligand which binds at the same site as NAD, but they did not suppress the reactivity with ADP. It is proposed that at pH 9.0 potassium phosphate and other electrolytes alter glutamate dehydrogenase so that the regulatory site which binds NAD or GTP is no longer available to these ligands.

1. The oxidation of NADH and NADPH catalysed by the soluble supernatant from the hepatopancreas of Octopus vulgaris is due to a single enzyme, which has been purified approximately 100-fold. The enzyme reacts rapidly with potassium ferricyanide, and more slowly with 2,6-dichlorophenol-indophenol. No activity is obtained with oxygen, cytochrome c, lipoic acid, vitamin K1, vitamin K3, ubiquinone-30, p-benzoquinone, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride or methylene blue. 2. GSH, cysteine and mercaptoethanol stimulate the enzymic activity up to fivefold. GSSG is without any apparent effect. When stimulated by GSH the enzyme becomes sensitive to dicoumarol, which produces an inhibition competitive with respect to the activator. 3. The purified enzyme contains an acid-removable flavine component, which has been identified as FMN by spectrofluorimetry and chromatography in three solvent systems. After acid ammonium sulphate treatment the enzymic activity is lost, but it can be almost fully restored by incubation with FMN. FAD produces only a partial reactivation.

Two glutamate dehydrogenases appear to be present in rat liver, one associated with nuclei, and the other with mitochondria. The two enzymes differ in regard to activation by inorganic phosphate, in the effects of pH on activity, in apparent Km values for some of the substrates and in the shape of curve obtained by a plot of NAD concentration versus activity.

Antarctic fish of the family Nototheniidae usually have a single major hemoglobin (Hb 1), often a second, minor component (Hb 2, about 5% of the total), and traces of another component (Hb C, less than 1%). These are functionally similar Bohr and Root effect hemoglobins. All species of other highly endemic fish families so far investigated also have one single major hemoglobin. The hematological features of the nototheniid Trematomus newnesi are remarkably different. It is the only Antarctic species in which Hb 1 and Hb 2 display only a very weak Bohr effect and no Root effect. Perhaps consequentially, Hb C (the only component showing regulation of oxygen binding by protons and other effectors) is not present in traces but accounts for 20-25% of the total. The primary structure of the three hemoglobins of T. newnesi and of Root effect HbC present in trace amounts in another nototheniid (Pagothenia bernacchii) is discussed in relationship with oxygen binding and in terms of molecular and stereochemical models. The hemoglobin multiplicity, the oxygen binding features of Hb 1 and Hb 2, and the presence of functionally distinct components, thus reveal that the oxygen transport of T. newnesi has unique characteristics.

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

Spectroscopic and crystallographic evidence of endogenous (His) ligation at the sixth coordination site of the heme iron has been reported for monomeric, dimeric, and tetrameric hemoglobins (Hbs) in both ferrous (hemochrome) and ferric (hemichrome) oxidation states. In particular, the ferric bis- histidyl adduct represents a common accessible ordered state for the β chains of all tetrameric Hbs isolated from Antarctic and sub-Antarctic fish. Indeed, the crystal structures of known tetrameric Hbs in the bis-His state are characterized by a different binding state of the α and β chains. An overall analysis of the bis-histidyl adduct of globin structures deposited in the Protein Data Bank reveals a marked difference between hemichromes in tetrameric Hbs compared to monomeric/dimeric Hbs. Herein, we review the structural, spectroscopic and stability features of hemichromes in tetrameric Antarctic fish Hbs. The role of bis-histidyl adducts is also addressed in a more evolutionary context alongside the concept of its potential physiological role. © 2011 IUBMB IUBMB Life, 63(5): 295–303, 2011

The dominant perciform suborder Notothenioidei is an excellent study group for assessing the evolution and functional importance of biochemical adaptations to temperature. The availability of notothenioid taxa in a wide range of latitudes (Antarctic and non-Antarctic) provides a tool to enable identification of physiological and biochemical characteristics gained and lost during evolutionary history. Non-Antarctic notothenioids belonging to the most basal families are a crucial source for understanding the evolution of hemoglobin in high-Antarctic cold-adapted fish. This paper focuses on the structure, function and evolution of the oxygen-transport system of Cottoperca gobio, a sub-Antarctic notothenioid fish of the family Bovichtidae, probably derived from ancestral species that evolved in the Antarctic region and later migrated to lower latitudes. Unlike most high-Antarctic notothenioids, but similar to many other acanthomorph teleosts, C. gobio has two major hemoglobins having the β chain in common. The oxygen-binding equilibria and kinetics of the two hemoglobins have been measured. Hb1 and Hb2 show strong modulation of oxygen-binding equilibria and kinetics by heterotropic effectors, with marked Bohr and Root effects. In Hb1 and Hb2, oxygen affinity and subunit cooperativity are slightly higher than in most high-Antarctic notothenioid hemoglobins. Hb1 and Hb2 show similar rebinding rates, but also show significant dynamic differences that are likely to have functional consequences. Molecular dynamic simulations of C. gobio Hb1 were performed on the dimeric protein in order to obtain a better understanding of the molecular basis of structure/function relationships. Fil: Giordano, Daniela. Consiglio Nazionale delle Ricerche; Italia Fil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Vergara, Alessandro. Università degli Studi di Napoli Federico II; Italia Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Samuni, Uri. City University of New York; Estados Unidos Fil: Dantsker, David. Albert Einstein College of Medicine; Estados Unidos Fil: Grassi, Luigi. Consiglio Nazionale delle Ricerche; Italia Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Friedman, Joel M.. Albert Einstein College of Medicine; Estados Unidos Fil: Mazzarella, Lelio. Università degli Studi di Napoli Federico II; Italia Fil: Di Prisco, Guido. Consiglio Nazionale delle Ricerche; Italia Fil: Verde, Cinzia. Consiglio Nazionale delle Ricerche; Italia

Spontaneous autoxidation of tetrameric hemoglobins leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric hemoglobins isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the X-ray crystal structure of oxidized Trematomus bernacchii hemoglobin, re-determined at high resolution. The oxidation of these hemoglobins leads to formation of states that were not usually detected in previous analyses of tetrameric hemoglobins. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes co-exist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by X-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish hemoglobins examined, hemichrome I is the major form. EPR shows that for the cathodic hemoglobin component of T. newnesi (HbCTn), the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, never reported before for a wild-type tetrameric hemoglobin, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the present data show that ferric states commonly associated with monomeric and dimeric hemoglobins are also found in tetrameric hemoglobins.

The Root effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen-binding ability at acidic pH. Here, we report the high-resolution structure of the deoxy form of Hb isolated from the Antarctic fish Trematomus bernacchii (HbTb) crystallized at pH 6.2 and 8.4. The structure at acidic pH has been previously determined at a moderate resolution (Ito et al., J Mol Biol 1995;250:648-658). Our results provide a clear picture of the events occurring upon the pH increase from 6.2 to 8.4, observed within a practically unchanged crystal environment. In particular, at pH 8.4, the interaspartic hydrogen bond at the alpha(1)beta(2) interface is partially broken, suggesting a pK(a) close to 8.4 for Asp95alpha. In addition, a detailed survey of the histidine modifications, caused by the change in pH, also indicates that at least three hot regions of the molecule are modified (Ebeta helix, Cbeta-tail, CDalpha corner) and can be considered to be involved at various levels in the release of the Root protons. Most importantly, at the CDalpha corner, the break of the salt bridge Asp48alpha-His55alpha allows us to describe a detailed mechanism that transmits the modification from the CDalpha corner far to the alpha heme. More generally, the results shed light on the role played by the histidine residues in modulating the strength of the Root effect and also support the emerging idea that the structural determinants, at least for a part of the Root effect, are specific of each Hb endowed with this property.

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

The Root effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α(1)β(2) and α(2)β(1) interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.

Recent studies have demonstrated that hemoglobins isolated from Antarctic fish undergo peculiar oxidation processes. Here we show, by combining crystallographic and spectroscopic data, that the oxidation pathway of Trematomus bernacchii hemoglobin (HbTb) is distinct from that observed for the major component of Trematomus newnesi (Hb1Tn), despite the high sequence identity of the two proteins and structural similarity of their ferrous and fully oxidized states. Resonance Raman analysis of HbTb autoxidation upon air-exposure reveals the absence of the oxidized pentacoordinated state that was observed for Hb1Tn. The HbTb oxidation pathway is characterized by two ferric species: an aquo hexacoordinated high spin state and a bis-histidyl hexacoordinated low spin form, which appear in the early stages of the oxidation process. The high resolution structure of an intermediate along the oxidation pathway has been determined at 1.4 A resolution. The analysis of the electron density of the heme pocket shows, for both the α and the β iron, the coexistence of multiple binding states. In this partially oxidized form, HbTb exhibits significant deviations from the canonical R state both at the local and global level. The analysis of these modifications highlights the structural correlation between key functional regions of the protein. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1117–1125, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Gill cytoplasmic carbonic anhydrase of the haemoglobinless Antarctic icefish Chionodraco hamatus (Ice-CA) was directly sequenced and consists in 259 residues with an acetylated N-terminus. The molecular mass, deduced from the sequence, was 28.45 kDa, while mass spectrometry analysis of the native protein gave higher values. Treatment with dithiothreitol abolished this difference, indicating possible post-translational modifications. Isoelectric focusing analysis of Ice-CA suggested S-thiolation, which was identified as S-glutathionylation by immunostaining. Deglutathionylated Ice-CA maintained the anhydrase activity but showed higher susceptibility to hydrogen peroxide, suggesting that glutathione binding to Cys residues may have a role in the defence against oxidative damage. Ice-CA is characterized by lower thermal stability, higher activity and lower activation energy than its homologue gill CA of the temperate European eel, confirming the adaptation of the catalytic capacity to low temperatures. Alignment of Ice-CA with homologous enzymes from other fish shows high identity; the enzyme is grouped with a previously described fish CA monophyletic clade although Ice-CA shows several characteristics that can increase protein-solvent interaction and structural flexibility.

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.