Antarctic ecosystems in transition - life between stresses and opportunities.

Important findings from the second decade of the 21st century on the impact of environmental change on biological processes in the Antarctic were synthesised by 26 international experts. Ten key messages emerged that have stakeholder-relevance and/or a high impact for the scientific community. They address (i) altered biogeochemical cycles, (ii) ocean acidification, (iii) climate change hotspots, (iv) unexpected dynamism in seabed-dwelling populations, (v) spatial range shifts, (vi) adaptation and thermal resilience, (vii) sea ice related biological fluctuations, (viii) pollution, (ix) endangered terrestrial endemism and (x) the discovery of unknown habitats. Most Antarctic biotas are exposed to multiple stresses and considered vulnerable to environmental change due to narrow tolerance ranges, rapid change, projected circumpolar impacts, low potential for timely genetic adaptation, and migration barriers. Important ecosystem functions, such as primary production and energy transfer between trophic levels, have already changed, and biodiversity patterns have shifted. A confidence assessment of the degree of 'scientific understanding' revealed an intermediate level for most of the more detailed sub-messages, indicating that process-oriented research has been successful in the past decade. Additional efforts are necessary, however, to achieve the level of robustness in scientific knowledge that is required to inform protection measures of the unique Antarctic terrestrial and marine ecosystems, and their contributions to global biodiversity and ecosystem services.

Regulation of globin expression in Antarctic fish under thermal and hypoxic stress.

In the freezing waters of the Southern Ocean, Antarctic teleost fish, the Notothenioidei, have developed unique adaptations to cope with cold, including, at the extreme, the loss of hemoglobin in icefish. As a consequence, icefish are thought to be the most vulnerable of the Antarctic fish species to ongoing ocean warming. Some icefish also fail to express myoglobin but all appear to retain neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X. Despite the lack of the inducible heat shock response, Antarctic notothenioid fish are endowed with physiological plasticity to partially compensate for environmental changes, as shown by numerous physiological and genomic/transcriptomic studies over the last decade. However, the regulatory mechanisms that determine temperature/oxygen-induced changes in gene expression remain largely unexplored in these species. Proteins such as globins are susceptible to environmental changes in oxygen levels and temperature, thus playing important roles in mediating Antarctic fish adaptations. In this study, we sequenced the full-length transcripts of myoglobin, neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X from the Antarctic red-blooded notothenioid Trematomus bernacchii and the white-blooded icefish Chionodraco hamatus and evaluated transcripts levels after exposure to high temperature and low oxygen levels. Basal levels of globins are similar in the two species and both stressors affect the expression of Antarctic fish globins in brain, retina and gills. Temperature up-regulates globin expression more effectively in white-blooded than in red-blooded fish while hypoxia strongly up-regulates globins in red-blooded fish, particularly in the gills. These results suggest globins function as regulators of temperature and hypoxia tolerance. This study provides the first insights into globin transcriptional changes in Antarctic fish.

Structural and functional properties of Antarctic fish cytoglobins-1: Cold-reactivity in multi-ligand reactions.

While the functions of the recently discovered cytoglobin, ubiquitously expressed in vertebrate tissues, remain uncertain, Antarctic fish provide unparalleled models to study novel protein traits that may arise from cold adaptation. We report here the spectral, ligand-binding and enzymatic properties (peroxynitrite isomerization, nitrite-reductase activity) of cytoglobin-1 from two Antarctic fish, Chaenocephalus aceratus and Dissostichus mawsoni, and present the crystal structure of D. mawsoni cytoglobin-1. The Antarctic cytoglobins-1 display high O2 affinity, scarcely compatible with an O2-supply role, a slow rate constant for nitrite-reductase activity, and do not catalyze peroxynitrite isomerization. Compared with mesophilic orthologues, the cold-adapted cytoglobins favor binding of exogenous ligands to the hexa-coordinated bis-histidyl species, a trait related to their higher rate constant for distal-His/heme-Fe dissociation relative to human cytoglobin. At the light of a remarkable 3D-structure conservation, the observed differences in ligand-binding kinetics may reflect Antarctic fish cytoglobin-1 specific features in the dynamics of the heme distal region and of protein matrix cavities, suggesting adaptation to functional requirements posed by the cold environment. Taken together, the biochemical and biophysical data presented suggest that in Antarctic fish, as in humans, cytoglobin-1 unlikely plays a role in O2 transport, rather it may be involved in processes such as NO detoxification.

Conformational Flexibility Drives Cold Adaptation in Pseudoalteromonas haloplanktis TAC125 Globins.

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states.

Adaptations to environmental change: Globin superfamily evolution in Antarctic fishes.

The ancient origins and functional versatility of globins make them ideal subjects for studying physiological adaptation to environmental change. Our goals in this review are to describe the evolution of the vertebrate globin gene superfamily and to explore the structure/function relationships of hemoglobin, myoglobin, neuroglobin and cytoglobin in teleost fishes. We focus on the globins of Antarctic notothenioids, emphasizing their adaptive features as inferred from comparisons with human proteins. We dedicate this review to Guido di Prisco, our co-author, colleague, friend, and husband of C.V. Ever thoughtful, creative, and enthusiastic, Guido spearheaded study of the structure, function, and evolution of the hemoglobins of polar fishes - this review is testimony to his wide-ranging contributions. Throughout his career, Guido inspired younger scientists to embrace polar biological research, and he challenged researchers of all ages to explore evolutionary adaptation in the context of global climate change. Beyond his scientific contributions, we will miss his warmth, his culture, and his great intellect. Guido has left an outstanding legacy, one that will continue to inspire us and our research.

Enzymes from Marine Polar Regions and Their Biotechnological Applications.

The microorganisms that evolved at low temperatures express cold-adapted enzymes endowed with unique catalytic properties in comparison to their mesophilic homologues, i.e., higher catalytic efficiency, improved flexibility, and lower thermal stability. Cold environments are therefore an attractive research area for the discovery of enzymes to be used for investigational and industrial applications in which such properties are desirable. In this work, we will review the literature on cold-adapted enzymes specifically focusing on those discovered in the bioprospecting of polar marine environments, so far largely neglected because of their limited accessibility. We will discuss their existing or proposed biotechnological applications within the framework of the more general applications of cold-adapted enzymes.

Biotechnological Applications of Bioactive Peptides From Marine Sources.

This review is an overview on marine bioactive peptides with promising activities for the development of alternative drugs to fight human pathologies. In particular, we focus on potentially prolific producers of peptides in microorganisms, including sponge-associated bacteria and marine photoautotrophs such as microalgae and cyanobacteria. Microorganisms are still poorly explored for drug discovery, even if they are highly metabolically plastic and potentially amenable to culturing. This offers the possibility of obtaining a continuous source of bioactive compounds to satisfy the challenging demands of pharmaceutical industries. This review targets peptides because of the variety of potent biological activities demonstrated by these molecules, including antiviral, antimicrobial, antifungal, antioxidant, anticoagulant, antihypertensive, anticancer, antidiabetic, antiobesity, and calcium-binding bioactivities. Several of these peptides have already gained recognition as effective drug agents in recent years. We also focus on cutting-edge omic approaches for the discovery of novel compounds for pharmacological applications. With rapid depletion of natural resources, omic technologies may be the solution to efficiently produce a vast variety of novel peptides with unique pharmacological potential.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

The Greenland shark Somniosus microcephalus-Hemoglobins and ligand-binding properties.

A large amount of data is currently available on the adaptive mechanisms of polar bony fish hemoglobins, but structural information on those of cartilaginous species is scarce. This study presents the first characterisation of the hemoglobin system of one of the longest-living vertebrate species (392 ± 120 years), the Arctic shark Somniosus microcephalus. Three major hemoglobins are found in its red blood cells and are made of two copies of the same α globin combined with two copies of three very similar ß subunits. The three hemoglobins show very similar oxygenation and carbonylation properties, which are unaffected by urea, a very important compound in marine elasmobranch physiology. They display identical electronic absorption and resonance Raman spectra, indicating that their heme-pocket structures are identical or highly similar. The quaternary transition equilibrium between the relaxed (R) and the tense (T) states is more dependent on physiological allosteric effectors than in human hemoglobin, as also demonstrated in polar teleost hemoglobins. Similar to other cartilaginous fishes, we found no evidence for functional differentiation among the three isoforms. The very similar ligand-binding properties suggest that regulatory control of O2 transport may be at the cellular level and that it may involve changes in the cellular concentrations of allosteric effectors and/or variations of other systemic factors. The hemoglobins of this polar shark have evolved adaptive decreases in O2 affinity in comparison to temperate sharks.

Marine Microbial Secondary Metabolites: Pathways, Evolution and Physiological Roles.

Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology.

Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 Å resolution) shows typical features of group-II truncated hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.

The globins of cold-adapted Pseudoalteromonas haloplanktis TAC125: from the structure to the physiological functions.

Evolution allowed Antarctic microorganisms to grow successfully under extreme conditions (low temperature and high O2 content), through a variety of structural and physiological adjustments in their genomes and development of programmed responses to strong oxidative and nitrosative stress. The availability of genomic sequences from an increasing number of cold-adapted species is providing insights to understand the molecular mechanisms underlying crucial physiological processes in polar organisms. The genome of Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct truncated globins exhibiting the 2/2 α-helical fold. One of these globins has been extensively characterised by spectroscopic analysis, kinetic measurements and computer simulation. The results indicate unique adaptive structural properties that enhance the overall flexibility of the protein, so that the structure appears to be resistant to pressure-induced stress. Recent results on a genomic mutant strain highlight the involvement of the cold-adapted globin in the protection against the stress induced by high O2 concentration. Moreover, the protein was shown to catalyse peroxynitrite isomerisation in vitro. In this review, we first summarise how cold temperatures affect the physiology of microorganisms and focus on the molecular mechanisms of cold adaptation revealed by recent biochemical and genetic studies. Next, since only in a very few cases the physiological role of truncated globins has been demonstrated, we also discuss the structural and functional features of the cold-adapted globin in an attempt to put into perspective what has been learnt about these proteins and their potential role in the biology of cold-adapted microorganisms.

Ligand-rebinding kinetics of 2/2 hemoglobin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Kinetic studies were performed on ligand rebinding to a cold-adapted globin of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO). This 2/2 hemoglobin displays a rapid spectroscopic phase that is independent of CO concentration, followed by the standard bimolecular recombination. While the geminate recombination usually occurs on a ns timescale, Ph-2/2HbO displays a component of about 1µs that accounts for half of the geminate phase at 8°C, indicative of a relatively slow internal ligand binding. The O2 binding kinetics were measured in competition with CO to allow a short-time exposure of the deoxy hemes to O2 before CO replacement. Indeed Ph-2/2HbO is readily oxidised in the presence of O2, probably due to a superoxide character of the FeO2 bond induced by of a hydrogen-bond donor amino-acid residue. Upon O2 release or iron oxidation a distal residue (probably Tyr) is able to reversibly bind to the heme and as such to compete for binding with an external ligand. The transient hexacoordinated ferrous His-Fe-Tyr conformation after O2 dissociation could initiate the electron transfer from the iron toward its final acceptor, molecular O2 under our conditions. The hexacoordination via the distal Tyr is only partial, indicating a weak interaction between Tyr and the heme under atmospheric pressure. Hydrostatic high pressure enhances the hexacoordination indicating a flexible globin that allows structural changes. The O2 binding affinity for Ph-2/2HbO, poorly affected by the competition with Tyr, is about 1Torr at 8°C, pH7.0, which is compatible for an in vivo O2 binding function; however, this globin is more likely involved in a redox reaction associating diatomic ligands and their derived oxidative species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Antarctic bacterial haemoglobin and its role in the protection against nitrogen reactive species.

In a cold and oxygen-rich environment such as Antarctica, mechanisms for the defence against reactive oxygen and nitrogen species are needed and represent important components in the evolutionary adaptations. In the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, the presence of multiple genes encoding 2/2 haemoglobins and a flavohaemoglobin strongly suggests that these proteins fulfil important physiological roles, perhaps associated to the peculiar features of the Antarctic habitat. In this work, the putative role of Ph-2/2HbO, encoded by the PSHAa0030 gene, was investigated by in vivo and in vitro experiments in order to highlight its involvement in NO detoxification mechanisms. The PSHAa0030 gene was cloned and then over-expressed in a flavohaemoglobin-deficient mutant of Escherichia coli, unable to metabolise NO, and the resulting strain was studied analysing its growth properties and oxygen uptake in the presence of NO. We here demonstrate that Ph-2/2HbO protects growth and cellular respiration of the heterologous host from the toxic effect of NO-donors. Unlike in Mycobacterium tuberculosis 2/2 HbN, the deletion of the N-terminal extension of Ph-2/2HbO does not seem to reduce the NO scavenging activity, showing that the N-terminal extension is not a requirement for efficient NO detoxification. Moreover, the ferric form of Ph-2/2HbO was shown to catalyse peroxynitrite isomerisation in vitro, confirming its potential role in the scavenging of reactive nitrogen species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Biophysical characterisation of neuroglobin of the icefish, a natural knockout for hemoglobin and myoglobin. Comparison with human neuroglobin.

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe(2+) form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.

Understanding and protecting the world's biodiversity: the role and legacy of the SCAR programme "Evolution and Biodiversity in the Antarctic".

Current global changes are prompting scientists and governments to consider the risk of extinction of species inhabiting environments influenced by ice. Concerted, multidisciplinary, international programmes aimed at understanding life processes, evolution and adaptations in the Polar Regions will help to counteract such an event by protecting polar life and ecosystems. There is a long tradition of international scientific cooperation in Antarctica that provides a strong foundation for such approaches. While basic understanding is emerging, we still largely lack predictive biological models, and need to achieve further integration amongst biological and non-biological disciplines. The ongoing SCAR Science Research Programme, "Evolution and Biodiversity in the Antarctic (EBA)" has successfully carried out its crucial role of providing an overarching umbrella for SCAR research in Life Sciences. Now is the time for aiming to progress beyond this important role, and the Antarctic biology community is proposing two programmes, focussed on distinct but complementary aspects of polar biology and working across marine, freshwater and terrestrial environments: "State of the Antarctic Ecosystem (AntEco)", and "Antarctic Thresholds--Ecosystem Resilience and Adaptation (AnT-ERA)". These programmes are the legacy of EBA, and they are key to understanding and protect Antarctic biodiversity.

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus.

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.

Molecular adaptations in Antarctic fish and marine microorganisms.

The Antarctic marine environment is one of the most extreme on Earth due to its stably low temperature and high oxygen content. Here we discuss various aspects of the molecular adaptations evolved by Antarctic fish and marine microorganisms living in this environment. This review will in particular focus on: (i) the genetic/genomic bases of adaptation in Antarctic notothenioid fish; (ii) the role of neuroglobin recently identified in the brain of Antarctic icefish; (iii) the structural and functional features of globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes.

BACKGROUND: Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity. RESULTS: To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin. CONCLUSIONS: These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity.