Background: Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.Results: A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematodeCaenorhabditishas the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such asEncephalitozoon, Entamoeba, PlasmodiumandTrypanosoma.Conclusion: Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.

Although essentially unknown, several functions are hypothesized for neuroglobin and cytoglobin, two new members of the globin family. In this article, we try to shed more light on their possible roles in hypoxia and detoxification of reactive oxygen species in vivo. The relative transcriptional changes of neuroglobin and cytoglobin in a situation of chronic hypoxia in mice were examined using real‐time quantitative PCR. The kinetics of the hypoxic expression of neuroglobin (brain, eyes) and cytoglobin (brain, eyes, liver, heart, skeletal muscle) is organ‐specific. Moreover, reactive oxygen species production is higher in liver than in the other tissues. In eyes, the negative correlation, after reoxygenation, between neuroglobin protein level and H₂O₂ concentration is a first proof of a reactive oxygen species‐scavenging function for neuroglobin. In addition, apoptotic cell death after hypoxia is for the first time demonstrated in heart and liver.

Background: The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. In silico analysis of the genome of C. elegans identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in C. elegans. Results: All 33 C. elegans putative globin genes are transcribed. The translated sequences have the essential signatures of single domain bona fide globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of C. elegans. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in daf-2(e1370)/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates hif-1 transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of C. elegans, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect. Conclusion: We found that all 33 putative globins are expressed, albeit at low or very low levels, perhaps indicating cell-specific expression. They show wide diversity in gene structure and amino acid sequence, suggesting a long evolutionary history. Ten globins are responsive to oxygen deprivation in an interacting HIF-1 and DAF-16 dependent manner. Globin ZK637.13 is not responsive to oxygen deprivation and regulated by the Ins/IGF pathway only suggesting that this globin may contribute to the life maintenance program.

Background: Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. Results: In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Conclusion: Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.

The techniques of high applied pressure and flash photolysis have been combined to study ligand rebinding to neuroglobin (Ngb) and tomato Hb, globins that may display a His-Fe-His hexacoordination in the absence of external ligands. High pressure induces a moderate decrease in the His association rate and a large decrease in His dissociation rate, thus leading to an enhancement of the overall His affinity. The overall structural difference between penta- and hexacoordinated globins may be rather small and can be overcome by external modifications such as high pressure. Over the pressure range 0.1–700 MPa (7 kbar), the globins may show a loss of over a factor of 100 in the amplitude of the bimolecular rebinding phase after photodissociation. The kinetic data show that pressure induces a moderate increase of the rate for ligand binding from the correlated pair state (just after photodissociation) and a large (factor of 1000) decrease in rate for migration through the protein. The effect on the ligand migration phase was similar for both the external ligands (such as oxygen) as for the internal (histidine) ligand, suggesting the dominant role of protein fluctuations, rather than specific chemical barriers. Thus high pressure efficiently closes the protein migration channels; however, contrary to the effect of high viscosity, high pressure induces a greater decrease in rate for ligand migration toward the exterior (heme to the solvent) versus inward migration, as if the presence of the ligand itself induces an additional steric constraint.

Background: The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. Results: We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O₂ binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. Conclusion: The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.