Distribution and hypoxia-regulation of haemocyanin in springtails (Collembola).

Haemocyanin is the copper-containing respiratory protein present in many arthropods. In the hexapods, respiratory proteins had long been considered unnecessary as sufficient O2 was thought to be obtained via the trachea. Nevertheless, many ametabolous and hemimetabolous hexapod species actually possess haemocyanin. Here we investigated the occurrence of haemocyanin in Collembola (springtails). Haemocyanin was found in 22 collembolan species of the suborders Symphypleona, Tomoceroidea and Entomobryomorpha, demonstrating its widespread occurrence. No haemocyanin was identified in 16 species of these taxa, and it appears to be absent in Poduromorpha. The presence of haemocyanin does not correlate with either the phylogenetic history or lifestyle of the investigated species. We further investigated the function of haemocyanin in Folsomia candida (Entomobryomorpha) by applying different hypoxia regimes. Whereas short-term (1 h) and mild (10% O2 ) hypoxia led to a decrease in haemocyanin mRNA, strong hypoxia (24 h, 1.5% O2 ) resulted in a ∼4300-fold increase in haemocyanin expression. Hypoxia induction of haemocyanin could not be demonstrated in evolutionarily more advanced Hexapoda, where it is restricted to the embryo. The results indicate (1) an important role of haemocyanin in the oxygen supply of F. candida, which may be adaptive in the potentially hypoxic environment in the soil, and (2) a change in haemocyanin function in hexapod evolution.

The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per µg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood.

Function and evolution of vertebrate globins.

Globins are haem-proteins that bind O2 and thus play an important role in the animal's respiration and oxidative energy production. However, globins may also have other functions such as the decomposition or production of NO, the detoxification of reactive oxygen species or intracellular signalling. In addition to the well-investigated haemoglobins and myoglobins, genome sequence analyses have led to the identification of six further globin types in vertebrates: androglobin, cytoglobin, globin E, globin X, globin Y and neuroglobin. Here, we review the present state of knowledge on the functions, the taxonomic distribution and evolution of vertebrate globins, drawing conclusions about the functional changes underlying present-day globin diversity.

Neuroglobin of seals and whales: evidence for a divergent role in the diving brain.

Although many physiological adaptations of diving mammals have been reported, little is known about how their brains sustain the high demands for metabolic energy and thus O(2) when submerged. A recent study revealed in the deep-diving hooded seal (Cystophora cristata) a unique shift of the oxidative energy metabolism and neuroglobin, a respiratory protein that is involved in neuronal hypoxia tolerance, from neurons to astrocytes. Here we have investigated neuroglobin in another pinniped species, the harp seal (Pagophilus groenlandicus), and in two cetaceans, the harbor porpoise (Phocoena phocoena) and the minke whale (Balaenoptera acutorostrata). Neuroglobin sequences, expression levels and patterns were compared with those of terrestrial relatives, the ferret (Mustela putorius furo) and the cattle (Bos taurus), respectively. Neuroglobin sequences of whales and seals only differ in two or three amino acids from those of cattle and ferret, and are unlikely to confer functional differences, e.g. in O(2) affinity. Neuroglobin is expressed in the astrocytes also of P. groenlandicus, suggesting that the shift of neuroglobin and oxidative metabolism is a common adaptation in the brains of deep-diving phocid seals. In the cetacean brain neuroglobin resides in neurons, like in terrestrial mammals. However, neuroglobin mRNA expression levels were 4-15 times higher in the brains of harbor porpoises and minke whales than in terrestrial mammals or in seals. Thus neuroglobin appears to play a specific role in diving mammals, but seals and whales have evolved divergent strategies to cope with cerebral hypoxia. The specific function of neuroglobin that conveys hypoxia tolerance may either relate to oxygen supply or protection from reactive oxygen species. The different strategies in seals and whales resulted from a divergent evolution and an independent adaptation to diving.

Old proteins - new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells.

AIM: The unexpected identification of myoglobin (MB) in breast cancer prompted us to evaluate the clinico-pathological value of MB, haemoglobin (HB) and cytoglobin (CYGB) in human breast carcinoma cases. We further screened for the presence of neuroglobin (NGB) and CYGB in tumours of diverse origin, and assessed the O(2) -response of HB, MB and CYGB mRNAs in cancer cell lines, to better elicit the links between this ectopic globin expression and tumour hypoxia. METHODS: Breast tumours were analysed by immunohistochemistry for HB, MB and CYGB and correlated with clinico-pathological parameters. Screening for CYGB and NGB mRNA expression in tumour entities was performed by hybridization, quantitative PCR (qPCR) and bioinformatics. Hypoxic or anoxic responses of HB, MB and CYGB mRNAs was analysed by qPCR in human Hep3B, MCF7, HeLa and RCC4 cancer cell lines. RESULTS: 78.8% of breast cancer cases were positive for MB, 77.9% were positive for HB and 55.4% expressed CYGB. The closest correlation with markers of hypoxia was observed for CYGB. Compared to the weakly positive status of MB in healthy breast tissues, invasive tumours either lost or up-regulated MB. Breast carcinomas showed the tendency to silence CYGB. HB was not seen in normal tissues and up-regulated in tumours. Beyond breast malignancies, expression levels of NGB and CYGB mRNAs were extremely low in brain tumours (glioblastoma, astrocytoma). NGB was not observed in non-brain tumours. CYGB mRNA, readily detectable in breast cancer and other tumours, is down-regulated in lung adenocarcinomas. Alpha1 globin (α1 globin) and Mb were co-expressed in MCF7 and HeLa cells; CYGB transcription was anoxia-inducible in Hep3B and RCC4 cells. CONCLUSIONS: This is the first time that HB and CYGB are reported in breast cancer. Neither NGB nor CYGB are systematically up-regulated in tumours. The down-regulated CYGB expression in breast and lung tumours is in line with a tumour-suppressor role. Each of the screened cancer cells expresses at least one globin (i.e. main globin species: CYGB in Hep3B; α1 globin + MB in MCF7 and HeLa). Thus, globins exist in a wide variety of solid tumours. However, the generally weak expression of the endogenous proteins in the cancer argues against a significant contribution to tumour oxygenation. Future studies should consider that cancer-expressed globins might function in ways not directly linked to the binding and transport of oxygen.

A putative hexamerin from a Campodea sp. suggests an independent origin of haemocyanin-related storage proteins in Hexapoda.

Haemocyanins are copper-containing respiratory proteins in the arthropod haemolymph. In hexapods, haemocyanins gave rise to hexamerins, which have lost the ability to bind copper and thus oxygen. Hexamerins are thought to act mainly as storage proteins in nonfeeding periods. So far, hexamerins have only been identified in ectognathan hexapods, but not in Entognatha. Here we report the identification of a putative hexamerin from Campodea sp. (Diplura). The full-length cDNA of Campodea sp. hexamerin 1 (CspHex1) measures 2188 bp and translates into a native polypeptide of 667 amino acids. As in other hexamerins, the six copper-coordinating histidines are not conserved. However, sequence comparison and phylogenetic analyses demonstrated that CspHex1 is not closely related to other hexapod hexamerins, which derive from hexapod type 1 haemocyanin subunits in the ectognathan lineage, but rather resembles a derivative of hexapod type 2 haemocyanin subunits. Hence, haemocyanin-related storage proteins emerged at least two times independently in Hexapoda.

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O(2) affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives.

Marsupial uncoupling protein 1 sheds light on the evolution of mammalian nonshivering thermogenesis.

Brown adipose tissue expressing uncoupling protein 1 (UCP1) is responsible for adaptive nonshivering thermogenesis giving eutherian mammals crucial advantage to survive the cold. The emergence of this thermogenic organ during mammalian evolution remained unknown as the identification of UCP1 in marsupials failed so far. Here, we unequivocally identify the marsupial UCP1 ortholog in a genomic library of Monodelphis domestica. In South American and Australian marsupials, UCP1 is exclusively expressed in distinct adipose tissue sites and appears to be recruited by cold exposure in the smallest species under investigation (Sminthopsis crassicaudata). Our data suggest that an archetypal brown adipose tissue was present at least 150 million yr ago allowing early mammals to produce endogenous heat in the cold, without dependence on shivering and locomotor activity.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

The amphibian globin gene repertoire as revealed by the Xenopus genome.

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named 'globin Y') which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin alpha and beta genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction.

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.

Interspecies comparison of neuroglobin, cytoglobin and myoglobin: sequence evolution and candidate regulatory elements.

Neuroglobin and cytoglobin are two novel members of the vertebrate globin family. Their physiological role is poorly understood, although both proteins bind oxygen reversibly and may be involved in cellular oxygen homeostasis. Here we investigate the selective constraints on coding and non-coding sequences of the neuroglobin and cytoglobin genes in human, mouse, rat and fish. Neuroglobin and cytoglobin are highly conserved, displaying very low levels of non-synonymous nucleotide substitutions. An oxygen supply function predicts distinct modes of gene regulation, involving hypoxia-responsive transcription factors. To detect conserved candidate regulatory elements, we compared the neuroglobin and cytoglobin genes in mammals and fish. The myoglobin gene was included to test if it also contains hypoxia-responsive regulatory elements. Long conserved non-coding sequences, indicative of gene-regulatory elements, were found in the cytoglobin and myoglobin, but not in the neuroglobin gene. Sequence comparison and experimental data allowed us to delimit upstream regions of the neuroglobin and cytoglobin genes that contain the putative promoters, defining candidate regulatory regions for functional tests. The neuroglobin and the myoglobin genes both lack conserved hypoxia-responsive elements (HREs) for transcriptional activation, but contain conserved hypoxia-inducible mRNA stabilization signals in their 3' untranslated regions. The cytoglobin gene, in contrast, harbors both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation.

Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily.

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157-2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.

Molecular characterisation and evolution of the hemocyanin from the European spiny lobster, Palinurus elephas.

The hemocyanin of the European spiny lobster Palinurus elephas (synonym: Palinurus vulgaris) is a hexamer composed by four closely related but distinct subunits. We have obtained the full cDNA sequences of all four subunits, which cover 2275-2298 bp and encode for native polypeptides of 656 and 657 amino acids. The P. elephas hemocyanin subunits belong to the alpha-type of crustacean hemocyanins, whereas beta- and gamma-subunits are absent in this species. An unusual high ratio of non-synonymous versus synonymous nucleotide substitutions was observed, suggesting positive selection among subunits. Assuming a constant evolution rate, the P. elephas hemocyanin subunits emerged from a single hemocyanin gene around 25 million years ago. The alpha-type hemocyanins of P. elephas and the American spiny lobster Panulirus interruptus split around 100 million years ago. This is about five times older than the assumed divergence time of the species and suggests that the genera may have split with the formation of the Atlantic Ocean. The application of the Bayesian method for phylogenetic inference allows for the first time a solid reconstruction of the evolution of the decapod hemocyanins, showing that the beta-subunit types diverged first and that the crustacean pseudo-hemocyanins are associated with the gamma-type subunits.

Complete sequence, expression and evolution of two members of the hexamerin protein family during the larval development of the rice moth, Corcyra cephalonica.

Three distinct types of storage hexamerins are expressed in the "last-instar" larvae of the rice moth, Corcyra cephalonica. A cDNA expression library was constructed from fat body-RNA and screened with a polyclonal antibody raised against purified hexamerin (SP2) of Corcyra cephalonica. Two slightly different "full-length" hexamerin cDNA clones (Hex2a and Hex2b) were isolated and sequenced. Both include open reading frames of 2109 bp which are translated into polypeptides of 703 amino acids with 92.5% identity. Signal peptides of 19 amino acids are present at the N-termini. The 684 amino acids native proteins have a high content of aryl groups (17.6%). According to both the criteria for amino acid composition and the phylogenetic analysis, Hex2a and Hex2b belong to the lepidopteran arylphorins. Northern blot studies revealed that the Hex2 genes are species- and tissue-specifically expressed in fat body cells of "last-instar" (= 5th) larvae.

Expression analysis of neuroglobin mRNA in rodent tissues.

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is sufficient for its putative primary function as an oxygen-supplying protein.

Origin and evolution of arthropod hemocyanins and related proteins.

Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxidase-like enzyme. The evolution of distinct hemocyanin subunits, as well as the formation of multi-hexamers occurred independently within the arthropod subphyla. Hemocyanin subunit evolution is strikingly different in the Chelicerata, Myriapoda and Crustacea. Hemocyanins individually gave rise to two distinct copper-less storage proteins, the insect hexamerins and the crustacean pseudo-hemocyanins (cryptocyanins). The receptor responsible for the uptake of hexamerin by the larval fat body of the insects emerged from a hexamerin-precursor. Molecular phylogenetic analyses show a close relationship of the crustacean and insect proteins, providing strong support for a pancrustacean taxon, while structural data suggest a myriapod-chelicerate clade.

Neuroglobins from the zebrafish Danio rerio and the pufferfish Tetraodon nigroviridis.

Neuroglobin is a recently discovered respiratory, porphyrin-containing protein that is expressed in the brain of mouse and man. Here we show that neuroglobin is also present in the teleost fish. Complete cDNA sequences are reported from the pufferfish Tetraodon nigroviridis and the zebrafish Danio rerio. In addition, the neuroglobin gene of T. nigroviridis was sequenced, demonstrating the conservation of the B12.2, E11.0 and G7.0 introns plus the presence of an additional intron in the 5' noncoding region. The fish neuroglobins each comprise 159 amino acids and are 84.3% identical. Phylogenetic analyses show a basal position of the neuroglobins within the metazoan globin tree. An enhanced amino acid substitution rate was estimated for the fish neuroglobins ( approximately 0.93 x 10(-9) amino acid substitutions per site and year) compared with their mammalian proteins ( approximately 0.39 x 10(-9) replacements per site and year).

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda.

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.

The heme environment of mouse neuroglobin. Evidence for the presence of two conformations of the heme pocket.

Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe(2+)CO complex, two conformations of the Fe-CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex. For the Fe(2+)O(2) complex, we detect a single nu(Fe-OO) stretching mode at a frequency similar to that of oxymyoglobins and oxyhemoglobins of vertebrates (571 cm(-1)). Based on the Fe-C-O frequencies of the closed conformation of Ngb, a highly polar distal environment is indicated from which the O(2) off-rate is predicted to be lower than that of Mb. In the absence of exogenous ligands, a heme pocket residue coordinates to the heme iron, forming a six-coordinate complex, thereby predicting a low on-rate for exogenous ligands. These structural properties of the heme pocket of Ngb are discussed with respect to its proposed in vivo oxygen delivery function.