Sylvia Dewilde

Human Brain Neuroglobin Structure Reveals a Distinct Mode of Controlling Oxygen Affinity

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Neuroglobin and cytoglobin: Fresh blood for the vertebrate globin family

Neuroglobin and cytoglobin are two recently discovered members of the vertebrate globin family. Both are intracellular proteins endowed with hexacoordinated heme‐Fe atoms, in their ferrous and ferric forms, and display O2 affinities comparable with that of myoglobin. Neuroglobin, which is predominantly expressed in nerve cells, is thought to protect neurons from hypoxic–ischemic injury. It is of ancient evolutionary origin, and is homologous to nerve globins of invertebrates. Cytoglobin is expressed in many different tissues, although at varying levels. It shares common ancestry with myoglobin, and can be traced to early vertebrate evolution. The physiological roles of neuroglobin and cytoglobin are not completely understood. Although supplying cells with O2 is the likely function, it is also possible that both globins act as O2‐consuming enzymes or as O2 sensors. Here, we review what is currently known about neuroglobin and cytoglobin in terms of their function, tissue distribution and relatedness to the well‐known hemoglobin and myoglobin. Strikingly, the data reveal that O2 metabolism in cells is more complicated than was thought before, requiring unexpected O2‐binding proteins with potentially novel functional features.

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.

Small hemoproteins displaying amino acid sequences 20–40 residues shorter than (non‐)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non‐pathogenic unicellular organisms, and named ‘truncated hemoglobins’ (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a ‘two‐over‐two’ α‐helical sandwich, reflecting an unprecedented editing of the classical ‘three‐over‐three’ α‐helical globin fold. Based on specific Gly–Gly motifs the tertiary structure accommodates the deletion of the N‐terminal A‐helix and replacement of the crucial heme‐binding F‐helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

A phylogenomic profile of globins

BackgroundGlobins 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.ResultsA 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 nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.ConclusionAlthough 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.

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.

Nitric oxide binding properties of neuroglobin: A characterization by EPR and flash photolysis

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia colicell cultures with low O2 concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe2+-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k on) and low dissociation rates (k off) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.

Neuroglobin and cytoglobin expression in mice

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.

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c.

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b 5 is relatively slow (k = 6 × 102 M−1 s−1 at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k = 2 × 107 M−1 s−1) with an apparent overall equilibrium constant of 1 μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.

Review: Correlations between oxygen affinity and sequence classifications of plant hemoglobins

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full‐length globins with the classical eight helix Mb‐like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O2 binding (∼250 μM−1 s−1), moderately high rates of O2 dissociation (∼5–15 s−1), and high oxygen affinity (Kd or P50 ≈ 50 nM). These properties both facilitate O2 diffusion to respiring N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (KHisE7 ≈ 2), moderate rates of O2 binding (∼25 μM−1 s−1), very small rates of O2 dissociation (∼0.16 s−1), and remarkably high O2 affinities (P50 ≈ 2 nM), suggesting a function involving O2 and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (KHisE7 ≈ 100), low rates of O2 binding (∼1 μM−1 s−1), moderately low O2 dissociation rate constants (∼1 s−1), and moderate, Mb‐like O2 affinities (P50 ≈ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.