BACKGROUND
the increasing availability of genomic data shows that globin-like proteins/domains  are universal and arose very early in the evolution of life  <cit> .
recent studies reveal new globin types as well as new functions .
a general survey suggests that the majority of nematodes host globin-like proteins  <cit> .
based on their tissue localisation, these proteins can be grouped in three classes: pseudocoelomic fluid, body wall, and cuticular hemoglobins .
some nematode species may however lack a particular class  <cit> .
to date, the best characterized pseudocoelomic fluid hb is the hb from the ascarid ascaris suum.
this is an octameric molecule  consisting of two layers of four subunits stacked in an eclipsed orientation  <cit> .
each subunit of  <dig> kda consists of two covalently linked, highly similar globin domains  followed by a c-terminal polar zipper of  <dig> amino acids.
this hb has an exceptionally high o <dig> affinity  that is attributed to a very slow dissociation rate :  <dig>  s-1) and a normal association rate :  <dig>  μm-1s-1)  <cit> , whereby this hb will be in the oxy form even in the host's gut with a locally micro-oxygen concentration  <cit> .
this high o <dig> affinity can be explained structurally by the presence of three hydrogen bonds between tyrb <dig> and glne <dig> and the bound ligand  <cit> .
the function of the a. suum pseudocoelomic fluid hb is still a matter of debate .
due to its extremely high o <dig> affinity, it will rarely be deoxygenated in vivo and cannot therefore function as an o <dig> carrier or store.
several alternative functions have been proposed, such as o <dig> or no scavengers, iron and/or heme stores, catalyst in sterol biosynthesis, buffering, osmotic - or other unknown functions  <cit> .
the existence of globins in c. elegans was unexpected given the worm's small size, whereby sufficient amounts of o <dig> may reach the terminal oxidase sites by simple diffusion.
the complete c. elegans genome revealed  <dig> globin-like genes, all having orthologues in the sibling species c. briggsae  <cit> .
all  <dig> putative globin genes are expressed, albeit at low or very low levels, most likely indicating cell-specific expression.
they show wide diversity in gene structure and amino-acid sequence of the translated proteins, suggesting a long evolutionary history.
nevertheless, sequence similarity to standard vertebrate globins, such as hb α, β chains, myoglobin , neuro- and cyto-globin  can be detected  <cit> , despite the presence of additional interhelical, n- and/or c-terminal extensions.
the intron/exon patterns of these c. elegans globin-coding genes are unique in the number of introns and in their insertion positions, compared to the highly conserved intron/exon pattern of vertebrate and other non-vertebrate globin genes   <cit> .
given the large number of c. elegans globin genes it is unlikely that they all serve simple o <dig> metabolism.
c. elegans is a soil dwelling nematode; soil parameters such as temperature, moisture and o <dig> concentration fluctuate, creating temporary hypoxic/anoxic environments  <cit> .
living systems have developed, at the organism and cellular levels, various strategies to cope with reduced o <dig> levels, e.g.
increasing the glycolytic flux, reducing the aerobic metabolic rate and increasing the efficiency of o <dig> uptake from the environment.
however, nematodes including c. elegans lack both specialized respiratory systems and complex circulatory organs, and rely on respiratory adjustments at the molecular levels  <cit> .
specialized o2-sensing cells in the nervous system of c. elegans permit rapid behavioural responses to o <dig> availability.
the signal transduction pathway, detecting upper and lower o <dig> levels, involves the cgmp-gated channel tax-2/tax- <dig> and a soluble guanylate cyclase homologue, gcy- <dig>  the expressed n-terminal domain, gcy- <dig> displays the spectral characteristics of a ferrous high-spin hemoprotein capable of o <dig>  co and no binding  <cit> .
wild-type animals survive short periods of hypoxia/anoxia by greatly reducing metabolic rate and arresting movement and development.
on the other hand the metabolic rate of the animals is not affected by increasing o <dig> levels up to 100%  <cit> .
with the aim of shedding light on the roles played by c. elegans globins, we cloned several globin genes, and focussed our attention on two translation products .
we analyzed their expression patterns and alterations of transcript abundance under hypoxia.
we then analyzed the two expressed globins through optical and resonance raman spectroscopy, characterized the kinetics of ligand binding and o <dig> equilibrium properties, and determined the high resolution crystal structure of glb- <dig>  the results are discussed in the context of current views on multivariate globin function, of heme/ligand recognition mechanisms, and of the evolution of the globin fold through the phyla.
RESULTS
expression cloning of globins
eight c. elegans globins were selected for cloning and expression.
these were the canonical globin, glb- <dig>  three globins  that show sequence similarity to cygb, two globins displaying similarity to ngb , glb- <dig> that contains an exceptionally large n-terminal extension, and glb- <dig> in which ile substitutes the highly conserved phe at position cd <dig>  all globin genes were cloned into pet3a and expressed in vitro in e. coli.
after expression, only two globins, glb- <dig> and glb- <dig> were found in the cytosolic fractions with incorporated heme, whereas all the others were present as apo-proteins in inclusion bodies.
all attempts to reconstruct the purified apo-proteins with hemin in vitro using different methods  failed.
as many globins are easily reconstructed from hemin and their apo-protein we concluded that glb- <dig>  glb- <dig>  glb- <dig>  glb- <dig>  glb- <dig> and glb- <dig> under the conditions used are unable to bind heme and/or properly fold into a native hemoprotein.
based on previous experience with different globins, for crystallisation purposes, the cys residues in glb- <dig> were mutated to ser.
in glb- <dig> cys residues are present at positions a <dig>  e <dig> and gh <dig>  mutation of cysa <dig> and/or cyse <dig> to ser resulted in expression in inclusion bodies and in failure to incorporate hemin in vitro under the conditions used.
in contrast, mutation of cysgh <dig> into ser  resulted in expression in the cytosolic fraction as a heme-containing globin.
inspection of the 3d structure of glb-1*  shows that the cys residues at positions a <dig> and e <dig> are  <dig> Å apart and not linked by a disulphide bridge.
the intrinsic incorrect folding displayed by the a <dig> and e <dig> ser mutants suggests that the formation of a temporary disulfide bridge between these two residues is necessary during folding of the protein.
gel filtration chromatography on a calibrated superdex™  <dig> column equilibrated in  <dig> mm tris-hcl ph  <dig> ,  <dig> mm nacl and  <dig>  mm edta clearly shows that glb- <dig>  glb-1* and glb- <dig> behave in solution as non covalently bound dimers .
uv/vis and resonance raman spectra
uv/vis absorbance spectra of glb- <dig>  shows the soret band centered at  <dig> nm ; the β- and α- peaks at ~ <dig> and ~ <dig> nm, respectively, typical of an oxygenated globin.
the shoulder at  <dig> nm indicates that a considerable fraction of a high-spin  ferric species is also present.
after deoxygenation and reduction, the soret band shifts to  <dig> nm and the visible absorbance spectrum changes to that typical for the pentacoordinated hs ferrous form of globins.
in the absorption spectrum of co-ligated ferrous glb- <dig>  the soret band peak at ~ <dig> nm, and the β- and α-bands at ~ <dig> and ~ <dig> nm, respectively.
the uv/vis spectra of glb-1* match those of w.t.
glb- <dig> .
the high-frequency region of the rr spectrum of glb- <dig>  is in agreement with the uv/vis spectrum indicating a prevalence of the oxy form  ).
the weak line at  <dig> cm- <dig> indicates a small hs ferric fraction, in agreement with the uv/vis spectra.
after deoxygenation and reduction, the rr spectrum becomes typical of a  hs ferrous globin, again paralleling the uv/vis data ).
furthermore, the ν stretching mode is observed at  <dig> cm- <dig>  typical for hs globins ).
the rr spectra of glb-1* matched closely those of glb- <dig> .
the rr spectrum of the co-ligated form of glb- <dig> displays ν <dig>  ν <dig>  and ν <dig> at ~ <dig> cm- <dig>  ~ <dig> cm- <dig>  and ~ <dig> cm- <dig> respectively ).
upon increase of the laser power, a second component appears in the rr spectrum ).
this component  is typical of hs ferrous globins, indicating that photodissociation of co has taken place.
a strong peak around ~ <dig> cm- <dig> is observed that decreases upon strong photo-dissociation , indicative of the ν mode ).
fe-co stretching modes at higher frequencies cannot be observed.
however, it should be noted that, since no isotopic labelling of the co ligand was performed, weak fe-co stretching modes may escape attention and the current results should be interpreted cautiously.
the soret band of glb- <dig>  is found at  <dig> nm , and the β- and α- bands display maxima at  <dig> and  <dig> nm, respectively, indicating that the globin is in a hexacoordinated low-spin  ferric state.
upon reduction, the soret band shifts to  <dig> nm, indicative of ferrous globins, with an intense α band at  <dig> nm indicating a hexacoordinated ls ferrous heme protein.
the co-ligated ferrous glb- <dig> exhibits a typical uv/vis spectrum, as also observed for co-ligated glb- <dig>
the rr spectra of glb- <dig>  are typical of hexacoordinated ls ferric heme proteins , in agreement with the uv/vis data ).
in the low-frequency region, the out-of-plane mode γ <dig> is hardly detectable, indicating no significant out-of-plane distortion ).
after reduction of glb- <dig>  the rr spectra become typical for hexacoordinated ls ferrous globins  ).
in the rr spectrum of the co-ligated ferrous form of glb- <dig>  the ν mode is again situated at ~ <dig> cm- <dig> ).
no clear ν modes were observed at higher frequencies.
the 3d structural model of glb-1*
the structure of glb-1* gh <dig> → ser mutation to avoid protein aggregates during crystallization) was solved by mad methods based on the anomalous signal of the heme iron atom.
diffraction data were collected at three wavelengths at the id <dig> esrf beamline , for the tetragonal p <dig> form .
refinement of the crystal structure  converged at a general rfactor value of  <dig> % , with ideal stereochemical parameters  <cit> .
the final model contains  <dig>  protein atoms ,  <dig> heme,  <dig> dioxygen molecule and  <dig> ordered solvent atoms  .
the tertiary structure of glb-1* conforms to the three-over-three α-helical sandwich of the classical globin fold  <cit> , where the seven/eight helices building up the globin fold are conventionally labelled, a, b, ..., h, according to their sequential order .
for comparison, a rmsd value of  <dig>  Å is calculated  in a structural overlay on sperm whale mb, the largest structural deviation matching a 4-residue insertion at the ab loop, the cd interhelical region , a 3-residue insertion at the ef hinge, and a 2-residue deletion in the gh region.
residues 17- <dig> , 43- <dig>  and 102- <dig>  of glb-1* display a  <dig> conformation.
the diffraction data collected at  <dig>  Å resolution on glb-1* crystals grown in the trigonal p <dig> space group  were also refined, yielding rfactor and rfree values of  <dig> % and  <dig> %, respectively, with ideal stereochemical parameters .
superposition of  <dig> cα atoms of the two independent molecules, in the trigonal crystal form, yielded a rmsd of  <dig>  Å. comparison of the glb-1* structures from the tetragonal and trigonal crystal forms yielded rmsd of  <dig>  Å and  <dig>  Å, for  <dig> cα atoms, depending on the chains superposed.
the only backbone differences observed in the two crystal forms are located at the n- and c-terminal residues of the polypeptide chain , and at residues 124- <dig> of the gh hinge.
all results discussed below apply to both tetragonal and trigonal crystal forms unless otherwise stated.
in the high resolution tetragonal crystal form the crystallographic two-fold axis gives rise to a glb-1* homodimer, whose  <dig> Å <dig> association interface is mostly built by residues belonging to the e- and f helices , and to the ab  and ef  corners of both chains.
the crystallographic dimer is identical to the quaternary assembly found in the trigonal crystal form, where two crystallographically independent glb-1* chains fill the asymmetric unit.
the rmsd calculated over the whole dimer in the two crystal forms is  <dig>  Å. the heme groups belonging to the two subunits lie  <dig> Å far apart .
in particular, the d-propionates of each heme group are stabilized by an intramolecular salt bridge with the nh <dig> of residue argf <dig> , and can establish an intermolecular salt bridge with argf7' belonging to the facing subunit .
the dimerization interface observed for the glb- <dig> dimer is tightly packed, the stability of the assembly being granted by several hydrophobic interactions, hydrogen bonds  and salt bridges.
such a highly symmetrical homodimer  is predicted to be energetically stable by the protein interfaces, surfaces and assemblies  detection software  <cit> .
remarkably, the glb-1* dimeric assembly is strictly reminiscent of the quaternary assembly observed in scapharca inaequivalvis hb  <cit>  and in caudina arenicola hb  <cit> .
stabilization of the heme group within the glb-1* fold occurs through  <dig> van der waals contacts ; moreover, salt links are present between residue arge <dig> and heme a-propionate, and between residue argf <dig> and heme d-propionate.
these two arg residues, together with arge <dig>  lysc <dig>  lyse <dig>  and lys build an evident positively charged patch around the heme crevice, mostly located on the heme distal side .
the proximal hisf <dig> residue is properly coordinated to the heme-fe atom , and is fully staggered relative to the porphyrin pyrrole n-atoms, allowing in principle a short fe--hisf <dig> coordination bond, and in-plane location of the fe atom.
the hisf <dig> staggered orientation is defined by a strong hydrogen bond  of hisf <dig> nd <dig> atom to the carbonyl o atom of residue thrf <dig>
thanks to the high stability of the oxygenated form, the distal site of the crystallized glb- <dig> hosts an o <dig> molecule coordinated to the heme-fe atom , adopting a moderately bent geometry  to tyrb <dig>  that in turn is hydrogen bonded  to the ne <dig> atom of glne <dig> .
tyrb <dig> and glne <dig> are the only polar residues in the heme distal site, which is essentially lined by aromatic residues b <dig>  pheb <dig>  phec <dig>  phee <dig>  ilee <dig>  and pheg8).
likely related to the overall apolarity, but also to the steric constraints posed by the above mentioned residues, no water molecules are observed in the heme distal site.
the glb-1* heme distal site resembles that of a. suum hb, with tyrb <dig> directly stabilizing the o <dig> molecule and glne <dig> making a hydrogen bond with the b <dig> residue  <cit> .
in a. suum hb the coordination bond fe---o <dig> is shorter  than that found in glb-1* and, due to the arg → phe residue substitution at the e <dig> topological site, the glb-1* salt bridge to the heme a-propionate is not present in a. suum hb.
ligand-binding: kinetic measurements and equilibrium data
the ligand-binding characteristics for both globins were measured using flash photolysis .
for glb- <dig>  the co-rebinding kinetics are monophasic  and the koff for o <dig> is two orders of magnitude lower than for mb, resulting in a high o <dig> affinity .
accordingly, o2-equilibrium experiments show that the o <dig> affinity of glb- <dig> is high and ph-independent , with p <dig> values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  torr at ph  <dig>  and  <dig> , respectively.
hill coefficients below unity  indicate that the subunits of this dimeric globin have different o <dig> affinity .
n.d.: not detectable
note: ko <dig>  obs = ko <dig>  penta/ = //khis, off)
the same experiments were performed for glb- <dig>  it was difficult to measure the kinetics of glb- <dig>  because this globin is readily oxidized to the ferric species in the presence of o <dig>  several reducing systems were tried, but even then it was not possible to keep the heme iron of this protein reduced in the presence of o <dig>  however, glb- <dig> is easily reduced anaerobically by sodium dithionite or by nadph under light exposure.
the co-rebinding kinetics to the hexacoordinated glb- <dig> are characterized by a biphasic trace  with a slow phase of replacement due to the slow dissociation of the distal residue.
the characterization of this slow phase was confirmed by measurement of the full absorption spectra versus time .
based on the kon for the co ligand, the kon-value for the distal ligand his was calculated to be  <dig>  <dig> s- <dig>
in order to attempt to measure the o2-dissociation rate of glb- <dig>  mixed atmosphere o2/co experiments were performed.
the oxy complex of glb- <dig> is unstable .
after co dissociation, a first phase of rebinding was measured where three ligands are in competition .
a slow phase  was measured at equimolar ligand concentration .
this phase does not correspond to the o <dig> replacement by co, but rather to the o2-induced oxidation of the heme, because the initial co-globin signal  decreases after each rebinding cycle.
although a small fraction of transient o2-heme complex is replaced by co, the koff  is undistinguishable from that of the oxidation rate.
o <dig> equilibrium measurements were tentatively run in the presence of the hayashi reducing system.
even then, only ferric and deoxy globins were detected when changing o <dig> tensions, confirming that a stable oxy-derivative is not formed .
in situ localisation
analysis of tissue-specific expression patterns revealed that glb- <dig> is expressed exclusively in the head mesodermal cell  and in the stomato-intestinal muscle .
the function of the head mesodermal cell is currently unknown.
the stomato-intestinal muscle connects the surfaces of the posterior intestinal cells to the ventral epidermis and is coupled to the anal sphincter and anal depressor muscles via gap junctions.
contraction of these muscles promotes defecation.
glb- <dig> is expressed mainly in a subset of neuronal cells and in head muscular tissue .
both globin genes clearly display distinct expression patterns.
notably, the majority of c. elegans globins are expressed in the nervous system in highly cell-specific patterns  <cit> .
globin expression under hypoxic conditions
to investigate hypoxic response, worms were subjected to hypoxia  for  <dig> h in a hypoxic chamber, and globin gene expression levels were determined by quantitative real-time rt-pcr.
analysis of the expression profiles revealed significant upregulation for glb- <dig>  whereas expression levels of glb- <dig>  remain unaffected by hypoxia .
discussion
gas exchange in c. elegans relies completely on diffusion from the surrounding environment into the tissues  <cit> .
c. elegans has an extended network of  <dig> neurons.
neuronal signal transduction consumes high amounts of energy, which mainly originates from aerobic metabolism.
neurons have therefore developed various adaptation strategies against hypoxic stress, for example the expression of globin-like proteins.
c. elegans globin-like proteins are mainly present in neurons.
it is therefore possible that c. elegans globin-like proteins play a role in the defence against the toxic effects of hypoxia/anoxia by functioning in detoxification of ros and rns as well as in o <dig> sensing and/or that they may have a role in redox reactions, rather than functioning as o <dig> carriers, as proposed for ngb  <cit> .
although the c. elegans genome codes for more than  <dig> globin-like proteins, which are all expressed as demonstrated by rt-pcr, no characteristic uv/vis globin absorption spectrum can be detected in a crude c. elegans extract.
this suggests that the overall in vivo protein concentrations are very low  <cit> , which tallies with the possibility that some of the globin-like molecules  are expressed only in a small subset of cells.
expression of a selected set of c. elegans globins in an e. coli system in vitro, demonstrates that, under the conditions used, only glb- <dig> and glb- <dig> can be recovered as hemoproteins in the cytosolic fraction of the cell.
refolding of the others, starting from the apoprotein, purified from inclusion bodies, and adding hemin in vitro failed.
when inspecting the sequences of the c. elegans globins, a correlation was found between the presence of long n- and/or c-terminal extensions and the expression in inclusion bodies as was also seen previously for cygb  <cit> .
the inability to refold several of the globin-like proteins may also suggest that some of these may not incorporate a heme group in vivo.
hence, they could support a function that does not require heme.
such consideration, however, does not exclude that these proteins may play a physiological role.
indeed, the n-terminal domain of rsbr, a protein of the bacterial stressosomes  clearly displays the globin fold but is unable to bind heme  <cit> .
moreover, the n-terminal domain of rsbr dimerizes in a fashion identical to that of the globin-coupled sensor, hemat of bacillus subtilis and geobacter sulfurreducens  <cit> .
it is hypothezised that in rsbr the globin-folded domain evolved from an ancestral sensor globin related to the globin-coupled sensors, but lost its ability to bind heme in later evolutionary stages  <cit> .
the uv/vis and rr spectra of glb- <dig> indicate that this globin is pentacoordinated in the deoxy form.
the ν stretching mode, observed at ~ <dig> cm- <dig> has previously been observed in other globins, e.g.
sperm whale mb   <cit> .
in mb, this conformer becomes dominant at low ph values, when the distal hise <dig> residue swings out of the heme pocket.
in glb- <dig> it is ascribed to an open conformation of the heme pocket, with a weak interaction between co and the surrounding residues.
no fe-co stretching modes at higher frequencies can be observed, in agreement with earlier observations on paramecium caudatum hb  <cit> .
however, it should be noted that, since no isotopic labelling of the co ligand was performed, weak fe-co stretching modes may escape attention.
unlike the co form, the 3-d structure shows a stabilization of bound o <dig> through a hydrogen bond with tyrb <dig>  same is seen for a suum hb  <cit> .
glb- <dig> displays a quite high o <dig> affinity, mainly due to the slow koff for o <dig>  because of the presence of the glne7/tyrb <dig> heme distal pair, a high o <dig> affinity was expected.
nevertheless, the o <dig> affinity is almost  <dig> times lower than that of a. suum .
this difference is mainly due to a slower o <dig> dissociation process in a. suum hb  than in glb- <dig> , related to strong stabilization of the bound o <dig> by two hydrogen bonds, a short one with tyrb <dig>  and a weaker one with glne <dig>   <cit> .
in glb- <dig> tyrb <dig> also stabilizes the bound oxygen , but the fe---o <dig> coordination bond is longer than that in a. suum hb.
therefore it is easier to break, resulting in a lower o <dig> affinity compared with a. suum hb.
the nemertean c. lacteus mini-hb also displays a tyrb10/glne <dig> pair, but displays high rates of o <dig> dissociation  ~200- <dig> s-1) and hence a moderate o <dig> affinity  as a result of a third polar residue, thre <dig>  in its distal site.
in fact, the presence of thr at position e <dig> modulates the formation of a hydrogen bond between tyrb <dig> and the heme-bound o <dig>  <cit> .
uv/vis spectra similar to those of ferrous glb- <dig> were observed for the ferrous forms of ngb  <cit> , and cygb  <cit> .
the spectra are typical of ferrous globins exhibiting bis-histidine heme coordination.
the ν stretching mode of co-ligated ferrous glb- <dig> is situated at ~ <dig> cm- <dig>  again typical of an open conformation of the heme pocket.
no clear ν modes were observed at higher frequencies, in contrast to other heme proteins exhibiting bis-histidine coordination, such as ngb, which exhibits three intense fe-co stretching modes   <cit> .
the biphasic plot observed for the glb- <dig> co-rebinding kinetics results from the competition between co and the distal hise <dig> ligand.
the slow phase of replacement indicates that the dissociation of the distal e <dig> residue is slow.
for most hexacoordinated globin samples equilibrated under  <dig> atm co, this phase is only a few percent.
the calculated kon value for the distal ligand his  is the highest ever measured for a hexacoordinated globin   <cit> .
also note that the kco of glb- <dig> is about one order of magnitude lower than that of ngb .
this is in agreement with the rr results indicating an open conformation of the glb- <dig> co-ligated form.
in ngb, closed conformations have been identified  <cit> .
concerning the rates of o2-binding, both equilibrium and flash photolysis data confirm that glb- <dig> is not capable of reversible o <dig> binding, and hence cannot act as an o <dig> store.
other globins, like ngb, cygb, 2-on- <dig>  hemoglobins and globin-coupled sensors, clearly illustrate different possible functions for members of the globin superfamily  <cit> .
the tissue distribution of ngb, cygb and the hypoxia-inducible myoglobin expression in non-muscle tissues in fish supports the hypothesis that globins may occur in every cell  <cit> .
additional in vivo analysis underscores the different behaviour of glb- <dig> and glb- <dig>  gfp reporter assays revealed a clearly distinct expression pattern of glb- <dig> and glb- <dig>  in addition both globins behave differently under hypoxic conditions ; glb- <dig> is significantly upregulated under hypoxia, whereas expression of glb- <dig> remains unchanged.
strikingly, the opposite was observed under conditions of total anoxia; whereas the expression level of glb- <dig> is unaffected, expression of glb- <dig> is induced.
CONCLUSIONS
glb- <dig> and glb- <dig> are fundamentally different relative to their tissue localisation, expression pattern under hypoxia and anoxia, ligand-binding mechanism and affinities.
ferrous glb- <dig> binds o <dig> with a high affinity and is pentacoordinated in the absence of exogenous heme ligands.
taken together, the observed functional data suggest for glb- <dig> a putative role in vivo related to o <dig> binding, no scavenging or o <dig> storage.
this globin could serve to maintain the o <dig> concentration at a constant level provided that its cellular concentration is high enough.
the induced glb- <dig> expression under hypoxia conditions is consistent with a possible role in oxygen-dependent metabolism.
similar hypoxia-dependent inductions have been reported for globins putatively involved in oxygen transport or storage  <cit> .
interestingly, several hypoxia-tolerant organisms including daphnia, danio rerio and carassius auratus display upregulation of pentacoordinated globins upon hypoxic exposure  <cit> .
furthermore, the oxygenated globin could play a role in regulating no-homeostasis.
ferrous glb- <dig> globin cannot serve such functions due to its inability to form a stable oxygenated species.
it can be speculated that at high o <dig> levels glb- <dig> will readily reduce o2; the reduced species may then participate in redox reactions or simply dismutate into o <dig> and h2o <dig>  glb- <dig> may thus resemble oxidases.
under anaerobic conditions, this globin might participate in other redox reactions.
indeed, the very high glb- <dig> heme affinity for the distal hise <dig> residue points to roles other than binding gaseous ligands.
interestingly, an n-terminal myristoylation site is predicted http://expasy.org/ with very high confidence  for glb- <dig>  which may indicate membrane anchoring, an unprecedented globin function  <cit> .

 the increasing availability of genomic data shows that globin-like proteins/domains are universal and arose very early in the evolution of life. recent studies reveal new globin types as well as new functions. a general survey suggests that the majority of nematodes host globin-like proteins. based on their tissue localisation, these proteins can be grouped in three classes: pseudocoelomic fluid, body wall, and cuticular hemoglobins. some nematode species may however lack a particular class. to date, the best characterized pseudocoelomic fluid hb is the hb from the ascarid ascaris suum. this is an octameric molecule consisting of two layers of four subunits stacked in an eclipsed orientation. each subunit of kda consists of two covalently linked, highly similar globin domains followed by a c-terminal polar zipper of amino acids. this hb has an exceptionally high o affinity that is attributed to a very slow dissociation rate: s-1) and a normal association rate: μm-1s-1), whereby this hb will be in the oxy form even in the host's gut with a locally micro-oxygen concentration. this high o affinity can be explained structurally by the presence of three hydrogen bonds between tyrb and glne and the bound ligand. the function of the a. suum pseudocoelomic fluid hb is still a matter of debate. due to its extremely high o affinity, it will rarely be deoxygenated in vivo and cannot therefore function as an o carrier or store. several alternative functions have been proposed, such as o or no scavengers, iron and/or heme stores, catalyst in sterol biosynthesis, buffering, osmotic - or other unknown functions. the existence of globins in c. elegans was unexpected given the worm's small size, whereby sufficient amounts of o may reach the terminal oxidase sites by simple diffusion. the complete c. elegans genome revealed globin-like genes, all having orthologues in the sibling species c. briggsae. all putative globin genes are expressed, albeit at low or very low levels, most likely indicating cell-specific expression. they show wide diversity in gene structure and amino-acid sequence of the translated proteins, suggesting a long evolutionary history. nevertheless, sequence similarity to standard vertebrate globins, such as hb α, β chains, myoglobin, neuro- and cyto-globin can be detected, despite the presence of additional interhelical, n- and/or c-terminal extensions. the intron/exon patterns of these c. elegans globin-coding genes are unique in the number of introns and in their insertion positions, compared to the highly conserved intron/exon pattern of vertebrate and other non-vertebrate globin genes. given the large number of c. elegans globin genes it is unlikely that they all serve simple o metabolism. c. elegans is a soil dwelling nematode; soil parameters such as temperature, moisture and o concentration fluctuate, creating temporary hypoxic/anoxic environments. living systems have developed, at the organism and cellular levels, various strategies to cope with reduced o levels, e.g. increasing the glycolytic flux, reducing the aerobic metabolic rate and increasing the efficiency of o uptake from the environment. however, nematodes including c. elegans lack both specialized respiratory systems and complex circulatory organs, and rely on respiratory adjustments at the molecular levels. specialized o2-sensing cells in the nervous system of c. elegans permit rapid behavioural responses to o availability. the signal transduction pathway, detecting upper and lower o levels, involves the cgmp-gated channel tax-2/tax- and a soluble guanylate cyclase homologue, gcy- the expressed n-terminal domain, gcy- displays the spectral characteristics of a ferrous high-spin hemoprotein capable of o co and no binding. wild-type animals survive short periods of hypoxia/anoxia by greatly reducing metabolic rate and arresting movement and development. on the other hand the metabolic rate of the animals is not affected by increasing o levels up to 100%. with the aim of shedding light on the roles played by c. elegans globins, we cloned several globin genes, and focussed our attention on two translation products. we analyzed their expression patterns and alterations of transcript abundance under hypoxia. we then analyzed the two expressed globins through optical and resonance raman spectroscopy, characterized the kinetics of ligand binding and o equilibrium properties, and determined the high resolution crystal structure of glb- the results are discussed in the context of current views on multivariate globin function, of heme/ligand recognition mechanisms, and of the evolution of the globin fold through the phyla.  expression cloning of globins eight c. elegans globins were selected for cloning and expression. these were the canonical globin, glb- three globins that show sequence similarity to cygb, two globins displaying similarity to ngb, glb- that contains an exceptionally large n-terminal extension, and glb- in which ile substitutes the highly conserved phe at position cd all globin genes were cloned into pet3a and expressed in vitro in e. coli. after expression, only two globins, glb- and glb- were found in the cytosolic fractions with incorporated heme, whereas all the others were present as apo-proteins in inclusion bodies. all attempts to reconstruct the purified apo-proteins with hemin in vitro using different methods failed. as many globins are easily reconstructed from hemin and their apo-protein we concluded that glb- glb- glb- glb- glb- and glb- under the conditions used are unable to bind heme and/or properly fold into a native hemoprotein. based on previous experience with different globins, for crystallisation purposes, the cys residues in glb- were mutated to ser. in glb- cys residues are present at positions a e and gh mutation of cysa and/or cyse to ser resulted in expression in inclusion bodies and in failure to incorporate hemin in vitro under the conditions used. in contrast, mutation of cysgh into ser resulted in expression in the cytosolic fraction as a heme-containing globin. inspection of the 3d structure of glb-1* shows that the cys residues at positions a and e are Å apart and not linked by a disulphide bridge. the intrinsic incorrect folding displayed by the a and e ser mutants suggests that the formation of a temporary disulfide bridge between these two residues is necessary during folding of the protein. gel filtration chromatography on a calibrated superdex™ column equilibrated in mm tris-hcl ph, mm nacl and mm edta clearly shows that glb- glb-1* and glb- behave in solution as non covalently bound dimers. uv/vis and resonance raman spectra uv/vis absorbance spectra of glb- shows the soret band centered at nm; the β- and α- peaks at ~ and ~ nm, respectively, typical of an oxygenated globin. the shoulder at nm indicates that a considerable fraction of a high-spin ferric species is also present. after deoxygenation and reduction, the soret band shifts to nm and the visible absorbance spectrum changes to that typical for the pentacoordinated hs ferrous form of globins. in the absorption spectrum of co-ligated ferrous glb- the soret band peak at ~ nm, and the β- and α-bands at ~ and ~ nm, respectively. the uv/vis spectra of glb-1* match those of w.t. glb-. the high-frequency region of the rr spectrum of glb- is in agreement with the uv/vis spectrum indicating a prevalence of the oxy form ). the weak line at cm- indicates a small hs ferric fraction, in agreement with the uv/vis spectra. after deoxygenation and reduction, the rr spectrum becomes typical of a hs ferrous globin, again paralleling the uv/vis data ). furthermore, the ν stretching mode is observed at cm- typical for hs globins ). the rr spectra of glb-1* matched closely those of glb-. the rr spectrum of the co-ligated form of glb- displays ν ν and ν at ~ cm- ~ cm- and ~ cm- respectively ). upon increase of the laser power, a second component appears in the rr spectrum ). this component is typical of hs ferrous globins, indicating that photodissociation of co has taken place. a strong peak around ~ cm- is observed that decreases upon strong photo-dissociation, indicative of the ν mode ). fe-co stretching modes at higher frequencies cannot be observed. however, it should be noted that, since no isotopic labelling of the co ligand was performed, weak fe-co stretching modes may escape attention and the current results should be interpreted cautiously. the soret band of glb- is found at nm, and the β- and α- bands display maxima at and nm, respectively, indicating that the globin is in a hexacoordinated low-spin ferric state. upon reduction, the soret band shifts to nm, indicative of ferrous globins, with an intense α band at nm indicating a hexacoordinated ls ferrous heme protein. the co-ligated ferrous glb- exhibits a typical uv/vis spectrum, as also observed for co-ligated glb- the rr spectra of glb- are typical of hexacoordinated ls ferric heme proteins, in agreement with the uv/vis data ). in the low-frequency region, the out-of-plane mode γ is hardly detectable, indicating no significant out-of-plane distortion ). after reduction of glb- the rr spectra become typical for hexacoordinated ls ferrous globins ). in the rr spectrum of the co-ligated ferrous form of glb- the ν mode is again situated at ~ cm- ). no clear ν modes were observed at higher frequencies. the 3d structural model of glb-1* the structure of glb-1* gh → ser mutation to avoid protein aggregates during crystallization) was solved by mad methods based on the anomalous signal of the heme iron atom. diffraction data were collected at three wavelengths at the id esrf beamline, for the tetragonal p form. refinement of the crystal structure converged at a general rfactor value of %, with ideal stereochemical parameters. the final model contains protein atoms, heme, dioxygen molecule and ordered solvent atoms. the tertiary structure of glb-1* conforms to the three-over-three α-helical sandwich of the classical globin fold, where the seven/eight helices building up the globin fold are conventionally labelled, a, b,..., h, according to their sequential order. for comparison, a rmsd value of Å is calculated in a structural overlay on sperm whale mb, the largest structural deviation matching a 4-residue insertion at the ab loop, the cd interhelical region, a 3-residue insertion at the ef hinge, and a 2-residue deletion in the gh region. residues 17-, 43- and 102- of glb-1* display a conformation. the diffraction data collected at Å resolution on glb-1* crystals grown in the trigonal p space group were also refined, yielding rfactor and rfree values of % and %, respectively, with ideal stereochemical parameters. superposition of cα atoms of the two independent molecules, in the trigonal crystal form, yielded a rmsd of Å. comparison of the glb-1* structures from the tetragonal and trigonal crystal forms yielded rmsd of Å and Å, for cα atoms, depending on the chains superposed. the only backbone differences observed in the two crystal forms are located at the n- and c-terminal residues of the polypeptide chain, and at residues 124- of the gh hinge. all results discussed below apply to both tetragonal and trigonal crystal forms unless otherwise stated. in the high resolution tetragonal crystal form the crystallographic two-fold axis gives rise to a glb-1* homodimer, whose Å association interface is mostly built by residues belonging to the e- and f helices, and to the ab and ef corners of both chains. the crystallographic dimer is identical to the quaternary assembly found in the trigonal crystal form, where two crystallographically independent glb-1* chains fill the asymmetric unit. the rmsd calculated over the whole dimer in the two crystal forms is Å. the heme groups belonging to the two subunits lie Å far apart. in particular, the d-propionates of each heme group are stabilized by an intramolecular salt bridge with the nh of residue argf, and can establish an intermolecular salt bridge with argf7' belonging to the facing subunit. the dimerization interface observed for the glb- dimer is tightly packed, the stability of the assembly being granted by several hydrophobic interactions, hydrogen bonds and salt bridges. such a highly symmetrical homodimer is predicted to be energetically stable by the protein interfaces, surfaces and assemblies detection software. remarkably, the glb-1* dimeric assembly is strictly reminiscent of the quaternary assembly observed in scapharca inaequivalvis hb and in caudina arenicola hb. stabilization of the heme group within the glb-1* fold occurs through van der waals contacts; moreover, salt links are present between residue arge and heme a-propionate, and between residue argf and heme d-propionate. these two arg residues, together with arge lysc lyse and lys build an evident positively charged patch around the heme crevice, mostly located on the heme distal side. the proximal hisf residue is properly coordinated to the heme-fe atom, and is fully staggered relative to the porphyrin pyrrole n-atoms, allowing in principle a short fe--hisf coordination bond, and in-plane location of the fe atom. the hisf staggered orientation is defined by a strong hydrogen bond of hisf nd atom to the carbonyl o atom of residue thrf thanks to the high stability of the oxygenated form, the distal site of the crystallized glb- hosts an o molecule coordinated to the heme-fe atom, adopting a moderately bent geometry to tyrb that in turn is hydrogen bonded to the ne atom of glne. tyrb and glne are the only polar residues in the heme distal site, which is essentially lined by aromatic residues b pheb phec phee ilee and pheg8). likely related to the overall apolarity, but also to the steric constraints posed by the above mentioned residues, no water molecules are observed in the heme distal site. the glb-1* heme distal site resembles that of a. suum hb, with tyrb directly stabilizing the o molecule and glne making a hydrogen bond with the b residue. in a. suum hb the coordination bond fe---o is shorter than that found in glb-1* and, due to the arg → phe residue substitution at the e topological site, the glb-1* salt bridge to the heme a-propionate is not present in a. suum hb. ligand-binding: kinetic measurements and equilibrium data the ligand-binding characteristics for both globins were measured using flash photolysis. for glb- the co-rebinding kinetics are monophasic and the koff for o is two orders of magnitude lower than for mb, resulting in a high o affinity. accordingly, o2-equilibrium experiments show that the o affinity of glb- is high and ph-independent, with p values of ± and ± torr at ph and, respectively. hill coefficients below unity indicate that the subunits of this dimeric globin have different o affinity. n.d.: not detectable note: ko obs = ko penta/ = //khis, off) the same experiments were performed for glb- it was difficult to measure the kinetics of glb- because this globin is readily oxidized to the ferric species in the presence of o several reducing systems were tried, but even then it was not possible to keep the heme iron of this protein reduced in the presence of o however, glb- is easily reduced anaerobically by sodium dithionite or by nadph under light exposure. the co-rebinding kinetics to the hexacoordinated glb- are characterized by a biphasic trace with a slow phase of replacement due to the slow dissociation of the distal residue. the characterization of this slow phase was confirmed by measurement of the full absorption spectra versus time. based on the kon for the co ligand, the kon-value for the distal ligand his was calculated to be s- in order to attempt to measure the o2-dissociation rate of glb- mixed atmosphere o2/co experiments were performed. the oxy complex of glb- is unstable. after co dissociation, a first phase of rebinding was measured where three ligands are in competition. a slow phase was measured at equimolar ligand concentration. this phase does not correspond to the o replacement by co, but rather to the o2-induced oxidation of the heme, because the initial co-globin signal decreases after each rebinding cycle. although a small fraction of transient o2-heme complex is replaced by co, the koff is undistinguishable from that of the oxidation rate. o equilibrium measurements were tentatively run in the presence of the hayashi reducing system. even then, only ferric and deoxy globins were detected when changing o tensions, confirming that a stable oxy-derivative is not formed. in situ localisation analysis of tissue-specific expression patterns revealed that glb- is expressed exclusively in the head mesodermal cell and in the stomato-intestinal muscle. the function of the head mesodermal cell is currently unknown. the stomato-intestinal muscle connects the surfaces of the posterior intestinal cells to the ventral epidermis and is coupled to the anal sphincter and anal depressor muscles via gap junctions. contraction of these muscles promotes defecation. glb- is expressed mainly in a subset of neuronal cells and in head muscular tissue. both globin genes clearly display distinct expression patterns. notably, the majority of c. elegans globins are expressed in the nervous system in highly cell-specific patterns. globin expression under hypoxic conditions to investigate hypoxic response, worms were subjected to hypoxia for h in a hypoxic chamber, and globin gene expression levels were determined by quantitative real-time rt-pcr. analysis of the expression profiles revealed significant upregulation for glb- whereas expression levels of glb- remain unaffected by hypoxia. discussion gas exchange in c. elegans relies completely on diffusion from the surrounding environment into the tissues. c. elegans has an extended network of neurons. neuronal signal transduction consumes high amounts of energy, which mainly originates from aerobic metabolism. neurons have therefore developed various adaptation strategies against hypoxic stress, for example the expression of globin-like proteins. c. elegans globin-like proteins are mainly present in neurons. it is therefore possible that c. elegans globin-like proteins play a role in the defence against the toxic effects of hypoxia/anoxia by functioning in detoxification of ros and rns as well as in o sensing and/or that they may have a role in redox reactions, rather than functioning as o carriers, as proposed for ngb. although the c. elegans genome codes for more than globin-like proteins, which are all expressed as demonstrated by rt-pcr, no characteristic uv/vis globin absorption spectrum can be detected in a crude c. elegans extract. this suggests that the overall in vivo protein concentrations are very low, which tallies with the possibility that some of the globin-like molecules are expressed only in a small subset of cells. expression of a selected set of c. elegans globins in an e. coli system in vitro, demonstrates that, under the conditions used, only glb- and glb- can be recovered as hemoproteins in the cytosolic fraction of the cell. refolding of the others, starting from the apoprotein, purified from inclusion bodies, and adding hemin in vitro failed. when inspecting the sequences of the c. elegans globins, a correlation was found between the presence of long n- and/or c-terminal extensions and the expression in inclusion bodies as was also seen previously for cygb. the inability to refold several of the globin-like proteins may also suggest that some of these may not incorporate a heme group in vivo. hence, they could support a function that does not require heme. such consideration, however, does not exclude that these proteins may play a physiological role. indeed, the n-terminal domain of rsbr, a protein of the bacterial stressosomes clearly displays the globin fold but is unable to bind heme. moreover, the n-terminal domain of rsbr dimerizes in a fashion identical to that of the globin-coupled sensor, hemat of bacillus subtilis and geobacter sulfurreducens. it is hypothezised that in rsbr the globin-folded domain evolved from an ancestral sensor globin related to the globin-coupled sensors, but lost its ability to bind heme in later evolutionary stages. the uv/vis and rr spectra of glb- indicate that this globin is pentacoordinated in the deoxy form. the ν stretching mode, observed at ~ cm- has previously been observed in other globins, e.g. sperm whale mb. in mb, this conformer becomes dominant at low ph values, when the distal hise residue swings out of the heme pocket. in glb- it is ascribed to an open conformation of the heme pocket, with a weak interaction between co and the surrounding residues. no fe-co stretching modes at higher frequencies can be observed, in agreement with earlier observations on paramecium caudatum hb. however, it should be noted that, since no isotopic labelling of the co ligand was performed, weak fe-co stretching modes may escape attention. unlike the co form, the 3-d structure shows a stabilization of bound o through a hydrogen bond with tyrb same is seen for a suum hb. glb- displays a quite high o affinity, mainly due to the slow koff for o because of the presence of the glne7/tyrb heme distal pair, a high o affinity was expected. nevertheless, the o affinity is almost times lower than that of a. suum. this difference is mainly due to a slower o dissociation process in a. suum hb than in glb-, related to strong stabilization of the bound o by two hydrogen bonds, a short one with tyrb and a weaker one with glne. in glb- tyrb also stabilizes the bound oxygen, but the fe---o coordination bond is longer than that in a. suum hb. therefore it is easier to break, resulting in a lower o affinity compared with a. suum hb. the nemertean c. lacteus mini-hb also displays a tyrb10/glne pair, but displays high rates of o dissociation ~200- s-1) and hence a moderate o affinity as a result of a third polar residue, thre in its distal site. in fact, the presence of thr at position e modulates the formation of a hydrogen bond between tyrb and the heme-bound o. uv/vis spectra similar to those of ferrous glb- were observed for the ferrous forms of ngb, and cygb. the spectra are typical of ferrous globins exhibiting bis-histidine heme coordination. the ν stretching mode of co-ligated ferrous glb- is situated at ~ cm- again typical of an open conformation of the heme pocket. no clear ν modes were observed at higher frequencies, in contrast to other heme proteins exhibiting bis-histidine coordination, such as ngb, which exhibits three intense fe-co stretching modes. the biphasic plot observed for the glb- co-rebinding kinetics results from the competition between co and the distal hise ligand. the slow phase of replacement indicates that the dissociation of the distal e residue is slow. for most hexacoordinated globin samples equilibrated under atm co, this phase is only a few percent. the calculated kon value for the distal ligand his is the highest ever measured for a hexacoordinated globin. also note that the kco of glb- is about one order of magnitude lower than that of ngb. this is in agreement with the rr results indicating an open conformation of the glb- co-ligated form. in ngb, closed conformations have been identified. concerning the rates of o2-binding, both equilibrium and flash photolysis data confirm that glb- is not capable of reversible o binding, and hence cannot act as an o store. other globins, like ngb, cygb, 2-on- hemoglobins and globin-coupled sensors, clearly illustrate different possible functions for members of the globin superfamily. the tissue distribution of ngb, cygb and the hypoxia-inducible myoglobin expression in non-muscle tissues in fish supports the hypothesis that globins may occur in every cell. additional in vivo analysis underscores the different behaviour of glb- and glb- gfp reporter assays revealed a clearly distinct expression pattern of glb- and glb- in addition both globins behave differently under hypoxic conditions; glb- is significantly upregulated under hypoxia, whereas expression of glb- remains unchanged. strikingly, the opposite was observed under conditions of total anoxia; whereas the expression level of glb- is unaffected, expression of glb- is induced.  glb- and glb- are fundamentally different relative to their tissue localisation, expression pattern under hypoxia and anoxia, ligand-binding mechanism and affinities. ferrous glb- binds o with a high affinity and is pentacoordinated in the absence of exogenous heme ligands. taken together, the observed functional data suggest for glb- a putative role in vivo related to o binding, no scavenging or o storage. this globin could serve to maintain the o concentration at a constant level provided that its cellular concentration is high enough. the induced glb- expression under hypoxia conditions is consistent with a possible role in oxygen-dependent metabolism. similar hypoxia-dependent inductions have been reported for globins putatively involved in oxygen transport or storage. interestingly, several hypoxia-tolerant organisms including daphnia, danio rerio and carassius auratus display upregulation of pentacoordinated globins upon hypoxic exposure. furthermore, the oxygenated globin could play a role in regulating no-homeostasis. ferrous glb- globin cannot serve such functions due to its inability to form a stable oxygenated species. it can be speculated that at high o levels glb- will readily reduce o2; the reduced species may then participate in redox reactions or simply dismutate into o and h2o glb- may thus resemble oxidases. under anaerobic conditions, this globin might participate in other redox reactions. indeed, the very high glb- heme affinity for the distal hise residue points to roles other than binding gaseous ligands. interestingly, an n-terminal myristoylation site is predicted http://expasy.org/ with very high confidence for glb- which may indicate membrane anchoring, an unprecedented globin function. 
we expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance raman spectroscopy, measured the kinetics and equilibria of o binding and determined the crystal structure of glb-1*. within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including tyrb and glne glb- exhibits high ligand affinity, which is, however, lower than in other globins with the same distal tyrb10-glne amino-acid pair. 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. furthermore, we studied the expression patterns of glb- and glb- 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.
 the genome of the nematode caenorhabditis elegans contains more than 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.  we expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance raman spectroscopy, measured the kinetics and equilibria of o binding and determined the crystal structure of glb-1*. furthermore, we studied the expression patterns of glb- and glb- 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 tyrb and glne glb- exhibits high ligand affinity, which is, however, lower than in other globins with the same distal tyrb10-glne amino-acid pair. in the absence of external ligands, the heme ferrous iron of glb- is strongly hexacoordinated with hise 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.  the presented data indicate that glb- and glb- belong to two functionally-different globin classes. 