The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

The recent discovery of globin proteins in the nerve cells of mammals and other vertebrates (1) has created much interest in unraveling the cellular functions and potential biomedical implications of these oxygen binding molecules in the metabolism of the vertebrate nervous system (2). Historically, however, "nerve hemoglobins" (nHbs) 4 were first observed in invertebrate taxa. In 1872, Lankester had already recorded the brilliant red color of the ganglia of the polychaete annelid Aphrodite aculeata (3). Since then, nHbs have also been found in, or associated with, nerve tissues of several other invertebrate taxa, such as molluscs, arthropods, nemerteans, and nematode species (4,5). Of these, only the nHbs of A. aculeata (6) and the nemertean worm Cerebratulus lacteus (7) have been characterized at the molecular level.
With few exceptions (e.g. in air-breathing gastropods), the invertebrate nHbs are expressed at high concentrations (in the mM range) and have therefore been suggested to support nerve function by mediating O 2 storage and/or transport during temporary hypoxia (4). For example, in the gastropod mollusc Aplysia depilans, the oxygenation state of the nHb was correlated with the electrical activity of the neural ganglia, and firing was shown to be proportional to the degree of oxygenation of the globin (8). The nHb in the nerve bundles of the bivalve mollusc Tellina alternata was reported to extend the time of O 2 delivery to the nerves during anoxic periods by as much as 30 min, whereas this effect was not demonstrable in the nerves of a related clam species Tagelius plebeius, which lack the nHb (9). In many cases, the nHbs appear to be specifically expressed in the glial cells surrounding neurons, as reported for the clams T. alternata and Spisula solidissima (10), the water-dwelling gastropods Lymnaea stagnalis and Planorbis corneus (11), the nemertean C. lacteus (7), and the annelid A. aculeata (12). In Aplysia and in the air-breathing gastropods Helix pomatia and Cepaea nemoralis, however, the nHbs have been detected within the neurons themselves (11,13). The physiological implications of this cell type-specific expression are unclear.
With P 50 values between 1.1 and 4 torr (12), the oxygen affinities of nHbs are moderate and quite similar to those of vertebrate myoglobins (Mbs), in agreement with the proposed oxygen supply function. However, spectral analyses revealed two different types of nHbs based on the coordination at the heme iron. The nHbs of Aplysia spec., A. aculeata and C. lacteus are pentacoordinate, like vertebrate hemoglobins (Hbs) and Mbs, whereas those of the bivalves T. alternata and S. solidissima are hexacoordinate and exhibit a cytochrome b-type absorption spectrum (9,14). In hexacoordination, an internal protein ligand (mostly the distal E7 histidine residue) occupies the sixth coordinating position of the heme iron and has to be displaced before an external gaseous ligand can be bound. This results in complex ligand binding kinetics, of which the functional implications still have to be revealed (15,16).
Several of these features (e.g. hexacoordination, moderate oxygen affinity) of invertebrate nHbs are shared with the neuroglobins (Ngb), which have been identified in many vertebrate taxa, including mammals, birds, amphibians, and fishes (17). Ngb is expressed in the central and peripheral nervous system, the retina, and in endocrine tissue, but it is specifically found in neurons and not in astrocytes or other glial cells (18 -22). In contrast to most nHbs, Ngb is expressed at lower concentrations (M) in the vertebrate brain, whereas it is more strongly expressed in the neuronal retina (20). Ngb also displays a bis-His-Fe hexacoordinate heme structure (1,(23)(24)(25). Ngb is thought to protect neurons against hypoxic damage in vitro and against ischemia/reperfusion injury in vivo (26,27). However, its function is unclear, and several putative physiological roles of Ngb are still discussed (2).
For further understanding of both vertebrate Ngb and invertebrate nHb functions, we have aimed at the molecular characterization of the nHb of the Atlantic surf clam S. solidissima (14,28,29). This protein is an example of a hexacoordinate nHb, which is abundantly expressed in glial cells and lends the nerves of this organism a bright red appearance.

MATERIALS AND METHODS
Animals-S. solidissima (Mollusca, Bivalvia) were obtained from the Aquatic Resources Division, Marine Biological Laboratory, Woods Hole, MA. Nerve cords and foot and contractor muscle were dissected, frozen in liquid nitrogen, and stored at Ϫ80°C until use.
Determination of the Partial Amino Acid Sequence of S. solidissima nHb-Nerves were thawed and homogenized in 50 mM Tris-HCl, pH 7.5, containing the Complete TM protease inhibitor mixture (Roche Diagnostics) and centrifuged 10 min at 10,000 rpm; the red supernatant was saved. The nHb was precipitated by differential ammonium sulfate precipitation at 40 and 90% saturation, respectively. Desalted samples were made up to 50 mM Tris-HCl, pH 7.5, 6 M guanidinium hydrochloride, 1% 2-mercapto-ethanol and heated for 5 min at 100°C. Samples were acidified with 0.1% trifluoroacetic acid and separated by reversed phase chromatography on a Vydac C4 column (4.1 mm ϫ 25 cm) using a gradient 0.1% trifluoroacetic acid, 0.5% CH 3 CN to 0.1% trifluoroacetic acid, 75% CH 3 CN in 45 min at a flow rate of 1 ml/min. The nHbcontaining fractions were vacuum dried and digested with trypsin at an enzyme/substrate ratio of 1/50. The resulting peptides were separated on a Vydac C18 column (2.1 mm ϫ 25 cm; flow rate, 200 l/min) using the same gradient as for globin purification. Purified peptides were sequenced by automated Edman degradation using an ABI Prosice protein sequencer operated according to the manufacturer's instructions. Peptide sequences were aligned using the primary structure of sperm whale Mb and A. aculeata nHb as templates.
cDNA Library Construction-Freshly dissected nerve cords of S. solidissima were immediately submerged and stored in RNAlater RNA stabilization reagent (Qiagen, Hilden, Germany) at 4°C. RNA extraction was performed on 40 nerve cords using the RNeasy kit (Qiagen). Poly(A) ϩ mRNA was purified from ϳ100 g of total RNA according to the protocol of the Oligotex mRNA kit (Qiagen). ϳ0.5 g of poly(A) ϩ mRNA was employed for cDNA synthesis using the ZAP Express cDNA synthesis kit (Stratagene, La Jolla, CA) and Sephacryl S-500 spin columns (Stratagene) for size fractionation. The resulting double-stranded XhoI-EcoRI cDNA fractions were ligated to 1 g of ZAP Express vector each. Packaging of the directionally cloned cDNA library was performed using the Gigapack III Gold extract (Stratagene). The resulting phage library, consisting of 10 5 clones, was amplified prior to the isolation of globin gene cDNA sequences by vector-anchored PCR (see below).
S. solidissima nHb cDNA Sequence-Degenerated primers were designed based on the partial sequence obtained at the protein level. S. solidissima total RNA was prepared using the TRIzol method (Invitrogen). cDNA was synthesized using random hexamer primers and Superscript RT enzyme from Invitrogen. Primer SpiF1 (5Ј-AAR-CARGAYTGGAARACN) is a 20-mer with 64-fold redundancy, corresponding to the sense strand predicted by the peptide fragment KQD-WKTI. Primer SpiR1 (5Ј-GTYTGCATRTGNCCNCCDAT) is a 20-mer with 192-fold redundancy, corresponding to the antisense strand predicted by the peptide fragment IGGHMQ. PCR was carried out for 5 min at 94°C (1 min at 94°C, 1 min at 50°C, 1 min at 72°C) for 35 cycles and 10 min at 72°C. The amplified product was purified and sequenced. Based on this sequence a 5Ј-RACE was carried out using the 5Ј Rapid Amplification of cDNA Ends system for rapid amplification of cDNA ends (Invitrogen). The 3Ј-end of the S. solidissima globin cDNA was obtained from the amplified phage cDNA library by a PCR approach using the vector-specific oligonucleotide primer T3 and the gene-specific forward (5Ј-GATGCCGTCGCTAACAGTTG-3Ј) primer. Globin gene-specific primers were designed according to the partial cDNA obtained by RT-PCR and RACE. PCR amplicons were either sequenced directly or after cloning into the pGEM T-easy vector (Promega, Madison, WI) on both strands by DyeTerminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA). Sequencing reactions were loaded on an Applied Biosystems Prism 3730 capillary sequencer (GENterprise, Mainz, Germany). Sequences were further analyzed using Lasergene programs (DNAstar Inc.).
Isolation and Sequencing of the S. solidissima nHb Gene Region-Genomic DNA of S. solidissima foot and contractor muscle of several animals was prepared by the protocol of Schmidt et al. (30) with minor modifications. Sets of primer pairs were designed according to the complete nHb cDNA, and overlapping genomic fragments were amplified. Sequences were obtained as described above.
Expression of the Complete Coding Sequence of the nHb of S. solidissima in Escherichia coli-Recombinant S. solidissima nHb were expressed as described (15). In short, the cDNA of the nHb was cloned into the expression vector pET3a. After expression in E. coli BL21(DE3)pLysS cells, the S. solidissima nHb was purified to homogeneity by using: (i) ammonium sulfate precipitation (40 -90% saturation) for which the 90% pellet was dissolved and dialyzed against 5 mM Tris-HCl, pH 8.5, (ii) a DEAE-Sepharose fast flow column (step elution in 5 mM Tris-HCl, pH 8.5, 200 mM NaCl), and (iii) a Sephacryl S200 gel filtration column in 5 mM Tris-HCl, pH 8.5. The globin fractions were pooled and concentrated.
Mass Spectrometry of Native and Recombinant S. solidissima nHb-S. solidissima nHb in 5 mM Tris-HCl, pH 8.5, was analyzed by mass spectroscopy. The native or recombinant sample at 5 mM concentration was dissolved in 50:50 methanol:water in 5% formic acid and injected into custom-made nanoelectrospray needles. Nano-electrospray mass spectra were acquired on a Q-TOFII (Micromass, Wythwnshave, UK) over the mass-to-charge ratio (m/z) range 700 -2000. The multiply charged electrospray spectra were deconvoluted by MaxEnt1 software to produce the molecular mass of the protein. Before each measurement, the mass scale was calibrated with myoglobin.
Phosphoprotein Staining-Native and recombinant S. solidissima nHb were analyzed by standard SDS-PAGE (15%) and native gel electrophoresis. Pro-Q Diamond phosphoprotein gel staining (Invitrogen-Molecular Probes) was performed as described by the manufacturer. As positive controls for phosphorylated proteins, ovalbumin and the peppermint marker (supplied with the staining kit) were used. Negative controls were Mb and bovine serum albumin.
Optical and Resonance Raman Spectra-Optical measurements were done with a Cary-5 UV-VIS-NIR spectrophotometer. All optical spectra were measured in a range from 350 to 700 nm. Resonance-Raman (RR) measurements were carried out on an 80-cm Dilor XY-800 Raman scattering spectrometer consisting of a triple spectrograph operating in nor-mal mode and a liquid nitrogen-cooled CCD detector. The excitation source was a Kr-ion laser (Spectra Physics 2020) at 413.1 nm. The protein solution was stirred at 6,000 rpm to avoid local heating. Five spectra (120-s recording time) were acquired and averaged after the removal of cosmic ray spikes by a program developed in-house. Laser powers of 2 and 17 milliwatts were used.
Sample Preparation-The CO form of the sample was prepared by adding an excess of sodium dithionite and subsequently passing the sample through a PD10 column (Amersham Biosciences) equilibrated with CO-flushed Tris-HCl buffer (5 mM, pH 8.5). The deoxyferrous form was obtained by equilibration under nitrogen and by adding an excess of sodium dithionite. The concentration of the protein samples used for optical and RR measurements was typically ϳ60 M in Tris-HCl buffer (5 mM, pH 8.5).
Kinetic and Equilibrium Measurements of Ligand Binding to Native and Recombinant S. solidissima nHb-Oxygen affinity and cooperativity of recombinant nHb were measured in 0.1 M Tris buffer, 0.5 mM EDTA, at pH 7.5 and 20°C using a thin-layer gas diffusion chamber as previously described (25). Briefly, 4-l samples were allowed to equilibrate with various O 2 /N 2 gas mixtures, while the absorbance at 428 nm was continuously monitored using a UV-VIS Cary 50 Probe spectrophotometer equipped with optic fibers. pH was measured in 100-l subsamples using a BMS 2 MK 2 thermostated microelectrode (Radiometer). Values for O 2 affinity (P 50 , O 2 tension at half-saturation) and cooperativity (n 50 ) were interpolated from the zero-intercept and the slope, respectively, of Hill plots, log[Y/(1ϪY)] versus logPO 2 , where Y is the O 2 saturation.
Ligand recombination kinetics were measured by flash photolysis as previously described (31). Photolysis of the CO form was achieved with laser pulses at 532 nm (Quantel). The subsequent return to the CO bound form involves a competitive binding of the CO and the sixth (protein) ligand. Several detection wavelengths were used to better separate the different spectroscopic species. These kinetics allow the deter-mination of the CO on-rate, and both the on and off rates for the protein ligand. In a similar fashion, a mixed atmosphere of CO and oxygen was used to obtain the oxygen binding rates.
For neighbor-joining analyses (39), protein distances were calculated using the PAM substitution matrix (40). The programs NEIGHBOR from the PHYLIP package and MEGA version 2.1 were employed for tree reconstruction. In the case of applying the program NEIGHBOR, TREE-PUZZLE 5.2 was used for calculation of distance matrices. The reliability of the trees was tested by bootstrap analyses (41) with 1,000 replications.

RESULTS AND DISCUSSION
Primary Structure and Gene Organization-A part of the S. solidissima nHb primary structure was reconstructed from the sequence of relevant peptides generated by tryptic digestion of the purified protein (Fig. 1). The full sequence was completed by sequencing the corresponding cDNA of the nHb (GenBank TM accession number AM086629). Fig. 1 also shows the alignment of the S. solidissima nHb with related globin sequences. Key amino acid positions of the globin structure such as NA2, B6, B14, C2, CD1, E7, F8, and H8 are conserved (42). At the N terminus there is an extension of ϳ6 amino acid residues, the fourth one being a cysteine. A second cysteine is present at position E12. Otherwise there are no remarkable amino acid substitutions compared with other mollusc hemoglobins (see Fig. 1). Conceptual translation of the 162-amino acid-long sequence predicted a molecular mass of 18,191 Da, excluding the N-terminal methionine. However, mass spec-trometry data on the intact native nerve globin showed a molecular mass of 18,235 Da, which confirms the absence of the N-terminal methionine and suggests an N-terminal mono-acetylation (ϩ42 Da). This is not so unusual as N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring on ϳ85% of the different varieties of eukaryotic proteins but rarely on prokaryotic proteins (43,44). We observed two additional molecular mass classes (18,333 and 18,430 Da) that differ from the native mono-acetylated protein by 98 and 196 Da, respectively (Fig. 2). These additional masses could agree with a binding of a mono-and a diphosphate to the nHb. Their physiological role, however, is unclear. The recombinant protein gave the single mass of 18,192 Da, with no additional acetylation. There was, however, an additional molecular mass class of 18,289 Da, suggesting again the presence of a phosphate. The binding of phosphate groups to both native and recombinant S. solidissima nHb was confirmed by ProQ Diamond phosphoprotein staining (data not shown).
Genomic DNA was extracted from S. solidissima foot and contractor muscle and pooled from multiple animals. The nHb gene was amplified by PCR and sequenced both directly and after cloning to resolve observed sequence heterogeneities. The obtained sequence (Gen-Bank TM accession number AM086630) contained differences in the intronic regions probably representing allelic polymorphisms, whereas only two variations in the coding exons were observed. We noted 51 single nucleotide polymorphisms and 4 insertion/deletion polymorphisms in the intronic regions, varying in length from 1 to 284 bp. The coding region of the S. solidissima nHb gene is distributed on three exons, consisting of 116, 226, and 144 bp (measured from the ATG start codon and excluding the stop codon), validating the results from the cDNA. The two introns contain standard splice donor and acceptor  MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5367 motifs, and they are located at positions B12.2 (i.e. between the second and third base of the 12th codon in helix B of the globin) and G7.0. The presence of the B12.2 and G7.0 intron positions is a conserved hallmark of vertebrate (and many invertebrate) globin genes. The S. solidissima nHb gene does not contain a third intron within the E helix region, which is characteristic for the Ngb gene lineage in vertebrates and urochordates (1,33,45).

Spisula solidissima Nerve Hemoglobin
Optical and RR Spectra-The optical spectrum of the ferrous oxy form of recombinant S. solidissima nHb (see Fig. 3) displays a Soret band at 414 nm. The ␣ and ␤ bands in the visible region are found at 537 and 573 nm, respectively, as reported earlier by Strittmatter and Burch (14). Similar values were observed for the oxy form of Paramecium Hb (46) and sperm whale Mb (47). For nHbCO, the Soret band is shifted to a slightly higher wavelength (420 nm), which is in agreement with the optical spectrum of the CO form of Paramecium Hb (46). The ␣ and ␤ bands of nHbCO are situated at 537 and 568 nm, again in good agreement with the CO form of Paramecium Hb (46). In the absorption spectrum of deoxyferrous Spisula nHb the Soret band appears at 426 nm. The ␣ and ␤ bands arise at 530 and 560 nm, respectively. These values were also found for non-symbiotic barley Hb (48), non-symbiotic rice Hb (49), Ngb (15), and cytoglobin (Cygb) (50). The values are typical for ferrous low spin heme complexes with a hexacoordination of the iron atom. The amino acid sequence of S. solidissima nHb indicates that the sixth ligand of the iron atom in the deoxy form is most likely E7His.
The RR spectra of nHbO 2 , deoxyferrous nHb, and nHbCO are shown in Fig. 4. It is well established that the marker lines 4 , 3 , and 2 in the high frequency region of the RR spectrum are sensitive to the oxidation state, spin state, and coordination number of the iron at the center of the heme pocket. In the RR spectrum of nHbO 2 (see Fig. 4, aA and bA), 4 , 3 , and 2 are situated at 1373, 1507, and 1578 cm Ϫ1 , respectively, in agreement with earlier studies on heme proteins in the oxy form, e.g. the oxy complex of Paramecium Hb (46). Upon increase of the laser power to 17 milliwatts, a second spectral component appears. The 4 peak of  this new component is positioned at 1363 cm Ϫ1 (see Fig. 4, aB and bB), which agrees with the formation of the photoproduct. This is confirmed by the RR spectrum of ferrous deoxy nHb, obtained after reduction with sodium dithionite (see Fig. 4, aC and bC). 4 , 3 , and 2 are situated at 1360, 1491, and 1580 cm Ϫ1 , respectively, typical for a hexacoordinate low spin Fe II heme form, confirming the absorption data (Fig. 3, dotted  lines). Furthermore, a high spin (HS) component is present, as is indicated by the lines at 1470 ( 3 ) and 1555 cm Ϫ1 ( 2 ). Note that the intensity of the HS 3 marker line is lower than the intensity of the LS 3 marker line. This suggests that there is only a very small HS population present, as the intensity of 3 is very high for pentacoordinated species in comparison with hexacoordinated species. A small HS component has been found for other bis-histidine-coordinated globins (51).
In the spectrum of S. solidissima nHbCO 4 , 3 , and 2 are located at 1373, 1500, and 1583 cm Ϫ1 , respectively, characteristic for a hexacoordinated low spin complex. Again, the lines at 1460 ( 4 ), 1469 ( 3 ), and 1556 cm Ϫ1 ( 2 ) indicate that there is a small population of an HS ferrous complex present, which agrees with a small fraction of photodissociation taking place. Two CO coordination configurations can be distinguished, one with the Fe-CO stretching mode at 494 cm Ϫ1 (75%) and one with this mode at 513 cm Ϫ1 (25%) (see supplemental information). The first configuration (494 cm Ϫ1 ) was observed previously, e.g. in sperm whale Mb (491 cm Ϫ1 ) (52). In Mb, this conformer becomes dominant at low pH values, when E7His swings out of the heme pocket. It is ascribed to an open conformation of the heme pocket, with a weak interaction between CO and the surrounding amino acids. The second conformer (513 cm Ϫ1 ) was also observed in Mb (508 cm Ϫ1 ) (52) and Cygb (510 cm Ϫ1 ) (50) and was assigned to a conformation in which the heme pocket is closed, due to the stabilization of CO by E7His. Note that no Fe-CO stretching modes at higher frequencies are observed, in con-trast to several other heme proteins capable of a bis-histidine coordination of the heme iron, such as Ngb (523 cm Ϫ1 ) (51), Cygb (518 cm Ϫ1 ) (50), or Barley Hb (534 cm Ϫ1 ) (53). Higher Fe-CO stretching frequencies correspond to a stronger stabilization of CO, e.g. due to interaction with several surrounding amino acid residues. The line at 586 cm Ϫ1 can be attributed to the Fe-C-O bending mode. Note that the bending mode is weak, which is typical for an open conformation of the heme pocket. It was previously shown that the conformation of the heme pocket can differ significantly for O 2 and CO binding (46). Also, it has been shown that when the distal His is able to interact with the bound oxygen closed conformation of the heme pocket autoxidation is greatly enhanced, as the ferrous O 2 acquires more ferric O 2 . (superoxide) character (25).
Kinetic and Equilibrium Measurements of Ligand Binding to Native and Recombinant S. solidissima nHb- Fig. 5 shows the oxygen equilibrium curve for recombinant S. solidissima nHb at 20°C, pH 7.5 and the data expressed as a Hill plot (inset). Under these conditions, the oxygen affinity is high, with a P 50 value of ϳ0.6 torr, and oxygen is bound in a non-cooperative manner as indicated by a slope of ϳ1 in the Hill plot. The P 50 and n values found in our study on the recombinant protein are significantly lower than those reported from in situ measurements on intact nerves at high protein concentrations, where a P 50 of 2.3 torr and an n value of 2.1 were found (28). The difference in P 50 and n values may arise because our measurements were carried out in vitro on a recombinant protein, whereas the previous data refer to the native protein in its natural environment, indicating in vivo aggregation at high concentration and/or the existence of in vivo allosteric effectors that lower oxygen affinity. Acetylation of the native protein, but not the recombinant one, may also contribute to the observed functional differences. Indeed Lacan et al. (54) showed that a new ␣-chain variant, Hb Lyon-Byron, is characterized by the replacement of the N-terminal valine by an alanine that is N-terminal acetylated. This resulted in a decreased oxygen affinity.    (15,23). In the case of the S. solidissima nHb the separation of the kinetic phases is less distinct, as the coefficient for His binding is only ϳ10 as compared with Ͼ1,000 for certain forms of Ngb.
The ligand binding rates for CO and oxygen are both quite high ( Table 1, Fig. 6), with oxygen having the higher rate, as generally observed for heme proteins. The value for oxygen association is among the highest observed, approaching the diffusion limit for the bimolecular globin ligand reaction.
The histidine association rate is an order of magnitude higher than that observed for other hexacoordinated globins, although there are relatively few values available for comparison. Binding of the histidine requires only 100 s compared with 1 ms for Ngb; Cygb is relatively slow, requiring ϳ10 ms (15,23). The His dissociation occurs in ϳ1 ms, resulting in a binding coefficient of ϳ10 for the hexacoordination of S. solidissima nHb. Because of these elevated rates for His binding, there is significant overlap with the CO binding phase and the two kinetic phases are less distinct than for the case of Ngb. In the case of human Ngb, there is an influence of an internal disulfide bond on the His kinetics (55), which can be probed by addition of dithiothreitol to break the bond. There was no effect of the dithiothreitol on the S. solidissima nHb binding kinetics. This was expected, as the 2 cysteine residues present (in positions NA4 and E12) are unlikely to form a disulfide bond.
The O 2 affinity obtained from the kinetic data (P 50 , 1.9 torr) is in fairly good agreement with the value determined by equilibrium measurements (P 50 , 0.6 torr). Small differences can be because of the difference in experimental approaches. From the kinetics, one can separate the intrinsic oxygen binding parameters from the contribution of the His affinity (Table 1).
We can conclude from the above described experiments (spectra and ligand binding properties) that S. solidissima nHb can be classified as FIGURE 7. Bayesian phylogenetic tree of vertebrate, invertebrate, and plant globins based on amino acid sequence data. The S. solidissima nHb (shaded) clearly groups with other mollusc hemoglobins, but not with vertebrate Ngbs or the Ngb-related nerve globin from the annelid A. aculeata. The posterior probabilities of clade support are shown at the branches. Three-letter species designations are specified under "Materials and Methods." normal intracellular hemoglobin having a "myoglobin-like" function. The major structural characteristic is the hexacoordinated nature of the iron atom. The structural differences between penta-and hexacoordinated hemoglobins might be, however, very small. Indeed, vertebrate Hb and Mb, normally pentacoordinated, shift toward hexacoordination (hemichrome) near their denaturation point because of a reorganization of their heme pocket (56 -58). Application of high pressure (0.1-700 MPa) to penta-or partially hexacoordinated Hbs can also increase the percentage of hexacoordination. This shift results in a difference of ligand binding characteristics (59).
Phylogenetic Analyses-The amino acid sequence of the S. solidissima nHb has an average identity of only 16 -20% and a similarity (calculated using the PAM250 matrix) of 30% to vertebrate Ngbs and Cygbs. In contrast, it displays 23-30% amino acid identity and 39 -50% similarity to other mollusc Hbs and Mbs. The amino acid sequence of the nHb was added to an alignment of selected vertebrate and invertebrate globin sequences (1,32,33), including representative mollusc globins. Phylogenetic trees were calculated using the Bayesian approach (Fig. 7) and the neighbor-joining distance method (not shown). Trees were rooted considering plant leghemoglobin as out-group. Regardless of the reconstruction method used, the S. solidissima nHb formed a common clade with the mollusc Hbs and consistently grouped with the gill Hbs of the bivalve mollusc L. pectinata. These cytoplasmic Hbs are specialized to supply either O 2 (Hb2 and 3) or sulfide (Hb1) for metabolism, probably playing a role in the symbiosis of these molluscs and chemoautotrophic bacteria (5).
In contrast to the S. solidissima nHb, the nHb of the annelid A. aculeata (6) grouped with vertebrate Ngb sequences as described before (1). The highly derived and phylogenetically less informative nHb sequence from the nemertean worm C. lacteus (7) tended to group at various positions basal to the Ngb gene lineage and was therefore excluded from the final tree reconstruction (data not shown). This behavior is most likely based on its structural shortening of the globin rather than on its phylogenetic position (7).
Implications for Vertebrate and Invertebrate Nerve Globin Function-The nervous system of both vertebrates and invertebrates requires huge amounts of metabolic energy and thus oxygen. It is therefore not surprising that neurons are highly vulnerable to any shortage in oxygen supply as it occurs under pathological (e.g. ischemia) or natural hypoxic conditions (60). There is no doubt that respiratory proteins that either enhance oxygen flow under normoxic conditions or store oxygen for short-term hypoxic states should be evolutionarily advantageous for the survival of neurons and thus are frequently found in the nervous systems of various invertebrate taxa (4). Strongly expressed, glial nHbs like the one in the bivalve mollusc S. solidissima are present in those species that encounter substantial hypoxia in their natural habitats (4,5). Physiological experiments have convincingly demonstrated that glial nHbs enable these bivalves to maintain neuronal function while burrowing in O 2 -poor sediments (9,29). Our biochemical characterization of the S. solidissima nHb is generally in line with the proposed O 2 supply function, e.g. by confirming the Mb-like O 2 affinity of this respiratory protein. There are, however, additional unusual features of the S. solidissima nHb (phosphate binding, iron hexacoordination, etc.), the roles of which in the nHb respiratory function are not yet clear.
Originally, it had been proposed that invertebrate nHbs are phylogenetically orthologous to vertebrate Ngbs (1). This notion was based on the finding that in phylogenetic analyses the nHb of the annelid A. aculeata consistently groups with human and mouse Ngb (Ref. 1 and this report; Fig. 7). However, here we have shown that the nHb of the bivalve mollusc S. solidissima does not belong to the "Ngb plus A. aculeata nHb" clade but groups with the other mollusc Hbs and Mbs. This finding demonstrates that a classical Hb or Mb has been recruited during evolution to fulfill the task of providing O 2 to the nerve cells of S. solidissima. Invertebrate nHbs are therefore of polyphyletic origin. Consequently, mollusc glial nHbs will probably have only modest relevance as a model for studying the function of Ngb in the nervous system of vertebrates (2). We note, however, that less prominently expressed nHbs, coupled to a neuronal expression site, have been reported, e.g. for air-breathing gastropod molluscs (11), which like most vertebrates do not encounter substantial environmental hypoxia. This feature of a neuron-specific, low-expressed nHb is a clear parallel to vertebrate Ngbs. It will therefore be interesting to investigate the phylogenetic affiliation, biochemical features, and cellular function of invertebrate "neuronal" nHbs in comparison with the invertebrate glial nHbs and neuronal vertebrate Ngb.
Independent Origin of Hb Hexacoordination-From a biochemical perspective, it is noteworthy that the S. solidissima nHb represents the first true respiratory Hb to be found in an iron-hexacoordinated form, resembling in this respect the vertebrate Ngb proteins (15,61). This confirms an earlier suggestion that a bis-His-Fe hexacoordinate heme structure has arisen independently in distinct globin lineages (45). The S. solidissima nHb will be instrumental as a model in studying the evolutionary determinants of this protein feature.