The unique structural features of carbonmonoxy hemoglobin from the sub-Antarctic fish Eleginops maclovinus

Tetrameric hemoglobins (Hbs) are prototypical systems for the investigations of fundamental properties of proteins. Although the structure of these proteins has been known for nearly sixty years, there are many aspects related to their function/structure that are still obscure. Here, we report the crystal structure of a carbonmonoxy form of the Hb isolated from the sub-Antarctic notothenioid fish Eleginops maclovinus characterised by either rare or unique features. In particular, the distal site of the α chain results to be very unusual since the distal His is displaced from its canonical position. This displacement is coupled with a shortening of the highly conserved E helix and the formation of novel interactions at tertiary structure level. Interestingly, the quaternary structure is closer to the T-deoxy state of Hbs than to the R-state despite the full coordination of all chains. Notably, these peculiar structural features provide a rationale for some spectroscopic properties exhibited by the protein in solution. Finally, this unexpected structural plasticity of the heme distal side has been associated with specific sequence signatures of various Hbs.

but elusive functional states that play a key role in the transition. In this scenario, we have undertaken the biochemical and biophysical characterization of the Hb isolated from the sub-Antarctic notothenioid fish Eleginops maclovinus (Hb1Em) that lives in the Beagle Channel surrounding the Tierra del Fuego (Argentina) by experiencing temperature ranges from 4 °C to 10 °C 32 . E. maclovinus diverged prior to the isolation of Antarctica and is phylogenetically the closest sister species to the modern Antarctic clade. Its genome, recently sequenced, is the best representative of the temperate character of the most recent common ancestor of the Antarctic notothenioids 33 . This species is a close relative of the Antarctic notothenioids such as Trematomus bernacchii 12,15,16,23,28,34 and Trematomus newnesi 11,13,19,35 whose Hbs have been extensively characterized. The spectroscopic characterization of E. maclovinus Hb1 has unraveled that the protein presents two distinct CO forms in solution 36,37 . We have previously reported the equilibrium and kinetic study of the oxygenation process for Hb1Em. Moreover, we also investigated the vibrational spectroscopy of the various ferrous and ferric states, along with the crystal structure corresponding to one of these carbomonoxy forms in a canonical R state 36 . Particularly, Hb1Em exhibits the Root effect (drastic drop of oxygen cooperativity at low pH) that is physiologically necessary to secrete O 2 against high O 2 pressures into the swimbladder or the retina, following local acidification of the blood in a counter-current capillary system 38 . Hb1Em also presents a biphasic CO dissociation kinetics and multiple CO vibrational frequencies in solution 36 . Here, we report the structure of a second carbomonoxy form that presents several structural properties that are unique for globin structures. These novel crystallographic data also provide some insights into Hb1Em functional properties as well as into the structural basis of some spectroscopic features that are common to the entire Hb superfamily.

Results
Binding state and secondary structure. The inspection of the electron density at the heme-binding pocket clearly indicates the presence of an exogenous CO molecule bound to the sixth coordination position in both α and β chains of the asymmetric unit (Fig. 1A,B). In both chains, the iron atom is in the plane of the heme group, in line with the general trends observed in hexa-coordinated states. Therefore, the heme binding state of this novel crystal form (hereafter denoted as Hb1EmCO_hexa) is identical to that observed in the carbonmonoxy Hb1Em previously reported (Hb1EmCO_ortho, PDB ID: 4ESA) 36 . Nevertheless, the analysis of Hb1EmCO_hexa indicates that this structure presents a number of striking differences when compared to Hb1EmCO_ortho. It also presents specific features that are rare or unique in the world of tetrameric Hbs. The comparative analysis of the secondary structure elements present in Hb1EmCO_hexa and Hb1EmCO_ortho indicates that the secondary structure of the β chains is rather well-preserved ( Fig. 2A). On the other hand, the analysis of Hb1EmCO_ hexa secondary structure shows that the α chain of this structure presents significant variations when compared to Hb1EmCO_ortho, although these crystal forms were obtained under the same crystallization conditions 37 . Indeed, as shown in Fig. 2B, the N-terminal end of the E helix (residues 54-56) is unfolded and a small 3 10 helix is observed for residues 47-49. Notably, this extra helix is in the sequence region that corresponds to the helix D of β chains of globins that is missing in globin α chains (Fig. 2C). The unfolding of the N-terminal region of the α-chain helix E has never been observed in the structures of tetrameric Hbs. Indeed, a survey of the Protein Data Bank (PDB) shows that none of the 1,519 Hb α/β chain structures reported in the PDB (see methods for details), with sequence identities with Hb1Em ranging from 45% to 100%, shows shortening of the helix E in the α chains as detected in Hb1EmCO_hexa. tertiary structure and binding pocket. The structural variations observed in Hb1EmCO_hexa have a remarkable impact on the tertiary structure and on the heme binding pocket of the protein. In the α chain a striking rearrangement of the heme pocket is observed: the side chain of the distal His59α, which assumes a canonical location in Hb1EmCO_ortho, is far from the heme group (Fig. S1A). Indeed, the distance between the N ε2 atom of His59α and the oxygen atom of the CO ligand, which are at hydrogen-bonding distance in canonical www.nature.com/scientificreports www.nature.com/scientificreports/ carbonmonoxy Hbs, is 10.3 Å in Hb1EmCO_hexa (Table S1). This unique location of His59α is favored by the helix E distortion and stabilized by the formation of several stabilizing interactions (Fig. 3). The most evident one is the strong electrostatic interaction established by His59α and Asp48α side chains (Fig. 3A). His59α side chain also interacts with the side chain of Val56α through CH-π interactions (Fig. 3B). Moreover, His59α takes part to a cluster of stacking interactions also involving His45α and Trp46α (Fig. 3C). The displacement of His59α produces a rearrangement of the distal side of the heme pocket characterized by a movement of the side chains of the hydrophobic residues Phe43α, Trp46α, and Leu29α that, compared to Hb1EmCO_ortho, become closer to the CO molecule (Fig. S2). Interestingly, the rearrangement of the overall architecture of the distal side observed in Hb1EmCO_hexa is unique among vertebrate globins. Indeed, the displacement of the His side chain from the heme pocket here observed never occurs in more than 1,500 Hb chains reported in the PDB (Fig. 4) and has  never been detected in the structures of other globins such as Mb, Cygb and Ngb (data not shown). Although the overall architecture of the distal side of the β-heme pocket is essentially preserved, it is worth mentioning that, as occasionally observed in other Hbs 11,39 , the distal His63β swings out of the heme pocket (Fig. S1B). Indeed, its χ1 angle (−77.9°) is rather different from that observed in both chains of Hb1EmCO_ortho (ranging from −163° to −156°) which represents the canonical R state CO form. We also evaluated the overall re-organization of the heme pockets by calculating the C α -C α distance between the distal and proximal His residues. This analysis provides further support to the unusual structural properties of Hb1EmCO_hexa as, in both chains, the distances of the C α atoms between the distal and proximal histidines in this structure are quite different from those observed in the canonical R state (Fig. S1). The higher value observed for the α chain (15.8 Å in Hb1EmCO_hexa versus 14.3 Å in Hb1EmCO_ortho) is clearly due to the displacement of the distal site (Table S1). On the other hand, the lower value detected for the β chain (12.8 Å in Hb1EmCO_hexa versus 14.1 Å in Hb1EmCO_ortho) is due to compression of the EF corner associated with the swinging out of the side chain of the distal histidine (Table S1).
Quaternary structure. To evaluate the impact on Hb1EmCO_hexa quaternary structure of the observed structural peculiarities at secondary and tertiary structure levels we applied a number of global and local indicators. These analyses were specifically designed to locate this non-canonical structure in the framework of the R-T transition characteristic of tetrameric Hbs. Taking into account the close structural analogy of Hb1EmCO_ ortho with canonical CO-bound forms of tetrameric Hbs, Hb1EmCO_ortho was used as the standard R-state of Hb1Em. On the other hand, the lack of structural characterizations of Hb1Em in its T state prompted us to use as a framework for the R-T transition the R (Hb1TnCO) 35 and the T (Hb1Tn deoxy) 40 states of the closely related Hb1 from T. newnesi (Hb1Tn). Pairwise comparisons of these structures were initially performed by considering root mean square deviation (RMSD) values, computed on the C α atoms of the isolated chains and the dimer/tetramer assembly ( Table 1). The inspection of the RMSD values shows that the quaternary structure of Hb1EmCO_hexa is quite different from that exhibited by Hb1EmCO_ortho (RMSD of 2.16 Å). Moreover, it is somehow intermediate between the R and the T state of Hb1Tn. A deeper analysis of the RMSD values indicates that Hb1EmCO_hexa is closer to Hb1Tn T state (RMSD of 1.18 Å) than to the R state (RMSD of 1.73 Å). To better localize the structure of Hb1EmCO_hexa in the R-T pathway we calculated difference distance matrices (DDMs). In line with the findings described above, the DDM obtained from the comparison of Hb1EmCO_hexa and Hb1EmCO_ortho clearly indicates that Hb1EmCO_hexa structure is rather different from the canonical R state, despite the identical binding state of all iron atoms (hexa-coordination with a CO molecule). Interestingly,  www.nature.com/scientificreports www.nature.com/scientificreports/ the difference matrix between Hb1EmCO_hexa and Hb1EmCO_ortho (Fig. 5A) resembles the one computed between the T and R states of Hb1Tn (Fig. 5B). Indeed, the inspection of these matrices indicates that the differences in the juxtaposition of the α1/β2 and β1/β2 subunits are rather similar in Hb1EmCO_ortho/Hb1EmCO_ hexa and in Hb1TnCO/Hb1TnT. On the other hand, the relative orientation of α1/α2 subunits is different in these two systems. The visual inspection of these DDM matrices also suggests that Hb1EmCO_hexa is closer to Hb1TnT than Hb1TnCO (Fig. 5C,D). This is line with the RMSD values obtained from the pair-wise comparisons (Table 1). Collectively, the analysis of the difference matrices generated by comparing Hb1EmCO_hexa versus the T state and the R state of Hb1Tn (Fig. 5C,D) confirms that this novel structure is shifted toward the T state in the R-T pathway, as also showed by the superimposition of these Hb structures reported in Fig. 6. The evidence in Hb1EmCO_hexa of a stable carbomonoxy form in a T quaternary state is in perfect agreement with a biphasic CO-dissociation kinetic and with the stopped-flow kinetic curves previously observed for the Hb of E. maclovinus 36 . The monitoring of specific structural features that differentiate T and R states of Hbs corroborates and strengths the picture emerging from the global analysis of Hb1EmCO_hexa quaternary structure. One of the key points that characterize the R-T transition is the relative position of His97β2 of the β2 FG corner and helix C of the α1 chain (Fig. S3). In Hb1EmCO_hexa, the location of the histidine is intermediate between that observed in T and R states. The T states of fish Hbs are frequently characterized by the formation of a peculiar interaction between the side chains of Asp95α1 and Asp101β2 that is stabilized by Asp99β2 16,17,41,42 . Although this carboxyl-carboxylate interaction is not observed in Hb1EmCO_hexa, these aspartic acid residues are closer in this structure when compared to Hb1EmCO_ortho (Fig. S4). It should be noted that Hb1EmCO_hexa crystals were obtained at a pH value (7.6) that does not favor this type of interaction and may contribute to impede the complete R-T transition of Hb1EmCO_hexa. Finally, Hb1EmCO_hexa also lacks the salt bridge formed by www.nature.com/scientificreports www.nature.com/scientificreports/ Lys40α1 side chain and the C-terminal carboxyl group of His146β2, which is considered a fingerprint of the T state in vertebrate Hbs, due to structural disorder observed for residues 145-146 of the β chain.

Discussion
The carbonmonoxy structure of Hb1EmCO_hexa here described presents a number of features that are unique in the universe of globins. These features are not artifact of the crystalline state since vibrational spectroscopy solution studies have shown that two distinct CO forms of Hb1EmCO coexist in solution 36 . Vibrational spectra provide a direct way to estimate the relative amount of these two CO conformers in solution. An abundance of roughly 95% and 5% is estimated for the band at 1954 and 1970 cm −1 , respectively. The most abundant form, which presents rather standard spectroscopic features (band at 1954 cm −1 ), is likely associated with previously described typical canonical R states, with CO that is hydrogen bonded to the distal histidine. The other form detected in solution, which presents a different higher vibration frequency (band at 1970 cm −1 ), could correspond to the non-canonical model here described where CO does not interact with the distal histidine, which swings out from the heme pocket. In support to this interpretation, there are several experimental and theoretical studies. In particular, an extra band at 1970 cm −1 has been previously observed in Hbs of temperate (carp) 43 , sub-Antarctic 36 , and Antarctic 11 fish, and of some rodents 43 . It has also been reported for wild-type HbA under denaturing conditions 43 , in phosphate-buffered solutions, and, in low amounts, even in red blood cells 44 . More interestingly, the CO stretching band around 1970 cm −1 was observed for several distal HbA mutants such as Tyr63βHis Emory 45 and Arg63βHis Zurich 46 . Finally, in support to our structural interpretation of the two CO vibrational bands at 1954 and 1970 cm −1 , a direct correspondence between CO coordination and band assignment is also in line with previous experimental theoretical studies on the occurrence of the 1970 cm −1 band in hemoproteins [47][48][49][50] . Indeed, according to these theoretical studies, the upshift in the CO stretching can well be assigned to the loss of the H-bond between N ε of the distal His imidazole ring and CO that leads to a more hydrophobic environment surrounding the CO ligand.
This structural assignment of the two vibrational bands at 1954 and 1970 cm −1 with a closed or an open local environment of the CO ligand perfectly fits with the displacement of the distal histidine from its canonical position and the consequent rearrangement of the local architecture of the distal side. It is important to note that this rearrangement involves a significant reorganization of the residues 43-48 of the α chain. This region embodies the fish-specific insertion in position 47 that makes the α chain of fish Hb one residue longer (Fig. S5). The presence of an additional residue in this region is important for the reorganization here observed. These considerations may explain the higher tendency of fish Hbs to exhibit the band around 1970 cm −1 in the FT-IR spectrum. Interestingly, an enhanced band around 1970 cm −1 is also displayed by rat Hb compared to human HbA 43 . Although the rat sequence, like all mammalian Hbs, does not contain extra residues in the 43-48 region of the α chain, its local sequence is significantly different from HbA. It is also worth to remind that relevant modifications in the CDα region in mammalian Hbs are tightly associated to the EFα contraction observed in bis-histidyl complex formation, crystallographically observed at acidic pH for horse Hbs 31 . Indeed, the sequence alignment reveals that, in rat Hb, Pro45 of HbA is replaced by a Ser residue, which is more flexible (Fig. S5). Moreover, in the human sequence His51 establishes a strong electrostatic interaction with Glu31 side chain that is absent in the rat Hb since His51 is replaced with a Pro residue. It is likely that these mutations endow the rat Hb with an increased flexibility of the 43-48 region that generates an enhanced band around 1970 cm −1 in the FT-IR and Resonance Raman spectrum. It is also noteworthy that the unusual alterations of the secondary and tertiary structures of the protein leads to a quaternary structure that still falls in the R-T functional transition of tetrameric Hbs. These findings suggest that although proteins may be endowed with local structural versatility, they likely possess a limited number of possible overall structural transitions. www.nature.com/scientificreports www.nature.com/scientificreports/ Besides the general implications on the possible CO conformers and on the R-T transitions, our study offers a structural interpretation for specific properties of Hb1Em. The observed biphasic CO dissociation 36 completely matches with the observation both in solution and in the crystal structures of two distinct CO conformers that are expected to present drastically different CO-dissociation rate constants. Moreover, the value of the faster k off for Hb1Em reveals a weak Fe-CO bond. This is clearly anticorrelated to the strong CO stretching at 1970 cm −1 due to the higher extent of π-back-bonding. Moreover, under the hypothesis that the two kinetic phases could be associated with the two CO conformers, the biphasic CO dissociation curves imply a quite slow interconversion rate between the two conformers. The slow interconversion, which implies a high free energy barrier, may well be related to the large marked changes observed between the previous R 36 and the current crystal structure of Hb1Em.
As observed for Hb1Em, autoxidation is very fast in fish and in particular in Antarctic and sub-fish Hbs 13,18 . This evidence can well be related to the presence in most of fish Hbs of significant amounts of the conformer(s) in which the CO ligand is not bound to the distal histidine (band around 1970 cm −1 ).
In conclusion, after sixty years of reports, structural characterization of Hb structures may still unravel unexpected findings. The structural characterization of Hb1EmCO here described shows an unexpected plasticity at the heme distal side, a key functional region that was believed to be strictly structured in tetrameric Hbs.

Materials and Methods
Protein purification, crystallization and data collection. Specimens of E. maclovinus were collected during the ICEFISH 2004 cruise close to the Falkland Islands. Blood samples were taken from the caudal vein. Separation of E. maclovinus hemoglobins was accomplished by FPLC anion exchange chromatography using a Mono Q-Tricorn column. Hb1Em was further purified by ion-exchange chromatography (see ref. 37 for further details about protein purification procedures).
A full description of the crystallization and data collection of Hb1Em in the carbonmonoxy form has been previously reported in detail 37 . Briefly, the protein was crystallized at 277 K in a CO atmosphere using the dialysis technique with microdialysis buttons. Na 2 S 2 O 4 was added to the crystallization solution to guarantee a reducing environment hampering autoxidation. The best crystals of Hb1Em were obtained using a protein concentration of 20 mg ml −1 and 1.8 M ammonium sulfate pH 8.0. Diffraction data were collected at the ELETTRA synchrotron and in-house using a Rigaku MicroMax-007 HF generator equipped with a Saturn944 CCD detector. Cryoprotection of the Hb1Em crystals for diffraction at 100 K was achieved by adding 20% glycerol and Na 2 S 2 O 4 to keep the ferrous state to the harvesting solution. The data sets were scaled and merged using the HKL2000 program package 51 . The crystal structure of Hb1Em_hexa was solved by molecular replacement using the program Phaser 52 and the structure of the αβ dimer of Hb1EmCO belonged to the space group P2 1 2 1 2 1 (PDB ID: 4ESA) as starting model. Then, an automatic rebuilding was performed using ARP/wARP 53 . Crystallographic refinement was carried out using 95% of the measured data with the ccp4i program. In order to monitor the progress of refinement, the remaining 5% of the observed data, which was randomly selected, was used to calculate the Rfree. The program REFMAC was used for the refinement 54 . The refinement details along with the statistics of the final protein models are given in Tables 2 and 3. As the two models are virtually identical, all of the analyses and descriptions reported in the text refer to the structure derived using the highest resolution synchrotron data. The stereochemistry of Hb1EmCO_hexa has been evaluated by using both standard protocols such as PROCHECK 55 and innovative approaches based on the monitoring of fine details of the protein backbone geometry [56][57][58] . In this latter approach, we evaluated the variability as function of local conformation of geometrical parameters like bond angles, the peptide bond planarity, and the carbon carbonyl pyramidalization and compared it to that observed in high resolution and well refined protein structures ( Fig. S6