The structure of the ferrous unligated form of CerHb (unligated-CerHb) was refined to a general R-factor value of 18.3% (R-free 22.1%) for data in the 25.0–1.9 Å resolution range, with ideal stereochemical parameters (root mean square deviation, r.m.s.d.) from ideal values for bond lengths 0.014 Å, and for bond angles 1.3°) [10]. The final model, restrained refined with isotropic B-factors, contains 819 protein atoms, corresponding to residues Met(0) to Leu(109)H20, one heme prostetic group, 115 ordered solvent atoms (nine in two alternate positions), one glycerol molecule, two sulfate and one acetate anions (Table 1): Asp(9)B8 side-chains has been refined in a double side-chain conformation. Analysis of the electron density map at the distal iron side confirmed the absence of any ligand bound to the heme-Fe²⁺ atom (Figure 1a).

Restrained refinement of the carbomonoxy-form of CerHb (CerHb-CO) converged to a final R-factor value of 14.6% (R-free 17.9%) for data in the 24.7–1.5 Å resolution range, with ideal stereochemical parameters (r.m.s. deviation from ideal values for bond length 0.010 Å and for bond angles 1.3°) [10]. The final model contains 836 protein atoms, corresponding to residues Met(0) to Leu(109)H20, one heme prostetic group, 123 ordered solvent atoms, one glycerol molecule, two sulfate and one acetate anions (Table 1). Residues Met0, Asp(9)B8, Ser(76)G2, Ser(91)G17, and Leu(109)H20 were refined with double side-chain conformations. Additionally, four water molecules and one sulphate ion were modelled in two alternate positions. B-factors were refined anisotropically. Analysis of the electron density map at the distal iron side clearly indicates the presence of a diatomic ligand bound to the heme-Fe²⁺ atom (Figure 1b).

When compared to each other, the unligated-CerHb and CerHb-CO tertiary structures are very similar in their backbone, displaying a r.m.s deviation value of 0.29 Å, calculated over 109 C_α atom pairs (Figure 2). Furthermore, both structures conform well to those previously reported in the literature [3,8] (see below). In particular, the CerHb-CO backbone is nearly identical to those of other ligated CerHb structures, such as oxygenated CerHb (CerHb-O₂), and aquo-met CerHb (CerHb-H₂O), with r.m.s deviation values over 109 C_α atom pairs of only 0.10 Å and 0.16 Å, respectively. On the contrary, the unligated-CerHb structure superimposed onto CerHb-O₂ and CerHb-H₂O yielded r.m.s deviation values (over 109 C_α atom pairs) of 0.30 Å and 0.31 Å, respectively. Thus, despite a very good overall structural conservation, the 3D structures of CerHb in its bound states appear to cluster together based on the r.m.s.d. values.

In general, the structural deviations between CerHb unligated and ligated state structures can be ascribed to a contained breathing mode of the protein, whose tertiary structure shrinks slightly upon ligand binding. Indeed, if the solvent-excluded volume (i.e. the volume inside the excluded protein surface, determined using a spherical probe of 1.4 Å for a water molecule) is calculated for each of the CerHb structures, the unligated-CerHb molecule displays a 1.5%–3% larger volume than CerHb in the ligated states. At the level of secondary structure, the only significant differences are localized at the C-terminus of the F helix and of the FG-hinge (residues 69–73), where the coordinate displacement of the unligated-CerHb C_α backbone relative to CerHb-CO is 0.45 Å, and at the GH-hinge (residues 90–97), with a coordinate displacement of 0.47 Å. The net result is a small shift of the F helix away from the heme in the unligated structure, with the length of the Fe–His(69)F8 NE2 bond increasing from 2.06 Å in CerHb-CO to 2.21 Å in unligated-CerHb, while maintaining the proximal His azimuthal angle essentially unperturbed. For reference, in the CerHb-O₂ and CerHb-H₂O structures the Fe–His(69)F8 NE2 bond length is 2.06 Å and 2.05 Å, respectively. In the CerHb-O₂ structure the heme group shifts slightly toward the outer rim of the globin pocket (0.29 Å) relative to its position in unligated-CerHb, and in CerHb-O₂ or CerHb-H₂O, however no significant differences are present for the iron position in the heme plane, with the iron atom only 0.08 Å out-of-plane at the proximal side in the unligated-CerHb.

The electron density clearly shows a diatomic ligand coordinated to the sixth coordination site of the heme Fe-atom in CerHb-CO (Figure 1b). On the basis of the shape of the electron density, and in view of the way crystals were prepared (see below), we identified and refined it as the exogenous CO molecule. The final refined model indicates an Fe–CO distance of 2.09 Å and an Fe–C–O angle of 172° for CerHb-CO (Table 2 and Figure 3a), comparable to that reported for Mb-CO [11,12]. Two main hydrogen bonds stabilize the heme Fe²⁺-bound carbon monoxide molecule. On the one hand, the carbon monoxide O atom is linked to the Tyr(11)B10 OH group (2.64 Å), on the other, it is hydrogen bonded to the Gln(44)E7 NE2 atom (3.10 Å). Additional polar interactions contribute to the overall structural organization of the heme distal site. Specifically, the Tyr(11)B10 OH group is connected to the Thr(48)E11 OG1 atom by a strong hydrogen bond (2.60 Å), while weaker interactions connect the Tyr(11)B10 OH group to the Gln(44)E7 NE2 atom (3.06 Å), and the Gln(44)E7 NE2 atom to the Thr(48)E11 OG1 atom (3.56 Å). Moreover, the close contact (3.43 Å) occurring between the heme Fe-bound CO molecule (through its O atom) and the rim of Phe(25)CD1 should be noted, indicative of an aromatic-electrostatic contact, which further contributes to stabilization of the ligand (Figure 3a). As a result of the interlacing of side chains and hydrogen bonds, the heme Fe-bound carbon monoxide molecule is fully buried in the distal site, being totally inaccessible to solvent. If we compare the binding mode of the diatomic ligands in CerHb-CO and CerHb-O₂ we notice that, despite the different binding geometry of the dioxygen molecule, the H-bonding network stabilizing the ligand is essentially the same in CerHb-CO and CerHb-O₂ (Table 2 and Figure 3b).

The distal site residues in the CerHb-CO and unligated-CerHb structures superimpose almost perfectly, not only in their C_α positions but also in their side-chains, with only few exceptions: in the presence of the ligand the Tyr(11)B10 side chain rotates about 4° away from the centre of the distal site, while Phe(10)B9 move slightly in by a rotation of about 9° around the C_α–C_β bond. At the same time the heme plane rotates about 4° around the CHB-CHD axis, with an inwards shift of about 0.3 Å (Figure 4a). Thus, we can conclude that ligand binding alters very marginally the structure of the distal site relative to the unbound state, and that the H-bonding network responsible for the stabilization of the bound ligand is substantially conserved independently of the nature of the diatomic ligand (either CO or O₂).

Ligand access to the heme takes place in CerHb through a wide hour-glass-shaped protein matrix tunnel connecting the distal site to a surface cleft located between the E and H helices [3,7,8]. The tunnel is lined with small hydrophobic residues, such as Tyr(51)E14, Ile(52)E15, Ala(55)E18, Leu(86)G12, Leu(98), Ala(101)H12, Ile(102)H13, and Ile(105)H16, and has a diameter that varies between 6.9 and 5.5 Å in the narrowest part, which is close to residue Leu(86)G12. The tunnel end at the heme distal site is surrounded by the side chains of Val(7)B6, Phe(10)B9, and Thr(48)E11.

The structural comparison of unligated-CerHb and CerHb-CO 3D structures indicates that ligand binding does not induce any relevant conformational change in the tunnel, whose lining residues match almost perfectly (Figure 4b). Comparable results, relative to the tunnel residues, are found when unligated-CerHb and CerHb-O₂ 3D structures are overlayed. Such findings are particularly relevant since the tunnel lining residues are all involved in determining the free energy profile for ligand entry, as calculated using dioxygen as a probe: (i) free energy barrier of ~5.5 kcal/mol due to steric restrictions posed by Val(7)B6, Phe(10)B9, Tyr(11)B10, and Thr(48)E11; (ii) small (≤1 kcal/mol) steric barrier near Leu(86)G12; and, (iii) a barrier of ~2 kcal/mol near Ala(55)E18 at the tunnel exit to solvent. Furthermore, mutational studies showed that increasing the side chain size at Leu(86)G12 and Ala(55)E18 increases the extent of CO geminate recombination (to ~50%), and decreases both the association rate k′_O2 and dissociation rate k_O2 with little change in affinity [7,8]. Based on our data we can conclude that no perturbation of the apolar tunnel occurs as a result of exogenous ligand binding (CO or O₂); the tunnel therefore maintains identical structure and, likely, dynamic properties, independently of the ligation state of the protein.

CerHb was expressed and purified as described previously using a synthetic gene with codon usage optimized for expression in Escherichia coli [3,13]. The oxygenated derivative of CerHb (CerHb-O₂) was crystallized by vapor diffusion techniques (protein concentration 27 mg/mL) under conditions matching those reported in the literature [3,13]. Elongated prismatic crystals (about 0.05 × 0.05 × 0.3 mm³) grew within 1 week. The unbound form of CerHb (unligated-CerHb) was prepared by soaking the CerHb-O₂ crystals in a stabilizing-cryo solution containing 2.8 M ammonium sulfate, 50 mM sodium acetate, pH 5.5, 15% glycerol and 50 mM dithionite, for 30 min in a sealed well. Unligated-CerHb crystals were then quickly frozen in liquid nitrogen for data collection, or transferred in a sealed well containing the same stabilizing-cryo solution but saturated with CO for preparation of the CO-bound derivative (CerHb-CO). After 30 min, the CerHb-CO crystals were quickly frozen in liquid nitrogen for data collection.

High resolution data (1.9 Å for unligated-CerHb, and 1.5 Å for CerHb-CO) were collected at 100 K at the European Synchrotron Radiation Facility, Grenoble, France (beam line ID23-1). These crystals belong to the orthorombic space group P2₁2₁2₁ (Table 1). All collected data were reduced and scaled using MOSFLM and SCALA [14,15], and phased by molecular replacement methods with the program MOLREP [16] by using the CerHb-O₂ structure as the starting model (PDB accession code 1KR7) [3]. The crystallographic refinement was performed using the program REFMAC [17], and the program COOT [18] was used for model building/inspection. The relevant data collection and refinement statistics are reported in Table 1. The program Procheck [10] was used to assess the stereochemical quality of the protein structures and the program 3V to calculate the solvent-excluded volume of the protein structures [19]. Atomic coordinates and structure factors of unligated-CerHb and CerHb-CO have been deposited with PDB accession codes 4AVE, and 4AVD, respectively.