2 hemoglobin from the plant pathogen Agrobacterium tumefaciens

Biochimica et Biophysica Acta 1814 (2011) 810–816

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p

Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens☆ Alessandra Pesce a, Marco Nardini b, Marie LaBarre c, Christian Richard c, Jonathan B. Wittenberg d, Beatrice A. Wittenberg d, Michel Guertin c, Martino Bolognesi b,⁎ a

Department of Physics, University of Genova, I-16146 Genova, Italy Department of Biomolecular Sciences and Biotechnology, and CIMAINA, University of Milano, I-20131 Milano, Italy Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec, Canada, G1K 7P4 d Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA b c

a r t i c l e

i n f o

Article history: Received 22 September 2010 Received in revised form 27 October 2010 Accepted 2 November 2010 Available online 9 November 2010 Keywords: 2/2 Hemoglobin Truncated hemoglobin Globin fold Heme stabilization Diatomic ligand recognition

a b s t r a c t Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1 Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogenbonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. © 2010 Elsevier B.V. All rights reserved.

1. Introduction 2/2 Hemoglobins (2/2Hbs), formerly known as truncated hemoglobins, build a wide family within the Hb superfamily. 2/2Hbs can be distinguished for their smaller size (20–25%) relative to vertebrate Hbs, and for a simplified fold (2-on-2) relative to the classical 3-on-3 α-helical globin fold [1]. The whole 2/2Hb family has been divided into three distinct groups (I, II, and III), identified by the N, O, and P suffixes, respectively. In particular, group II 2/2HbO belongs to four lineages represented by the Actinobacteria, Proteobacteria, Firmicutes, and plants [2–4]. To date, four 2/2HbO crystal structures have been solved, from Mycobacterium tuberculosis and from Thermobifida fusca (Mt-2/ 2HbO, Tf-2/2HbO; actinobacteria), from Bacillus subtilis and from Geobacillus stearothermophilus (Bs-2/2HbO, Gs-2/2HbO; Firmicutes)

Abbreviations: 2/2Hb, 2-on-2 hemoglobin; Mt-2/2HbO, Mycobacterium tuberculosis 2/2 hemoglobin O; Tf-2/2HbO, Thermobifida fusca 2/2 hemoglobin O; Bs-2/2HbO, Bacillus subtilis 2/2 hemoglobin O; Gs-2/2HbO, Geobacillus stearothermophilus 2/2 hemoglobin O; At-2/2HbO, Agrobacterium tumefaciens 2/2 hemoglobin O; Ce-2/2HbN, Chlamydomonas eugametos hemoglobin N; Cj-2/2HbP, Campylobacter jejuni 2/2 hemoglobin P ☆ This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. ⁎ Corresponding author. Department of Biomolecular Sciences and Biotechnology, University of Milano, Via Celoria 26, I-20133 Milano, Italy. Tel.: +39 02 5031 4893; fax: +39 02 5031 4895. E-mail address: [email protected] (M. Bolognesi). 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.11.001

[5–8]. The most distinctive structural feature characterizing 2/2HbOs, in relation to their heme distal ligand binding site, is the presence of group II-specific residues at the B10, CD1, E7, E11, and G8 positions, which may be of polar or apolar nature [1,3,5] (Fig. 1). These residues build intertwined H-bonding networks that stabilize the heme ligand and add an unusually polar character to the surrounding distal site. Sequence analyses show that Tyr and Trp are 2/2HbO invariant residues at the B10 and G8 sites, respectively. In animal and plant Hbs, the distal HisE7 residue is well-known for its stabilizing action on the heme-bound O2 through an H-bond. Conversely, apolar residues or Hbond donors occupy the E7 position in group II 2/2Hbs. Moreover, Tyr (Actinobacteria) and His (Proteobacteria) residues replace the almost invariant (through the Hb super-family) PheCD1 found in group I and III 2/2Hbs, as well as in vertebrate and plant Hbs (Fig. 1). The analyses of kinetic data have shown distinct ligand binding properties for Mt-2/2HbO, Tf-2/2HbO, Bs-2/2HbO, Gs-2/2HbO, and Arabidopsis thaliana 2/2HbO (plant), resulting in very different O2 affinities [6–11]. Bs- and Gs-2/2HbO display the highest O2 affinity (Kd = 0.15 and 0.075 nM, respectively), due to a high ligand association rate (14 and 76 μM−1 s−1, respectively), and to an extremely slow dissociation process (0.0021 and 0.0038 s−1, respectively). The affinities of Mt- and Tf-2/2HbO for O2 are significantly lower (Kd = 13 and 71 nM, respectively), due to low O2 association rates (both b1 μM−1 s−1) and dissociation rates (0.0014 and 0.07 s−1, respectively) comparable to or higher than those of Bs- and Gs-2/

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2HbO. Finally, Arabidopsis 2/2HbO shows the lowest affinity for O2, with a Kd value of 1500 nM, because of slow ligand association (0.2 μM−1 s−1) and a high O2 dissociation rate (0.3 s−1). Ligand binding studies on Mt-2/2HbO mutants have suggested that TyrCD1 and TrpG8 control O2 association and dissociation rates,

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TyrB10 playing a minor role [9,10]. The X-ray structure of cyano-met Mt-2/2HbO reveals that TyrCD1, not TyrB10, provides the main Hbond to the cyanide ligand, with which TrpG8 is also but more weakly H-bonded [5]. Conversely, residues PheCD1 and GlnE11 in Bs-2/2HbO alter the H-bonding scheme, such that the main ligand stabilizing

Fig. 1. Structure-based sequence alignment of At-2/2HbO with members of groups I, II and III 2/2Hbs. The globin fold topological positions, as defined in sperm whale Mb, are shown on the top of the sequence alignment. α-Helical and 310 regions are highlighted in blue and magenta, respectively. Gly motifs are highlighted in grey; residues conserved in all 2/2Hbs are highlighted in black; residues conserved in group II 2/2Hbs are highlighted in yellow; residues conserved only in proteobacteria group II 2/2Hbs are highlighted in green.

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H-bond is provided by TyrB10, while GlnE11 and TrpG8 are only very weakly linked to the ligand; residue ThrE7, remarkably, is not H-bonded to the heme-bound cyanide [7]. At present, no 2/2HbOs from proteobacteria have been studied. Here we present the crystal structure characterization of a group II 2/ 2Hb (At-2/2HbO) from the bacterium Agrobacterium tumefaciens, a soil and phyto-pathogenic bacterium causing crown gall tumors in a variety of plants. A. tumefaciens hosts two genes (AGR_C_4317 and AGR_C_428) that encode group II 2/2HbO and group III 2/2HbP. The structural properties of At-2/2HbO are discussed here at the light of current views on 2/2Hb structure-to-function relationships, on heme distal site structural and evolutionary plasticity, and on heme/ligand recognition and stabilization mechanisms in group II 2/2Hbs. 2. Methods 2.1. Cloning, expression, and purification of recombinant At-2/2HbO Recombinant At-2/2HbO was expressed and purified as previously described except that the cell extract was fractionated with (NH4)2SO4 (25–80% saturation) [10]. The polymerase chain reaction was used to amplify the AGR_C_4317 gene from A. tumefaciens C58 genomic DNA. The DNA primers used were 5′-GGAATTCCATATGAGCAGCGAGACCGTT-3′ (upper) and 5′-CGGGATCCTTTCATGGGTTATCCGCTTC-3′ (lower), the stop codon is underlined. The PCR reaction mixture contained 10 ng of C58 genomic DNA in a buffer containing 20 mM Tris–HCl, pH 8.8, 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% triton X-100, 0.1 mg/ml nuclease-free BSA, dNTPs (0.2 mM each), primers (0.2 μM each), DMSO (5% v/v), and Pfu Turbo DNA polymerase (1.25 U). The PCR cycling parameters included 4 steps: (a) 1 cycle (30 s at 94 °C), (b) 5 cycles (30 s at 94 °C, 30 s at 58 °C, 3 min at 72 °C), (c) 25 cycles (30 s at 94 °C, 30 s at 63 °C, 3 min at 72 °C), and (d) 1 cycle (25 min at 72 °C). The NdeI–BamHI-digested PCR fragment was purified and cloned into the pET3a prokaryotic expression vector (Stratagene) as described previously [12]. The purified protein was oxidized using ten-fold molar excess potassium ferricyanide. After completion of the reaction, the protein was desalted over a Bio-Gel P-6DG (Bio-Rad, USA) column equilibrated with 10 mM Tris–Cl pH 7.5 buffer containing 50 μM EDTA. The protein was stored in the same buffer in liquid nitrogen. 2.2. Crystallization and data collection Crystallization of aquo-met At-2/2HbO was achieved at 37 mg/ml protein concentration in a vapor diffusion setup, at 277 K. The crystallization conditions (32% w/v polyethylene glycol 4000, lithium chloride 0.8 M, and Tris–HCl 0.1 M at pH 8.5) were found after a wide screen (566 in-house-designed conditions) set up with a robotic apparatus (Genesis RSP100—Tecan). Small hexagonal single crystals grew in 2–3 weeks and were stored in a stabilizing solution containing 45% w/v PEG 4000, lithium chloride 0.8 M and Tris–HCl 0.1 M, pH 8.5. They were transferred to the same solution supplemented with 15% (v/v) glycerol, immediately prior to cryo-cooling and data collection (at 100 K). The crystals diffract up to 2.1 Å resolution, using synchrotron radiation (beamline ID14-3, ESRF, Grenoble, France), and belong to the monoclinic space group P21 (two molecules per asymmetric unit). All collected data were reduced and scaled using MOSFLM [13] and programs from the CCP4 suite [14] (Table 1). 2.3. Structure determination and refinement Structure solution was achieved through molecular replacement techniques using the program Phaser [15] and the G-subunit of the dodecameric Mycobacterium tuberculosis 2/2HbO (PDB entry-code 1NGK) as search model (deleting the C-helix, the CD and FG connecting loops, and truncating side chains to Ala for not-matching amino acids). A

Table 1 Data collection and refinement statistics for At-2/2HbO. Data collection Space group Cell dimensions Resolution limits (Å) Observed reflections Unique reflections Completeness (%) Mosaicity (°) R-mergeb (%) I/σ(I) Multiplicity Refinement statistics R-factorc/R-freed (%) Number of residues Number of heme groups Number of water molecules RMS deviation from ideality Bond lengths (Å) Bond angles (°) Ramachandran plote Residues in most favored regions (%) Residues in additional allowed regions (%)

P21 a = 53.2 Å, b = 44.5 Å, c = 57.6 Å, β = 112.2° 34.2–2.1 59,563 (8729) 14,775 (2158) 99.9 (99.9)a 0.8 12.7 (35.6) 12.4 (4.6) 4.0 (4.0)

17.7/23.9 129 (chain A), 130 (chain B) 2 99 0.015 1.377 96 4

a

Values in parentheses are for highest resolution shell (2.21–2.10 Å). R-merge = ΣhΣi|Ihi–〈 Ih〉|/ΣhΣi Ihi. R-factor= Σh||Fobs|−|Fcalc||/Σ|Fobs| where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. d R-free is calculated on 10% of the diffraction data, which were not used during the refinement. e Data produced using the program PROCHECK [19]. b c

prominent solution for the two At-2/2HbO molecules was found in the 25–3.5 Å resolution range, and initially refined using the program CNS [16], with a run of rigid body refinement for the two protein subunits, and then moving independently all the helices and the heme groups as rigid bodies. Model building/inspection was based on the program Coot [17]. The two At-2/2HbO chains were refined using the program REFMAC [18], locating 99 water molecules through inspection of difference Fourier maps. The final R-factor value was 17.7% (in the 34.2–2.1 Å resolution range), and R-free, 23.9% (Table 1). The programs Procheck [19] and Surfnet [20] were used to assess the model stereochemical quality and to search for protein matrix cavities. Atomic coordinates and structure factors have been deposited with the Protein Data Bank [21], with entry codes 2xyk, and r2xyksf, respectively.

3. Results 3.1. Overall structure The structure of aquo-met At-2/2HbO was solved using crystals belonging to the monoclinic space group P21, with unit cell edges: a = 53.2 Å, b = 44.5 Å, c = 57.6 Å, β = 112.2°; the estimated solvent content is 41%, corresponding to two At-2/2HbO molecules (chains A and B) per asymmetric unit. The At-2/2HbO structure was refined at 2.1 Å resolution, to final R-factor and R-free values of 17.7% and 23.9%, respectively, with ideal stereochemical parameters (Table 1). No electron density was observed for the last 4 C-terminal residues of chain A, and for the last 3 C-terminal residues of chain B. Superposition of 129 Cα atoms of the two independent At-2/2HbO chains yields a RMS deviation of 0.58 Å. At-2/2HbO chains A and B build a putative dimer based on a subunit contact interface of ~ 600 Å2, which, however, does not show specific interactions suggesting the formation of a stable assembly, in agreement with gel filtration experiments showing that the protein elutes as a monomer of 16.8 kDa (data not shown). The results discussed below equally apply to both At-2/2HbO chains A and B, unless otherwise stated.

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Fig. 2. Comparison of 2/2Hb folds. (a) Stereo view of the structural superposition of At-2/2HbO (cyan; group II) and Ce-2/2HbN (orange; group I) folds, represented as ribbon structures. (b) Structural superposition of At-2/2HbO (cyan) and Cj-2/2HbP (magenta; groupIII), in approximately the same orientation of panel a. In both panels, the heme group is displayed only for At-2/2HbO.

3.2. Structural comparison with 2/2Hbs The At-2/2HbO tertiary structure follows the typical features of the α-helical 2/2Hb-fold [1,3,4] (Fig. 2), with some structural modifications that are characteristic of group II [22]. Structural superposition (limited to 70 Cα atoms of the B, E, F, G, H helices) of At-2/2HbO with C. eugametos 2/2HbN (Ce-2/2HbN) and C. jejuni 2/2HbP (Cj-2/2HbP) structures, taken as prototypes of group I and group III 2/2Hbs, respectively, [23,24], yields RMS deviations in the 1.30–1.55 Å range (Fig. 2). The main differences are located in the N-terminal region, in the BC interhelical hinge (At-2/2HbO has a three-residue insertion), in the CD hinge (a one-residue and a six-residue deletion relative to Ce2/2HbN and Cj-2/2HbP, respectively), in the EF hinge, due to the presence of an additional six-residue α-helix (the Φ helix, from residues 66 to 71) typical of group II 2/2HbOs, in the FG and GH hinges, and in the C-terminal end of the H-helix (Figs. 1 and 2). The Cα backbone overlay of At-2/2HbO on Mt-2/2HbO, Tf-2/2HbO, Bs-2/ 2HbO, and Gs-2/2HbO yields RMS deviations in the 0.91–1.05 Å range, the main structural differences being located in the N-terminal region, in the BC (three-residue insertion in At-2/2HbO), in the CD and in the GH hinges and in the C-terminal region. The two Gly–Gly motifs present at the C-terminal end of A and E helices, typical of group I and II 2/2Hbs, are fully conserved in At-2/2HbO (Fig. 1), helping stabilization of the short A helix in a conformation locked onto helices B and E.

Contrary to what has been observed in group I 2/2Hbs [25], inspection of the At-2/2HbO structure does not show a proper protein matrix tunnel system. However, a medium polarity distal site cavity of about 45 Å3 volume is defined by the heme distal face, and by the distal residues Phe(25)B9, Tyr(26)B10, His(42)CD1, Ser(49)E7, Phe (53)E11, and Trp(93)G8; the cavity is shut on the solvent side by the Lys(52)E10—propionate-A salt link, and hosts at its center a water molecule that is part of a tight H-bonded network linking the heme distal residues and the heme ligand (see below). 3.3. Heme pocket Inspection of the electron density indicates that in both At-2/2HbO chains the heme group is rotated by 180° along the methinic α−γ meso axis, relative to (non)vertebrate globins [26], a structural feature that has been previously observed in several globins heterologously expressed in bacterial cells [27]. Stabilization of the bound heme in At-2/2HbO is achieved through direct Fe coordination to the proximal His(80)F8 residue, electrostatic interaction of the heme propionates, and van der Waals contacts (b4.0 Å) with 20 protein residues. Lys(52)E10 is at hydrogen-bonded salt bridge distance from both propionates-A and -D in chain A, while it makes an electrostatic interaction only with propionate-D in chain B. Furthermore, Arg(79)F7 (invariant in group II 2/2Hbs) falls at 3.5– 4 Å from propionate D, which is in turn hydrogen bonded to Tyr(67)

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Fig. 3. (a) Stabilization of the heme group in At-2/2HbO. The stereo figure shows the main residues involved in stabilization of the heme group through Fe coordination (HisF8), salt bridges, and/or hydrogen bonds (SerE7, LysE10, Tyr67, and ArgF7). For reference, the electron density (contoured at 1.0σ level) has been drawn for the heme and selected residues. (b) Heme distal site structure. The stereo figure displays the protein environment surrounding the heme Fe-atom coordinate water molecule (w49, shown as magenta sphere) in At2/2HbO subunit A. The side chains of residues involved in the stabilization of the liganded water molecule are shown and labeled. The figure shows two additional water molecules (w20 and w24), shown as cyan spheres) that are integral part of the distal site hydrogen bonded network stabilizing the heme ligand.

(Φ helix), strongly conserved in groups II and III 2/2Hbs (Figs. 1 and 3a). A similar heme-stabilizing interaction is present in group I 2/ 2HbNs from M. tuberculosis [28] and from Synechocystis sp. [29–31]. The architecture of the heme pocket in the proximal region is dominated by Leu(76)F4, Ile(85)FG5, Arg(90), and Met(125)H4, all of which contact the porphyrin ring. The solvent side of the proximal heme pocket is sealed by the Arg(79)F7—heme propionate-D salt bridge that renders His(80)F8 almost inaccessible to solvent. Analysis of the stereochemical parameters describing the heme Fe coordination indicates a regular Fe–HisF8 NE2 coordination bond (2.04 Å; all reported distances are averaged over the A and B chains), with the F8 imidazole ring lying in a staggered azimuthal orientation relative to the heme pyrrole N-atoms. The heme distal pocket residues Phe(25)B9, Tyr(26)B10, His(42) CD1, Ser(49)E7, Phe(53)E11, and Trp(93)G8 surround a heme-ligated water molecule (Fig. 3b). Part of these residues are conserved in more than one 2/2Hb group, while residues His(42)CD1, Ser(49)E7, and the neighbouring Tyr(56)E14 are peculiar of At-2/2HbO (Fig. 1). The His (42)CD1 imidazole ring is parallel to the heme, its orientation being set by a H bond (3.05 Å) between the ND1 atom and the carbonyl O atom of Cys(38)C4. Moreover, the orientation of the distal residue Ser (49)E7 is stabilized by an H bond between the OG atom and the heme propionate-A (Fig. 3a). At the dead end of the distal pocket, Trp(93) G8, conserved in 2/2Hb groups II and III, fills the inner part of the heme distal site, preventing further diffusion of ligands away from the Fe coordination site. Trp(93)G8 indole ring is parallel and in contact with the heme plane at the B and C pyrrole rings (Fig. 3b).

The heme distal site is characterized by the presence of a highly intertwined H-bonded network, involving residues Tyr(26)B10, His (42)CD1, Ser(49)E7, Trp(93)G8, and three water molecules (W20, W24, and W49 in chain A; W30, W10, and W84 in chain B, respectively; Fig. 3b). In particular, the heme-coordinated water molecule W49 in subunit A, and W84 in subunit B (with a Fe–O coordination bond of 2.05 Å) are directly hydrogen bonded to a distal site intermediate water molecule indicated as W24 in subunit A, and W10 in subunit B (distance 2.60 Å), and to the indole NE1 atom of Trp (93)G8 (distance 2.80 Å). The intermediate water molecule is fully buried in the distal site and is in turn hydrogen bonded to Tyr(26)B10 OH group (distance 2.54 Å) and to the NE2 atom of His(42)CD1 (distance 2.73 Å). The third distal site water molecule (W20 in subunit A and W30 in subunit B), located at the entrance of the distal pocket, is hydrogen bonded to the intermediate water molecule but also to the carbonyl O atom of Ser(49)E7 (distance 2.81 Å) (Fig. 3b). The presence of the heme-coordinated water molecule and of two additional distal site water molecules that may map the pathway from the solvent region to the heme distal site, together with the presence of a small distal site cavity, suggests that enough room is available for ligand access to the heme through the ”classical” E7–gate pathway, formerly characterized in vertebrate Hbs [32,33]. 4. Discussion One remarkable feature distinguishing 2/2Hbs from other globins is the structural variability in their distal heme pockets

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that display different combinations of polar residues [4]. Threedimensional structures and extensive spectroscopic studies have shown that the distal residues give rise to a “ligand-inclusive Hbond network” yielding a complex distal site structural organization. Sequence analyses show that different residue combinations have evolved in distinct group of bacteria, possibly reflecting adaptation to specific environmental conditions. As observed, such distinctive distal heme pockets translate into very different ligand binding properties. The results on the At-2/2HbO three-dimensional structure here reported highlight such distinctive properties, adding to the complex picture emerging from the currently available 2/2Hb structural data [1,3]. The distal site of At-2/2HbO is characterized by residues that are conserved in all group II 2/2Hbs, such as Phe(25)B9, Tyr(26)B10, and Trp(93)G8. However, the presence of His(42)CD1 is distinctive of group II 2/2Hbs from proteobacteria, whereas in the vast majority of 2/2Hbs and Hbs, the CD1 site is occupied by a strictly conserved Phe residue that shields the heme from solvent. The presence of His at site CD1 is thus very unusual and likely indicates functional adaptation. Another exception to the “PheCD1 rule” is found in group II 2/2Hbs from actinobacteria where Tyr occupies the CD1 position. As shown by sequence comparison (Fig. 1), group II 2/2Hbs that have a H-bond donor at CD1 position (Actinomycetes and Proteobacteria) bear an apolar residue at the E11 position, while those hosting PheCD1 (Firmicutes and plants) display Gln (a H-bonding residue) at site E11. Recent studies on Mt-2/2HbO mutants have revealed that TyrCD1 controls O2 binding but plays a minor role in stabilization of the bound ligand (Mt-2/2HbO hosts a Leu residue at the E11 site) [9,10]. In both Bs-2/2HbO and At-2/2HbO, the E7 residue (Thr and Ser, respectively) is H-bonded to a heme propionate and does not stabilize directly the heme ligand; absence of HisE7–heme ligand interaction is also observed in group III Cj-2/2HbP [24]. Instead, in group I 2/2Hbs from C. eugametos, P. caudatum, and Synechocystis sp., the E7 site is occupied by a H-bonding residue (Gln), which stabilizes the bound ligand [23,29–31]. Thus, the Ser(49)E7–heme propionate-A interaction, together with that of Lys(52)E10 with the same propionate, here reported for At-2/2HbO, may build a gate to the heme distal site, regulating the access/exit of ligands to/from the distal pocket (Fig. 3a). Similar E7, E10–propionate interactions are present in Bs-2/2HbO; however, the presence of a Gln residue at the Bs-2/2HbO E11 site (Phe(53)E11 in At-2/2HbO) provides a net distinction between the two distal sites. A main structural feature stressed by the At-2/2HbO crystallographic analysis is the network of distal site water molecules linking the heme coordination site to the outer solvent space. Such a structural feature, which may be representative of a functional path for ligand distal site diffusion to/from the heme, had been only partly noted before in the twelve subunits of Mt-2/2HbO and in Tf-2/2HbO, where a water molecule matches that located at the entrance of the At-2/2HbO distal pocket (W20 in subunit A and W30 in subunit B) [5,6]. Remarkably, neither a similar structuring effect nor the presence of distal site water molecules have been observed in group I and group III 2/2Hbs. 5. Conclusions A wide series of previous crystallographic studies have highlighted that protein matrix tunnels and/or core cavities differ substantially in their size and topologies among the 2/2Hb groups I, II, and III. Such structural effects may correlate with the different functional roles that have been ascribed at least to some 2/2Hbs (such as O2 storage and diffusion, NO-dioxygenase enzymatic activity, sensors, etc.). The results here reported for At-2/2HbO, brought in the more general context of the Hb superfamily, confirm that 2/2Hbs display very high levels of fine structural variability in their heme distal (“active”) sites. 2/2Hbs distal site residues vary dramatically, from apolar small/bulky

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to polar small/bulky, resulting in H-bonded networks that display entirely different topologies, extensions, and strategies for heme ligand stabilization. Such an observation finds very few comparable examples within the known protein families, where, in general, close conservation of the active site overall stereochemistry (e.g., within one protease family) is the pre-requisite for functional activity. The distal site structural variability displayed by 2/2Hbs, herein further stressed by At-2/2HbO structure, is even more striking if one considers that 2/2Hbs are very small heme proteins, whose size is close to a lower limit allowing to host properly a heme group. In fact, a small protein size should translate into strict structural restraints that would impose a rather conservative evolution of their distal site topology. In this respect, it is notable that the 2/2Hb distal site structural variability appears higher than the structural variability observed among vertebrate globins. The oxygenated At-trHbO undergoes rapid auto-oxidation, suggesting that this 2/2Hb may not act as an oxygen carrier or support its diffusion. Instead the structural changes that might occur upon reduction or oxidation of the heme iron may serve as a signal for growth under low O2 tension and could then regulate genes required for growth under such conditions. On the other hand, given similarities in spectral and in ligand binding properties with soluble guanylate cyclase and the heme domain of their prokaryotic homolog H-NOX [34], At-trHbO may bind NO to form a NO complex regulating expression of specific genes. At-trHbO may function as a stand-alone sensor or as a regulatory subunit of a multi-subunits sensor. In conclusion, all of the above concepts underline that the 2/2Hb family indeed may host a very high functional variability, likely higher than in the vertebrate globin family; such structure-based suggestions are in the enthusiastic wait for in vivo functional validation.

Acknowledgments This work was supported by the National Sciences and Engineering Research Council (NSERC) grant 46306-01 (2005-2010) to M.G. M.G. is supported by the NSERC grant 250073 and the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) grant 78927. Part of this study was supported by the University of Milano FIRST2005 grant (to M.B.).

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