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Heme-Ligand Tunneling in Group I Truncated Hemoglobins

2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês

10.1074/jbc.m401320200

1083-351X

Mario Milani, Alessandra Pesce, Yannick Ouellet, Sylvia Dewilde, Joel M. Friedman, Paolo Ascenzi, Michel Guertin, Martino Bolognesi,

Heme Oxygenase-1 and Carbon Monoxide

Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood. Crystallographic investigations have shown that the trHb fold (a two-on-two α-helical sandwich related to the globin fold) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to the heme distal site. The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure. Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under xenon pressure leads to binding of xenon atoms at specific (conserved) sites along the protein matrix tunnel. The crystallographic results are in keeping with data from molecular dynamics simulations, where a dioxygen molecule is left free to diffuse within the protein matrix. Modulation of xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles. In a parallel crystallographic investigation on M. tuberculosis trHbN, we show that butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the xenon binding experiments. These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity ligands/molecules within group I trHbs. Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood. Crystallographic investigations have shown that the trHb fold (a two-on-two α-helical sandwich related to the globin fold) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to the heme distal site. The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure. Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under xenon pressure leads to binding of xenon atoms at specific (conserved) sites along the protein matrix tunnel. The crystallographic results are in keeping with data from molecular dynamics simulations, where a dioxygen molecule is left free to diffuse within the protein matrix. Modulation of xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles. In a parallel crystallographic investigation on M. tuberculosis trHbN, we show that butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the xenon binding experiments. These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity ligands/molecules within group I trHbs. Truncated hemoglobins (trHbs) 1The abbreviations used are: trHb, truncated hemoglobin; Ce-trHb, group I trHb from C. eugametos; Mt-trHbN, group I trHb from M. tuberculosis; Mt-trHbO, group II trHb from M. tuberculosis; Pc-trHb, group I trHb from P. caudatum; Ss-trHb, group I trHb from Synechocystis sp.; Mb, myoglobin; MD, molecular dynamics; r.m.s.d., root mean square deviation. 1The abbreviations used are: trHb, truncated hemoglobin; Ce-trHb, group I trHb from C. eugametos; Mt-trHbN, group I trHb from M. tuberculosis; Mt-trHbO, group II trHb from M. tuberculosis; Pc-trHb, group I trHb from P. caudatum; Ss-trHb, group I trHb from Synechocystis sp.; Mb, myoglobin; MD, molecular dynamics; r.m.s.d., root mean square deviation. are small oxygen-binding hemoproteins, identified in bacteria, higher plants, and in certain unicellular eukaryotes, building a separate cluster within the hemoglobin superfamily. Based on amino acid sequence analysis, three trHb phylogenetic groups (groups I, II, and III) have been recognized (1Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). TrHbs display amino acid sequences that are 20-40 residues shorter than (non)vertebrate hemoglobins, to which they are scarcely related by sequence similarity. Notably, trHbs belonging to the different groups, but also within the same group, may share less then 20% amino acid sequence identity (1Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) (Fig. 1). TrHbs from more than one group can coexist in some bacteria, suggesting a wide diversification of functions. Possible trHb functions that are consistent with observed biophysical properties include long term ligand or substrate storage, NO detoxification, O2/NO sensor, redox reactions, and O2 delivery under hypoxic conditions (1Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 2Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Mukai M. Mills C.E. Poole R.K. Yeh S.R. J. Biol. Chem. 2001; 276: 7272-7277Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In Mycobacterium bovis BCG, trHbN promotes an efficient dioxygenase reaction whereby NO is converted to nitrate by the oxygenated heme (4Ouellet H. Ouellet Y. Richard C. Labarre M. Wittenberg B. Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5902-5907Crossref PubMed Scopus (235) Google Scholar).So far, four group I trHbs from Chlamydomonas eugametos (Ce-trHb), Paramecium caudatum (Pc-trHb), Mycobacterium tuberculosis (Mt-trHbN), and Synechocystis sp. (Ss-trHb) and one group II trHb from M. tuberculosis (Mt-trHbO) have been structurally characterized (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar, 7Falzone C.J. Vu B.C. Scott N.L. Lecomte J.T. J. Mol. Biol. 2002; 324: 1015-1029Crossref PubMed Scopus (53) Google Scholar, 8Hoy J.A. Kundu S. Trent III, J.T. Ramaswamy S. Hargrove M.S. J. Biol. Chem. 2004; 279: 16535-16542Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 9Milani M. Savard P.Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). The main structural features highlighted by these studies are an unprecedented two-on-two α-helical sandwich fold, a ligand-dependent hydrogen-bonding network within the distal heme pocket, and a protein matrix hydrophobic tunnel/cavity network connecting the solvent space to the heme distal pocket through one or two access sites (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar). Such tunnel/cavity network is topologically conserved in Ce-trHb, Pc-trHb, and in Mt-trHbN three-dimensional structures, being built by almost invariant apolar residues (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar) (Fig. 1). In contrast, the recently solved crystal structure of hexacoordinated Ss-trHb, where the sixth heme ligand is residue HisE10, 2TrHb residues are identified by their three-letter code, their globin fold topological position, and their sequence number. 2TrHb residues are identified by their three-letter code, their globin fold topological position, and their sequence number. did not show evidence of such cavity network, likely related to extensive conformational changes observed in the protein distal site region (8Hoy J.A. Kundu S. Trent III, J.T. Ramaswamy S. Hargrove M.S. J. Biol. Chem. 2004; 279: 16535-16542Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).In Mt-trHbN, the tunnel is composed of two orthogonal branches, yielding a L-shaped path through the protein matrix. The tunnel short branch (about 8 Å long) connects the heme distal site to the outer solvent space, at a location comprised between the central region of the G and H helices. The tunnel long branch stretches for about 20 Å through the protein matrix, from the heme distal cavity to a solvent access site located between the interhelical AB and GH loops. Overall, the tunnel volume is about 265 Å3 (6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar). A very similar but more open tunnel system displays a volume of about 400 Å3 in Ce-trHb, whereas in Pc-trHb the tunnel is restricted to three cavities, with an overall volume of 180 Å3 (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar). Small cavities have been observed in sperm whale myoglobin (Mb) and are recognized to act as temporary docking sites for small ligands such as O2, NO, and CO (10Tilton Jr., R.F. Kuntz Jr., I.D. Petsko G.A. Biochemistry. 1984; 23: 2849-2857Crossref PubMed Scopus (459) Google Scholar, 11Brunori M. Gibson Q.H. EMBO Rep. 2001; 2: 674-679Crossref PubMed Scopus (155) Google Scholar, 12Scott E.E. Gibson Q.H. Olson J.S. J. Biol. Chem. 2001; 276: 5177-5188Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 13Srajer V. Ren Z. Teng T.Y. Schmidt M. Ursby T. Bourgeois D. Pradervand C. Schildkamp W. Wulff M. Moffat K. Biochemistry. 2001; 40: 13802-13815Crossref PubMed Scopus (284) Google Scholar, 14Schotte F. Lim M. Jackson T.A. Smirnov A.V. Soman J. Olson J. Phillips Jr., G.N. Wulff M. Anfinrud P.A. Science. 2003; 300: 1944-1947Crossref PubMed Scopus (673) Google Scholar). However, the Mb cavities are much smaller (13-45 Å3) than the cavities found in trHbs and are topologically unrelated.The location and size of the hydrophobic tunnels in group I trHbs suggest important roles in controlling diffusion of small ligands to/from the heme distal pocket or in temporary ligand storage and/or accumulation (1Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar, 9Milani M. Savard P.Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar, 15Samuni U. Dankster D. Ray A. Wittenberg J.B. Wittenberg B.A. Dewilde S. Moens L. Ouellet Y. Guertin M. Friedman J. J. Biol. Chem. 2003; 278: 27241-27250Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To shed first light on such stimulating ideas, we have analyzed crystals of Mt-trHbN, Ce-trHb, and Pc-trHb treated with xenon or butyl isocyanide, two distinct probes expected to diffuse differently through the trHb tunnel system, and determined their three-dimensional structures. Our data, complemented by molecular dynamics (MD) simulations, show that diffusion to the heme distal site in the group I trHbs considered may follow a path matching the trHb hydrophobic tunnel system, where xenon and butyl isocyanide docking sites are experimentally identified.EXPERIMENTAL PROCEDURESMt-trHbN, Ce-trHb, and Pc-trHb were expressed, purified, and crystallized as previously reported (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar). To promote xenon diffusion within the protein matrix, the selected trHb crystals, in their cryo-protectant solutions, were treated in a high pressure chamber (Xcell, Oxford Cryo-system, UK). Mt-trHbN, Ce-trHb, and Pc-trHb crystals were exposed to 25 bar xenon, for 20-30 min. The x-ray diffraction data sets were collected at 100 K, using a MAR-Research 345 imaging plate detector coupled to a Rigaku RU-H3R rotating anode generator (copper Kα radiation; resolution limits of 2.43 Å for Mt-trHbN, 2.45 Å for Ce-trHb, and 2.40 Å for Pc-trHb). The diffraction data were processed using DENZO/SCALEPACK (16Otwinoski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38355) Google Scholar) or MOSFLM (17Leslie A.G.W. Joint CCP4/ESF-EACBM Newsl. Protein Crystallogr. 1992; 26Google Scholar) and programs from the CCP4 suite (18CCP4 (Collaborative Computational Project 4)Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19703) Google Scholar) (see Table I).Table IData collection and refinement statisticsData collectionMt-trHbN-butyl isocyn ESRF ID14-2Mt-trHbN-Xe CuKαCe-trHb-Xe CuKαPc-trHb-Xe CuKαWavelength (Å)0.9331.5421.5421.542Resolution (Å)20-2.1040-2.4330-2.4520-2.40Unique reflections13,7079,3454,8014,985Multiplicity3.9 (3.7)aOuter shell statistics Mt-trHbN-butyl isocyn (2.21-2.10 Å) within parentheses.4.3 (4.0)bOuter shell statistics Mt-trHbN-Xe (2.56-2.43 Å) within parentheses.3.6 (3.2)cOuter shell statistics Ce-trHb-Xe (2.49-2.45 Å) within parentheses.2.1 (1.9)dOuter shell statistics Pc-trHb-Xe (2.44-2.40 Å) within parentheses.Completeness (%)92.2 (92.2)94.9 (94.9)98.4 (95.4)98.3 (98.7)Mosaicity (°)1.30.90.981.08 4.8 (1.9)6.9 (2.8)8 (1.8)8 (2.6)Rsym (%)10.9 (35.1)9.8 (27.7)15.4 (43.2)11.2 (36.0)Space groupP212121P212121P212121P43Unit cell (Å)a = 43.5, b = 61.4, c = 91.8a = 44.3, b = 62.0, c = 90.7a = 34.5, b = 52.9, c = 67.0a = b = 61.4, c = 33.8RefinementR factor/Rfree (%)19.4/25.1eCalculated using 5% of the reflections.19.4/27.7eCalculated using 5% of the reflections.21.0/26.8fCalculated using 10% of the reflections.21.1/28.2fCalculated using 10% of the reflections.r.m.s.dBonds (Å)0.0120.0090.0030.011Angles (°)1.3351.2240.5721.390Ramachandran plotgGenerated using the program PROCHECK (32).Most favored (%)98.696.498.193.1Additionally allowed (%)1.43.61.96.9B factors (Å2) (atom occupancy)Protein29323147Solvent34272645Xe143 [1.0]44 [0.7]37 [0.8]Xe243 [0.6]35 [0.8]Xe331 [0.5]Xe444 [0.5]Xe561 [0.3]58 [0.4]Xe659 [0.7]Butyl isocyn51 [1.0]a Outer shell statistics Mt-trHbN-butyl isocyn (2.21-2.10 Å) within parentheses.b Outer shell statistics Mt-trHbN-Xe (2.56-2.43 Å) within parentheses.c Outer shell statistics Ce-trHb-Xe (2.49-2.45 Å) within parentheses.d Outer shell statistics Pc-trHb-Xe (2.44-2.40 Å) within parentheses.e Calculated using 5% of the reflections.f Calculated using 10% of the reflections.g Generated using the program PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab The available molecular models of Mt-trHbN, Ce-trHb, and Pc-trHb were relocated into their respective crystal unit cells by molecular replacement, using the EPMR program (19Kissinger C.R. Gehlhaar D.K. Smith B.A. Bouzida D. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1474-1479Crossref PubMed Scopus (72) Google Scholar). Mt-trHbN xenon adduct hosts two molecules per asymmetric unit (A and B subunits; space group P212121, unit cell constants a = 44.3 Å, b = 62.0 Å, c = 90.7 Å); the xenon adducts of Ce-trHb (space group P212121, unit cell constants a = 34.5 Å, b = 52.9 Å, c = 67.0 Å), and of Pc-trHb (space group P43, unit cell constants a = b = 61.4 Å, c = 33.8 Å) accommodate one protein molecule per asymmetric unit. After refinement using CNS (20Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar) and REFMAC5 (21Murshudov G.N. Lebedev A. Vagin A.A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1005) Google Scholar) and model inspection/rebuilding with O (22Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar), the refined Mt-trHbN subunits displayed a total of 8 xenon atoms, refined with occupancy values in the 30-100% range (R factor = 19.4% and Rfree = 27.7%, at 2.43 Å resolution). Monomeric Ce-trHb-refined (R factor = 21.0% and Rfree = 26.8%, at 2.45 Å resolution) and Pc-trHb-refined (R factor = 21.1% and Rfree = 28.2%, at 2.40 Å resolution) xenon adducts display 4 and 1 protein matrix xenon atoms, respectively.Mt-trHbN cyano-met crystals were soaked for about 30 min in a solution containing the stabilizing medium (1.46 m K2HPO4, 0.40 m KH2PO4, 0.001 m KCN, pH 8.1) and 0.038 m butyl isocyanide. X-ray diffraction data were collected at 100 K, at European Synchrotron Radiation Facility beam line ID14-2 (Grenoble, France), using the soaking solution supplemented with 20% (v/v) glycerol, as cryo-protectant. The diffraction data (to 2.10 Å resolution) were processed and scaled using MOSFLM (17Leslie A.G.W. Joint CCP4/ESF-EACBM Newsl. Protein Crystallogr. 1992; 26Google Scholar) and SCALA (18CCP4 (Collaborative Computational Project 4)Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19703) Google Scholar). Structure solution and refinement were carried over as described above for the xenon adduct; however, to minimize local bias, an Ala-trimmed model of Mt-trHbN was used as the starting model. A prominent difference electron density peak next to the A subunit heme distal cavity suggested the presence of butyl isocyanide and was refined accordingly (for the final model, R factor = 19.4% and Rfree = 25.1%, at 2.10 Å resolution).Similar experiments were performed on Ce-trHb and on Pc-trHb, soaking the crystals in stabilizing solutions containing 0.020 m butyl isocyanide, for 2 h. In the case of Ce-trHb, a 3.0 Å resolution data set was collected in house. Inspection of the difference Fourier maps showed an elongated difference electron density peak in the long branch of the protein matrix tunnel, allowing only a qualitative modeling of a bound butyl isocyanide molecule, because of low occupancy (data not shown). In the case of the butyl isocyanide Pc-trHb-soaked crystals (data collected in house, up to 2.4 Å resolution), no extra electron density could be located, indicating the lack of butyl isocyanide binding to this trHb. Table I summarizes all the data collection and refinement statistics. The atomic coordinates and structure factors for the above trHb adducts have been deposited with the Protein Data Bank with the entry codes 1s56 (Mt-trHbN xenon), 1uvx (Ce-trHb xenon), 1uvy (PctrHb xenon), and 1s61 (Mt-trHbN butyl isocyanide).The MD simulations of dioxygen diffusion within the Mt-trHbN tunnel system were performed using the program GROMACS and the GROMACS force field (23Berendsen H.J.C. van der Spoel D. van Drunen R. Comp. Phys. Comm. 1995; 91: 43-56Crossref Scopus (7001) Google Scholar, 24Lindahl E. Hess B. van der Spoel D. J. Mol. Mod. 2001; 7: 306-317Crossref Google Scholar). The crystal structure of Mt-trHbN subunit A (6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar) (Protein Data Bank 1IDR) was completed adding eight C-terminal residues in a random loop conformation. The protein was enclosed in a parallelepiped box (89.4 Å × 67.7 Å × 77.5 Å), solvated with 15,035 water molecules; the box was neutralized adding six Na+ ions (the overall number of atoms in the system is 45,998). A dioxygen molecule was added in a random position within the distal heme cavity. After application of an initial conjugate gradient minimization (until convergence to a maximum force within the system of 300 kJ mol-1nm-1) to relax the system, the MD cycles were performed in a NPT ensemble, with 1-fs time steps. The total simulation time was 1950 ps.RESULTSTrHb Xenon DerivativesMt-trHbN—Analysis of the Mt-trHbN xenon adduct allowed identification of 8 xenon atoms, with different occupancy levels, distributed between the two asymmetric unit molecules. The overall Mt-trHbN structure is little affected by xenon binding, the r.m.s.d. values calculated for comparisons of the xenon bound/unbound A and B subunits being 0.82 and 0.68 Å (on 122 Cα atom pairs), respectively. Three major xenon sites (named Xe1, Xe2, and Xe4; Fig. 2A) are equally present and located in both Mt-trHbN A and B subunits. The Xe1 site (100% xenon occupancy) is located in the tunnel long branch, at 13.3 Å from the heme iron atom, whereas site Xe2 (60% occupancy) falls in the short branch (at 6.3 Å from the heme iron atom). The Xe4 site (50% occupancy) is located at the tunnel short branch entrance, in a hydrophobic cleft between symmetry related molecules. In contrast, the Xe3 site (50% occupancy) is present in subunit B only, being located between the Xe2 and Xe4 sites, at the interface between the tunnel short branch and the solvent. Xe5, located at 9.0 Å from the heme iron atom, between the sites Xe1 and Xe2 in subunit A, displays only 30% occupancy, likely representing a transition site bridging between the Xe1 and Xe2 sites. Such a bridging role is also suggested by the elongated distribution of the residual Fo - Fc map, around Xe5.Fig. 2Stereo view of binding of xenon atoms and butyl isocyanide to trHbs. A, Mt-trHbN subunit B is portrayed together with the bound xenon atoms (magenta); the radius of the spheres representing each xenon atom is proportional to the occupancy of the site. The Xe5 atom, not observed in subunit B, is, however, reported here to highlight its location bridging between the Xe1 and Xe2 sites. The path of the protein matrix tunnel is defined by a gray mesh. The heme group is red, and PheE15(62) is gray. For all figures, the tunnel path has been calculated with SURFNET (29Laskowski R.A. J. Mol. Graph. 1995; 13: 323-330Crossref PubMed Scopus (814) Google Scholar), and figures have been drawn with BOBSCRIPT (30Esnousf B.M. J. Mol. Graph. 1997; 15: 138Google Scholar) and Raster3D (31Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar). B, Ce-trHb xenon adduct is shown in an orientation comparable with that in A, adopting the same graphical conventions. C, a view of Pc-trHb xenon adduct and of the associated cavities system, in an orientation and with graphical conventions as defined for A. D, Mt-trHbN subunit A structure in the presence of butyl isocyanide. The butyl isocyanide molecule (magenta) fills part of the tunnel volume available through selection of PheE15(62) conformation (orange). In the B subunit, where butyl isocyanide is scarcely present, PheE15(62) displays also the alternate conformation (yellow).View Large Image Figure ViewerDownload (PPT)The Xe2, Xe3, and Xe4 sites outline the path of the Mt-trHbN tunnel short branch from the heme distal cavity to the solvent (Fig. 2A) (6Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar). In particular, the Xe3 site protein surface location suggests that access to the heme distal site may occur through an entry site comprised between the G and H helices, defined by residues PheG5(91), AlaG9(95), LeuH8(116), IleH11(119), and AlaH12(120). Binding of xenon at the Xe3 site induces shifts of 0.4-1.2 Å at residues AlaG9(95) and IleH11(119), resulting in a wider solvent aperture for the tunnel short branch. The structural analysis also shows that the Mt-trHbN tunnel long branch is accessible from the solvent, in both A and B subunits, through an aperture defined by residues IleA15(19), IleB2(25), ValB5(28), and LeuG16(102). The location of a full occupancy xenon atom in the Xe1 cavity, at 5.5 Å from the long branch solvent aperture, is stabilized by van der Waals' contacts with residues ValB5(28), PheE15(62), LeuE19(66), LeuG12(98), and LeuG16(102) (Figs. 1 and 2A).Ce-trHb—The Ce-trHb structure displays the wider (∼5 Å in diameter) and more extended L-shaped tunnel observed so far in group I trHbs (Fig. 2B). The four xenon atoms found in the Ce-trHb xenon adduct follow the long tunnel branch, at mutual distances of 4-5 Å. In particular, three xenon atoms match the Xe1, Xe2, and Xe5 sites observed in Mt-trHbN (the MttrHbN-Xe site numbering scheme is kept for Ce-trHb and PctrHb, to identify topologically equivalent xenon sites within the trHb fold). The Xe1 site (70% occupancy) falls at 13.7 Å from the heme iron atom, contacting residues LeuE15(49), LeuE19(53), and LeuG16(91). The Xe2 site (occupancy of 80%) falls at 6.8 Å from the iron atom, being surrounded by residues AlaB5(15), PheB9(19), GlnE11(45), and LeuG12(87) and by the heme group. The third xenon site (40% occupancy) in Ce-trHb is located at a position matching the Xe5 site of Mt-trHbN (10.8 Å from the heme iron atom), next to residues PheB9(19), LeuE15(49), and LeuG12(87). Finally, the fourth xenon site (Xe6; 70% occupancy) does not match any xenon site in MttrHbN, being located at the protein-solvent interface at the exit of the tunnel long branch, contacting residues LeuA15(6), AlaB1(11), AlaB5(15), LeuG16(91), and Val(96) (Figs. 1 and 2B).Structural comparison of the Ce-trHb in the presence and in the absence of xenon atoms does not highlight significant main readjustments in the protein backbone (r.m.s.d. of 0.18 Å, calculated over 121 Cα pairs). However, xenon binding induces a 1.0 Å shift of LeuG12(87), such that the tunnel volume is increased from 320 Å3 in the absence of Xe to 380 Å3 in the presence of Xe, originating the Xe5 site. In contrast, the Xe1, Xe2, and Xe6 atoms are found in cavities that are already present in the Xe-free Ce-trHb structure (5Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar). The tunnel short branch of Mt-trHbN is more properly defined as a cavity in Ce-trHb (about 90 Å3), lined by residues PheG5(80), GlnG6(81), AlaG9(84), MetH8(105), ValH11(108), and AlaH12(109). Remarkably, upon xenon binding, such a cavity shrinks to about 50 Å3, because of the conformational readjustments at LeuG12(87) and MetH8(105), linked to occupation of the Xe5 site.Pc-trHb—The residue selection at the B5 and H7 topological sites (Fig. 1), together with a shift of the B6-B10 polypeptide segment, restrict the tunnel size in Pc-trHb and divide it into three cavities (overall volume 180 Å3), topologically distributed along the tunnel long branch described above (Fig. 2C). In agreement with such picture, only one xenon atom is bound by Pc-trHb, at the Xe1 site (80% occupancy, at 13.8 Å from the heme iron atom), in a 65 Å3 cavity lined by the hydrophobic residues ValB2(12), ValB5(15), ThrB6(16), LeuE15(49), LeuE19(53), LeuG12(85), LeuG16(89), and ThrH7(102). As for Mt-trHbN and Ce-trHb, the r.m.s.d. value, calculated for the protein backbones of the aquo-met Pc-trHb and its xenon adduct (0.28 Å, for 116 Cα pairs), is indicative of minor overall structural readjustments. Binding of the xenon atom, however, results in a wider Xe1 cavity (volume increase of 10 Å3), reflected by side chain shifts (of about 0.5 Å) of residues LeuE15(49) and LeuG12(85). In Pc-trHb, these residues build a narrow neck between the Xe1 site and the adjacent (smaller) cavity, corresponding to the Xe2 site in Ce-trHb. The small size of the potential Xe2 site in Pc-trHb (11 Å3) prevents xenon binding. However, xenon binding to Pc-trHb shifts the heme vinyl CBC atom by 0.7 Å to form an additional small cavity with a volume of 12 Å3. This additional cavity is not observed in the aquo-met Pc-trHb structure in the absence of Xe.Mt-trHbN-butyl Isocyanide DerivativeUnder the experimental conditions applied, binding of butyl isocyanide to Mt-trHbN occurs essentially only in the tunnel long branch of the A subunit (with 100% occupancy). The B subunit displays electron density compatible with very low occupancy binding only. The r.m.s.d. calculated between subunits A and B of the Mt-trHbN adduct is 0.93 Å, (over Cα pairs 3-125). Butyl isocyanide is completely buried within the short tunnel branch and the heme distal cavity (Figs. 1 and 2D), being stabilized by van der Waals' contacts with the heme, PheB9(32), PheE15(62), ValG8(94), LeuG12(98), and IleH11(119) and by dipole-dipole interaction with the amide group of GlnE11(58) (Fig. 2D).It has previously been shown that in Mt-trHbN the short and the long tunnel branches are separated by the two facing residues PheE15(62) and LeuG12(98). In particular, PheE15(62) was observed in two conformations (identified as "open" and "closed," resp