Portal de Periódicos da CAPES

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family

2000; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês

10.1093/emboj/19.11.2424

1460-2075

Alessandra Pesce,

Neonatal Health and Biochemistry

Article1 June 2000free access A novel two-over-two α-helical sandwich fold is characteristic of the truncated hemoglobin family Alessandra Pesce Alessandra Pesce Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Search for more papers by this author Manon Couture Manon Couture Departement de Biochimie et de Microbiologie, Pavillon Marchand, Universitè Laval, Quebec, G1K 7P4 Canada Present address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, NY, 10461 USA Search for more papers by this author Sylvia Dewilde Sylvia Dewilde Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Search for more papers by this author Michel Guertin Michel Guertin Departement de Biochimie et de Microbiologie, Pavillon Marchand, Universitè Laval, Quebec, G1K 7P4 Canada Search for more papers by this author Kiyoshi Yamauchi Kiyoshi Yamauchi Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka, 422-8529 Japan Search for more papers by this author Paolo Ascenzi Paolo Ascenzi Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Department of Biology, University of ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Roma, Italy Search for more papers by this author Luc Moens Luc Moens Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Search for more papers by this author Martino Bolognesi Corresponding Author Martino Bolognesi Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Search for more papers by this author Alessandra Pesce Alessandra Pesce Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Search for more papers by this author Manon Couture Manon Couture Departement de Biochimie et de Microbiologie, Pavillon Marchand, Universitè Laval, Quebec, G1K 7P4 Canada Present address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, NY, 10461 USA Search for more papers by this author Sylvia Dewilde Sylvia Dewilde Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Search for more papers by this author Michel Guertin Michel Guertin Departement de Biochimie et de Microbiologie, Pavillon Marchand, Universitè Laval, Quebec, G1K 7P4 Canada Search for more papers by this author Kiyoshi Yamauchi Kiyoshi Yamauchi Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka, 422-8529 Japan Search for more papers by this author Paolo Ascenzi Paolo Ascenzi Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Department of Biology, University of ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Roma, Italy Search for more papers by this author Luc Moens Luc Moens Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Search for more papers by this author Martino Bolognesi Corresponding Author Martino Bolognesi Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova Search for more papers by this author Author Information Alessandra Pesce1, Manon Couture2,3, Sylvia Dewilde4, Michel Guertin2, Kiyoshi Yamauchi5, Paolo Ascenzi1,6, Luc Moens4 and Martino Bolognesi 1 1Department of Physics – INFM and Advanced Biotechnology Center – IST, University of Genova, Largo Rosanna Benzi 10, 16132 Genova 2Departement de Biochimie et de Microbiologie, Pavillon Marchand, Universitè Laval, Quebec, G1K 7P4 Canada 3Present address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, NY, 10461 USA 4Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium 5Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka, 422-8529 Japan 6Department of Biology, University of ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Roma, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2424-2434https://doi.org/10.1093/emboj/19.11.2424 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Small hemoproteins displaying amino acid sequences 20–40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named ‘truncated hemoglobins’ (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a ‘two-over-two’ α-helical sandwich, reflecting an unprecedented editing of the classical ‘three-over-three’ α-helical globin fold. Based on specific Gly–Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced. Introduction The discovery of hemoglobins (Hbs) in virtually all kingdoms has shown that the gene for Hb is very ancient and that Hbs may serve functions other than as simple oxygen carriers (Hardison, 1998; Imai, 1999; Minning et al., 1999). Within unicellular organisms, two different Hbs, or Hb-related protein groups, are found. A first group, occurring in bacteria and fungi, includes single-chain flavohemoglobins, which consist of an N-terminal heme-containing domain displaying a conventional globin fold, and a C-terminal FAD or NADP+ binding domain structurally related to ferredoxin NADP+ reductase (Ermler et al., 1995). Moreover, a dimeric Hb, which may be non-covalently associated to a flavoprotein reductase, has been isolated from Vitreoscilla sp. The three-dimensional structure of the N-terminal heme domain of flavohemoglobin and of Vitreoscilla Hb conforms to the classical vertebrate and non-vertebrate Hb fold, based on seven or eight α-helices (Perutz, 1979; Holm and Sander, 1993); however, the homodimeric Vitreoscilla Hb adopts a quaternary structure not observed in any other known Hb (Bolognesi et al., 1997, 1999a; Tarricone et al., 1997). The second protein group includes small hemoproteins, refered to as truncated hemoglobins (trHbs), characterized so far in the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, in the unicellular alga Chlamydomonas eugametos, and in the eubacteria Nostoc commune and Mycobacterium tuberculosis (Iwaasa et al., 1989; Potts et al., 1992; Takagi, 1993; Couture et al., 1994, 1999a, b; Thorsteinsson et al., 1999). To these should be added those being discovered through the sequencing of microbial genomes (see Figure 1). In this regard, analysis of the currently available microbial genomic sequences indicates that certain eubacteria (Bacillus subtilis, Staphylococcus aureus, Campylobacter jejuni, Bordetella pertussis and Deinococcus radiodurans) contain both flavohemoglobin and trHb, which suggests specific functions for each of the two protein classes. Figure 1.Structure-based sequence alignment of P_trHb, C_trHb and 15 trHbs from different sources with respect to sperm whale (Physeter catodon) Mb and Vitreoscilla sp. Hb, the latter taken as reference vertebrate and bacterial globin molecules, respectively. The globin fold topological positions, as defined in sperm whale Mb, are shown on the top of the aligned sequences. Amino acid sequential numbering, as well as α-helical regions (indicated by |--| segments), refer to P_trHb. Heme-contacting residues are indicated by a bullet, and some key globin fold topological sites by a star. Residues that are conserved in all trHbs are highlighted in black boxes; yellow and green bars highlight amino acid sites, with conservative substitutions in trHbs, which are discussed in the text in relation to the achievement of the trHb globin fold. Sequence DDBJ/EMBL/GenBank accession numbers are: M.tuberculosis HbN, Z74020; M.tuberculosis HbO, AL021246; B.subtilis Hb, Z99110; N.commune Hb, L47979; Synechocystis PCC6803 Hb, D90910; P.caudatum Hb, M57542; T.pyriformis Hb, D13920; C.eugametos Hb, X72919. Preliminary sequence data for S.aureus, Shewanella putrefaciens, D.radiodurans and T.ferrooxidans Hbs were obtained from the Institute for Genomic Research website at http://www.tigr.org. Sequence data for B.pertussis, C.diphtheriae, C.jejuni and M.leprae were produced by the Sequencing Group at the Sanger Center and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/cj and ftp://ftp.sanger.ac.uk/pub/pathogens/bp, respectively. Sequence data for Legionella pneumophila were produced by the Columbia Genome Center, and can be obtained from the ‘Unfinished Microbial Genomes Database at NCBI’. Download figure Download PowerPoint TrHbs of 110–130 amino acids per protein chain (Figure 1) have been isolated as monomeric and dimeric species, displaying medium to extreme oxygen affinities, with a case of very high ligand binding cooperativity (Couture et al., 1999a,b). They all show very low amino acid sequence homology to vertebrate and non-vertebrate Hbs, with sequence identities <15% (Couture et al., 1994; Moens et al., 1996). Sequence alignments reveal substantial residue deletions at either N- or C-termini and in the CD–D region of the (non-)vertebrate globin fold. [α-helices building up the globin fold in vertebrate Hbs are conventionally labeled A, B…H according to their sequential order; topological sites are numbered sequentially within each α-helix (Perutz, 1979)]. The distal residue at the E7 position, which is almost invariably His or Gln in (non-)vertebrate Hbs, and which stabilizes the heme-bound ligand through hydrogen bonding, is often Gln in trHbs. However, residues not capable of hydrogen bonding may also be found at the E7 position (Figure 1). The other distal residue, at position B10, is invariably Tyr in trHbs. Its role in affecting ligand stability by complementing the hydrogen bonding capability of the E7 residue has been shown in non-vertebrate Hbs (Yang et al., 1995) and in the trHbs from C.eugametos and from M.tuberculosis (Couture et al., 1999a,b). Nostoc commune trHb, with a His residue at position B10, is the only exception. The structural implications of such a remarkable evolutionary divergence, with respect to the wide and highly characterized family of (non-)vertebrate Hbs, are unknown since no trHb three-dimensional structure has been determined so far. On the other hand, a recent NMR investigation on cyano-met N.commune trHb, focused on the heme crevice structural features, has suggested conservation of the F-, G- and H-helices and of the FG hinge region (Yeh et al., 2000). The functional roles of trHbs are virtually unknown and may be various. Nostoc commune trHb may be a component of a microaerobically induced terminal oxidase (Potts et al., 1992; Thorsteinsson et al., 1999). In the unicellular green alga C.eugametos a trHb is induced in response to activated photosynthesis and is localized, in part, along the thylakoid membranes (Couture et al., 1994). In M.tuberculosis, a trHb has been postulated to be involved in the protection of the bacilli against reactive nitrogen species produced by the host (Couture et al., 1999b). We report here the results of an X-ray crystallographic investigation on the trHbs isolated from the protist P.caudatum (P_trHb; 116 amino acids, in the aquo-met form) and from the green unicellular alga C.eugametos (C_trHb; 121 amino acids, in the cyano-met form), whose three-dimensional structures were determined independently. The emphasis of the discussion is on the unique modifications that are built onto the globin fold of both trHbs, on their heme distal binding sites, and on the structural requirements that allow heme stabilization within a very short, possibly minimal, Hb-like polypeptide chain. Results and discussion Structure determination The crystal structure of P_trHb was solved by means of multiple wavelength anomalous diffraction (MAD) based on the anomalous scattering of the heme Fe atom. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at 100 K. The final protein model was refined at 1.54 Å resolution, with close to ideal stereochemical parameters, to an R-factor of 13.3% (Rfree = 18.3%; see Table I). The electron density accounts for all 116 residues of recombinant P_trHb, and for 207 ordered water molecules. The C_trHb three-dimensional structure was solved by means of multiple isomorphous replacement, based on two heavy atom derivatives (see Table I), using a rotating anode X-ray source at room temperature. The structure was refined at 1.8 Å resolution, using a native data set collected at ESRF, at 100 K, to an R-factor of 17.6% (Rfree = 21.1%) with good overall stereochemistry. The protein model accounts for all 121 residues of the expressed protein, including 186 water molecules. Table 1. Data collection and refinement statistics for P_trHb and C_trHb (A) P_trHb MAD data collection statistics Absorption peak Inflection point Remote Wavelength (Å) 1.739 1.740 0.979 Resolution (Å) 30–2.7 30–2.7 30–1.54 Mosaicity (°) 0.31 0.31 0.31 Completeness (%) 97.6 (91.2)a 97.2 (86.6) 96.4 (88.2) Rmerge (%) 2.2 2.2 2.6 Independent reflections 3681 3723 19397 Average I/σ(I) 32 (13) 32 (13) 31 (7) (B) C_trHb MIR data collection statistics Native UO2(Ac)2 K2PtCl4 Wavelength (Å) 1.00 1.542 1.542 Resolution (Å) 30–1.80 16–3.1 16–3.1 Mosaicity (°) 0.47 0.24 0.76 Completeness (%) 98.6 (99.6) 98.7 (71.3) 95.8 (68.5) Rmerge (%) 5.6 21.7 19.2 Rderiv (%) – 37.0 31.1 Independent reflections 12839 2508 2358 Average I/σ(I) 21 (10) 9 (3) 8 (3) Phasing powerb – 1.59 1.18 Number of sites – 1 2 (C) Refinement statistics and model quality P_trHb C_trHb Resolution range (Å) 30–1.54 30–1.80 Total number of non-hydrogen atoms 881 978 Number of water molecules 207 186 R-factor 0.133 0.176 Rfreec 0.183 0.211 Space group P43 P212121 Unit cell (Å) a = 61.2 a = 34.6 b = 61.2 b = 53.1 c = 35.8 c = 67.2 R.m.s.d. from ideal geometry bond lengths (Å) 0.014 0.018 bond angles (°) 1.57 1.65 Ramachandran plotd most favoured region 96% 99% additional allowed region 4% 1% Averaged B-factors (Å2) main chain 11 15 side chain 14 17 solvent 27 31 heme 13 13 a Outer shell statistics are shown in parentheses. The outer shells are 2.75–2.70 Å for the absorption peak and inflection point, and 1.58–1.54 Å for remote point in P_trHb; and 1.86–1.80 Å for native and 3.15–3.10 Å for heavy atom derivatives in C_trHb. b Phasing power: r.m.s. (|FH|/E), where |FH| is the heavy atom structure factor amplitude and E is the lack of closure (|FPH – FP| – |FH|). c Calculated using 10% of the reflections. d Data produced using the program PROCHECK (Laskowski et al., 1993). The trHb fold P_trHb and C_trHb tertiary structures are very similar (the r.m.s.d. calculated over 107 Cα atom pairs is 0.97 Å), and display a molecular fold based on four main α-helices (see Figure 2). The α-helices around the heme group are arranged in a sort of bundle, composed of two antiparallel helix pairs (labeled B/E and G/H in Figure 2A), connected by an extended polypeptide loop. If attention is focused on the protein chain only, some structural resemblance to the ferritin subunit fold may be envisaged (Hempstead et al., 1997). Nevertheless, the secondary structure elements are arranged in different topological order in the two protein families. More properly, the trHb fold can be recognized as a subset of the classical ‘three-over-three’ α-helical sandwich (Holm and Sander, 1993), trimmed to a domain essentially composed of helices B, E, G and H, in a ‘two-over-two’ arrangement. Structural superpositions of P_trHb and C_trHb with sperm whale myoglobin (Mb; limited to the common α-helical regions, 59 Cα atom pairs) yield r.m.s.d. values in the 2.3 Å range (Figure 2B). On the other hand, superpositions with Vitreoscilla sp. Hb, the only other bacterial Hb structure known (Tarricone et al., 1997; Bolognesi et al., 1999a) (r.m.s.d. values of ∼2.8 Å; Figure 2C), are limited to 44 Cα pairs only, due to different α-helix orientations. Both sets of comparison highlight extensive modifications of the trHb fold with respect to more conventional Hb folds, particularly in the A-helix and in the CD–D regions, which are virtually absent, and in the EF–F regions (see below). Figure 2B and C show that from the tertiary structure viewpoint the protein region distal to the heme is more conserved in trHbs than the proximal half of the molecule, which appears substantially trimmed as compared with (non-)vertebrate and other bacterial Hbs. Figure 2.(A) A ribbon stereo view of P_trHb tertiary structure, including the heme group. α-helices are labeled according to the conventional globin fold nomenclature (Perutz, 1979). The protein loop identified as ‘pre-F’ in the text immediately follows the labeled E–F region, and precedes the F one-helical turn on the heme proximal side (left in the figure). (B) A stereo view of the structural overlay of C_trHb (green) and sperm whale Mb (red). The protein molecules are oriented approximately as in (A). The heme group of sperm whale Mb has been omitted for clarity. (C) Overlay of P_trHb (light blue) and the A chain of the homodimeric Vitreoscilla Hb (yellow, PDB code 1vhb), in approximately the same orientation as (A) and (B). The heme group of Vitreoscilla Hb is also included. All figures were drawn with MOLSCRIPT (Kraulis, 1991). Download figure Download PowerPoint Structural features of trHbs on the heme distal side In both trHbs, the regular structure achieved along the B- and E-helices allows one to identify several key residues relative to the heme distal ligand binding site. Residue TyrB10, which is buried in the inner part of the heme pocket and properly oriented through hydrogen bonds to residues GlnE7 and Thr/GlnE11 in P_trHb/C_trHb, respectively (in the following, unless specified otherwise, whenever residues belonging to the same topological position in P_trHb and C_trHb, respectively, are different they are explicitly listed and separated by a solidus), provides stabilization of the heme-bound distal ligand (see Figure 3). In fact, TyrB10 is strongly hydrogen bonded to a heme-coordinated water molecule in P_trHb (2.76 Å) and to the distal N atom of the heme iron-bound cyanide in C_trHb. However, the cyanide electron density and its coordination geometry (see Table II) suggest that the diatomic ligand may be present in more than one binding mode, indicative of X-ray-induced (partial) heme Fe reduction (Bolognesi et al., 1999b). The interaction of TyrB10 with the distal ligand(s) bears particular functional relevance because a distal HisE7 residue is absent in all trHbs, with the only exception being the trHb identified in the C.jejuni genome (see Figure 1). Figure 3.Stereo view of the heme distal site main residues, together with the B-, C- and E-helices, in aquo-met P_trHb (A) and cyano-met C_trHb (B). Hydrogen bonds within the distal residues cluster, including the heme ligand, are indicated with dashed lines; for reference, the PheCD1 residue is included. In both views the heme is edge on, and the protein moiety has been partly rotated along the horizontal axis with respect to the orientation adopted for Figure 2. Download figure Download PowerPoint Table 2. Coordination geometry at the Fe(III) heme centers Coordination bond Distance (Å) P_trHb average Fe–N(pyrrole) 2.01 HisF8 NE2 —Fe 2.14 Fe–O(ligand) 2.09 C_trHb average Fe–N(pyrrole) 1.98 HisF8 NE2–Fe 2.18 Fe–C(ligand)a 2.65 Angle (°) Fe–C–Na 130 a Owing to partial reduction of the heme Fe center, during the X-ray data collection stage, the cyanide coordination geometry reflects a structural average between a regularly Fe(III) coordinated ligand and Fe(II) unbound species, trapped in the distal site cavity. The B-factors for cyanide C and N atoms are 15 and 21 Å2, respectively. The GlnE7 side chain is pointing into the distal site cavity, providing one additional hydrogen bond to the heme-bound ligand in P_trHb [GlnE7 NE2–O(water) 2.73 Å] and in C_trHb [GlnE7 NE2–N(cyanide) 2.92 Å; see Figure 3]. Next, Thr/GlnE11, located one α-helical turn from the distal E7 residue and in contact with the heme, are moderately polar residues that are never found at this topological site in (non-)vertebrate Hbs, where they are invariantly of hydrophobic nature (Val, Leu, Ile) (Bashford et al., 1987; Kapp et al., 1995; Bolognesi et al., 1997). In both trHbs, the E11 residue is hydrogen bonded to the TyrB10 side chain (2.59 and 2.95 Å in P_trHb and C_trHb, respectively). Through minor fluctuations (<0.4 Å) from the observed conformation, residue E11 may further extend the interlaced distal site hydrogen-bonded network, which includes TyrB10, GlnE7, residue E11 and the heme-bound ligand. No other species (e.g. H2O or acetate) is present in the two distal sites. In both trHbs, residue LysE10 is solvent exposed, but bent towards the heme propionates, with which it is electrostatically linked. Particularly in C_trHb, LysE10 is also hydrogen bonded to the A propionate carboxylate (2.82 Å; see Figure 4). The strong conservation of a basic residue at this site may suggest a role in heme stabilization, a particularly relevant function in this Hb subfamily where the heme:protein interactions on the proximal side are substantially modified (see below). Indeed, heme dissociation from N.commune trHb has been evaluated as being ∼100-fold faster than in sperm whale Mb (Thorsteinsson et al., 1999). Figure 4.A stereo view of the hydrophobic cluster preventing solvent access to the heme pocket from the pre-F region, in C_trHb. The figure includes the heme group, the E-helix, the pre-F loop and the F α-helical turn. Residues LysE10 and Lys62, which are electrostatically linked to the heme propionates, are also displayed. Download figure Download PowerPoint Residue PheE14 is close to the EF interhelical region and is almost orthogonal to the porphyrin ring, at the heme CHD methinic bridge in both trHbs (Figure 4). This arrangement provides a rigid closure to the heme pocket, making use of a bulky aromatic residue that is strictly conserved throughout the known trHb amino acid sequences (Figure 1). Such a structural feature may be dictated by the specific orientation of the E-helix, caused by the extremely short segment following the C region, and by the virtual absence of the F-helix in trHbs (Figure 2). It can be suggested that PheE14 is serving a structural role comparable to that ascribed to PheCD1 in (non-)vertebrate and bacterial Hbs, shielding the heme from the solvent at a porphyrin ring location remote and almost opposite to CD1. Furthermore, residue Trp59 (in both trHbs), in the proximal pre-F segment, is in van der Waals contact with and orthogonal to the side chain of PheE14. Conservation of an aromatic residue at sequence site 59 is a key primary structure feature in all the trHbs (Figure 1), suggesting that both this and the E14 side chains play a relevant role in preventing solvent-induced heme oxidation. Despite the fact that both P_trHb and C_trHb display TyrB10 and GlnE7 distal residues, and share a comparable distal site structure, they display very different O2 affinities (P50 values are 0.49 and <0.005 mm Hg for P_trHb and C_trHb, respectively) (Couture et al., 1999a). This suggests that other factors may play a role in governing the overall ligand affinity of the two trHbs, such as kinetics (Couture et al., 1999a), or the ligand access, through protein motions, to secondary docking sites (Brunori et al., 1999a, b Chu et al., 2000). It may be noted that the hydrogen bonds and contacts within the C_trHb distal site are looser than in P_trHb despite the presence of a diatomic ligand (cyanide) bulkier than the distal water molecule of aquo-met P_trHb. This observation should be related to the known capability of ferric C_trHb to bind unusual ligands such as β-mercaptoethanol or dithiothreitol (Couture and Guertin, 1996), or to form a 6-coordinate low-spin heme complex with TyrB10, at alkaline pH (Das et al., 1999). Formation of the latter complex requires the TyrB10 phenolic O atom to shift by 3.5 Å with respect to its position in the cyano-met complex, indicating a remarkable distal site structure adaptability to the incoming ligand(s) in C_trHb. In this respect, engineering of distal site residues in sperm whale Mb (bearing LeuB10 in the wild-type species) has shown that the precise mutual orientation of the supporting α-helices is a primary factor in making TyrB10 available for distal ligand stabilization through direct hydrogen bonding (Brunori et al., 1999a). A distal site structural organization reminiscent of that of the two trHbs has previously been observed in Ascaris suum Hb, which displays TyrB10 and GlnE7 hydrogen bonded to the heme coordinated dioxygen, but hosts an apolar Ile residue at the E11 site (Yang et al., 1995). Remarkably, Ascaris Hb displays a very high oxygen affinity (P50 = 0.004 mm Hg) and has recently been proposed to be a true enzyme, acting as an NO-activated deoxygenase (Minning et al., 1999). Unique features in the trHb fold Among the many structural features of trHbs that deviate from the conventional globin fold, the deletion of the A-helix and the presence of an extended loop substituting for most of the F-helix deserve particular consideration. Indeed, these two features may be structurally correlated. A block deletion of 11 residues in the N-terminal A-helix region (see Figures 1 and 2) might appear to threaten protein stability, since anchoring of the N-terminal region to the EF corner through a conserved hydrophobic contact could be impaired (Lesk and Chothia, 1980; Bashford et al., 1987). Nevertheless, a compact trHb molecule with efficient sealing of the proximal side of the heme pocket is achieved through a hydrophobic cluster located in the AB topological region, previously identified in non-vertebrate globin structures (Bolognesi et al., 1997). In fact, the conservation of just the last A-helix turn allows the trHb N-terminus to lock onto the protein core, through clustering of residues LeuA11, PheA12, LeuA15, Val/AlaB5, LeuE15, LeuE19 and Val/IleH4. Inspection of the aligned sequences of trHbs shows that residue A12 is always either Phe or Tyr, and that a Gly–Gly motif, coding the required structural adaptability with respect to (non-)vertebrate Hbs, is strongly conserved in the AB region (Figure 1). In both trHbs the N-terminal dipeptide (topological sites A10–A11) runs antiparallel to the EF corner Leu(53)–Gly(54) segment, allowing hydrogen bonding between the protein backbone atoms of the two segments and further anchoring of the N-terminal region to the protein core. As a result of such antiparallel pairing, the protein backbone following the linear Leu(53)–Gly(54)–Gly(55)–Pro/Ala(56) EF peptide protrudes markedly from the protein core, giving rise to a unique proximal side structural arrangement (see Figures 2, 4 and 5). The Ramachandran φ,ψ angles adopted by residues 54–55 in both trHbs indicate the strict requirement for such a Gly–Gly sequence motif to code for their backbone conformation. The key structural role played by these residues in the trHb fold is further stressed by the conservation of the Gly–Gly–(Pro) motif in the EF region of the aligned amino acid sequences (see Figure 1). Figure 5.Proximal side of the heme in P_trHb, displaying the one-turn F-helix, residues LeuF4 and HisF8, together with the heme group. The figure portrays part of the heme crevice, defined by the C-helix (PheC7 and PheCD1 are shown), and by segments of the G-helix and of the H-helix (ValH18 and the C-terminus are shown). For reference, residue PheE14, in the lower part of the heme pocket, is included. Download figure Download PowerPoint The seven residues following the EF corner trace a wide loop structure (the ‘pre-F loop’), which provides contacts with the C-terminal half of the E-helix and specific interactions with the porphyrin group. A single turn of α-helical conformation is achieved only at residues Leu/MetF4–HisF8, proximal to the heme iron atom (see Figures 4 and 5). The absence of essentially the whole F-helix on the proximal side of the heme h