Martino Bolognesi

AIM: Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. METHODS AND RESULTS: In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. CONCLUSION: This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.

OBJECTIVES: Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. METHODS: Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. RESULTS: Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC₅₀ values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. CONCLUSIONS: The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

truncated hemoglobin hemoglobin myoglobin Truncated hemoglobins (trHbs)1 (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar) constitute a family of small oxygen-binding heme proteins distributed in eubacteria, cyanobacteria, protozoa, and plants (Table I, Supplemental Material) forming a distinct group within the hemoglobin (Hb) superfamily (2Moens L. Vanfleterin J. Van de Peer Y. Peeters K. Kapp O. Czelusniak J. Goodman M. Vinogradov S.N. Mol. Biol. Evol. 1996; 13: 324-333Google Scholar). They are nearly ubiquitous in the plant kingdom, occur in many aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been detected in the genomes of archaea or metazoa. Characteristically, trHbs occur at nano- to micromolar intracellular concentration, hinting at a possible role as catalytic proteins. Many trHbs display amino acid sequences 20–40 residues shorter than non-vertebrate hemoglobins to which they are scarcely related by sequence similarity. Crystal structures (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar) show that trHb tertiary structure is based on a 2-on-2 α-helical sandwich, which represents an unprecedented editing of the highly conserved globin fold. Moreover, an almost continuous hydrophobic tunnel, traversing the protein matrix from the molecular surface to the heme distal site, may provide a path for ligand diffusion to the heme. More than 40 putative trHb genes have been identified (Table I, Supplemental Material). A phylogenetic analysis (Fig.1) indicates that trHbs form a distinct family separate from bacterial flavohemoglobins,Vitreoscilla Hb, plant symbiotic and non-symbiotic hemoglobins, and animal hemoglobins. Notably, trHb genes and flavohemoglobin genes frequently coexist in the same bacterium (Table I, Supplemental Material), suggesting distinct functions for each. Three distinct groups (groups I, II, and III) can be distinguished within the trHb family with four clusters within group II. The extent of amino acid identity between members of the different groups,e.g. Mycobacterium tuberculosis trHbO (group II) and trHbN (group I), can be low (18%) (Fig. 1, Supplemental Material). However, identity rises dramatically when the M. tuberculosis trHbO sequence is compared with the orthologue sequences from Mycobacterium avium (84%),Mycobacterium leprae (83%), Cornybacterium diphterae (64%), and Streptomyces coelicolor (66%) or when the M. tuberculosis trHbN is compared with its M. avium orthologue (79%). The presence of trHb genes from groups I, II, and III in M. avium (Gram-positive) and inMethylococcus capsulatus (proteobacteria) may indicate that the different trHb lineages diverged from their last common ancestor before the separation of the main prokaryotic lineages (Fig. 1). An interesting progression is found in the genusMycobacterium. The genome of the opportunistic pathogen,M. avium, contains one trHb from each group of the family, trHbP (group III), trHbO (group II), and trHbN (group I) (Fig. 1). The facultative intracellular pathogen, M. tuberculosis, that infects man, has two, trHbN and trHbO, and the obligate intracellular pathogen, M. leprae, which has undergone extensive reductive evolution and is thought to have only a minimal gene set for a pathogenic mycobacterium (4Cole S.T. Eiglmeier K. Parkhill J. James X.D. Thomson N.R. Wheeler P.R. Honore N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin X. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.-A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Google Scholar), retains solely trHbO, which accordingly may play an essential role. Crystal structures of trHbs from Chlamydomonas eugametos, Paramecium caudatum, and M. tuberculosis show that their three-dimensional fold is based on a subset of the classical globin fold (the so-called 3-on-3 α-helical sandwich). In trHbs the antiparallel helix pairs B/E and G/H are the main secondary structure elements arranged in a 2-on-2 sandwich (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar) (Fig. 2). Within the trHb fold the N-terminal A helix is almost completely deleted, and the whole CD-D region is trimmed to about 3 residues, possibly the minimum polypeptide stretch to bridge between C- and E-helices. Moreover, most of the heme-proximal F-helix is substituted by a polypeptide segment (pre-F) in extended conformation, followed by the one-turn F-helix that properly supports the HisF8 residue, allowing heme iron coordination. Thus, the trHb polypeptide chain is not simply a truncated version of a conventionally folded globin. Rather, it owes its conformational stability to residue deletions and substitutions at specific sites, as compared with non-vertebrate globins (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar). Specific sequence motifs support attainment of the compact trHb fold. Among these, three glycine motifs (present in groups I and II), located at the AB and EF interhelical corners and immediately before the one-turn F-helix, help the pre-F segment to build a properly structured heme crevice within a very short polypeptide chain. Heme isomerism has been reported in trHbs (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 5Lecomte J.T.J. Scott N.L. Vu B.C. Falzone C.J. Biochemistry. 2001; 40: 6541-6552Google Scholar). Very few amino acids are strictly conserved throughout the known trHb sequences, the proximal HisF8 being the only invariant residue. A Phe-Tyr pair is strongly conserved at the B9-B10 sites, where Tyr-B10 participates in heme ligand stabilization. Site CD1, invariably Phe in non-vertebrate Hbs, hosts Phe, Tyr, or His, whereas at least six different residue types occupy the distal E7 position. Moreover, the almost invariant Phe-E14, located along the heme distal face, may be related to a heme/solvent shielding role together with apolar residues of the pre-F segment. Distal pocket residues may ligate the heme iron to form 6-coordinate, low spin, structures. Residue Tyr-B10 has been suggested to play this role in ferric C. eugametos trHb (6Das T.K. Couture M. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. Biochemistry. 1999; 38: 15360-15368Google Scholar); His-E10 of Synechocystis sp. trHb may ligate the iron of ferric and ferrous species (7Couture M. Das T.K. Savard P.Y. Ouellet Y. Wittenberg J.B. Wittenberg B.A. Rousseau D.L. Guertin M. Eur. J. Biochem. 2000; 267: 4770-4780Google Scholar, 8Scott N.L. LeComte J.T.J. Protein Sci. 2000; 9: 587-597Google Scholar, 9Hvitved A.N. Trent III, J.T. Premer S.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 34714-34721Google Scholar). These structures are in equilibrium with 5-coordinate or aquoferric forms with which exogenous ligands react. At high ligand concentration, where conversion of the 6- to the 5-coordinate form becomes rate-limiting, the rates of ligand combination give a measure of the rate of conversion (9Hvitved A.N. Trent III, J.T. Premer S.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 34714-34721Google Scholar, 10Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Google Scholar). If this rate is low, about 30 s−1 as in Synechocystissp. trHb (7Couture M. Das T.K. Savard P.Y. Ouellet Y. Wittenberg J.B. Wittenberg B.A. Rousseau D.L. Guertin M. Eur. J. Biochem. 2000; 267: 4770-4780Google Scholar, 9Hvitved A.N. Trent III, J.T. Premer S.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 34714-34721Google Scholar), the 6-coordinate form is strongly favored. In the instance of C. eugametos trHb, where the rate of this conversion in the ferrous alkaline form is 5-fold higher, 6- or 5-coordinate species prevail at different pH (10Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Google Scholar). Five-coordinate, high spin ferrous species of P. caudatum (11Das T.K. Weber R.E. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Van Hauwaert M.L. Moens L. Rousseau D.L. Biochemistry. 2000; 39: 14330-14340Google Scholar), M. tuberculosis trHbN (12Couture M. Yeh S-R. Wittenberg B.A. Wittenberg J.B. Ouellet Y. Rousseau D.L. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11223-11228Google Scholar), and M. tuberculosistrHbO 2M. Guertin and S. R. Yeh, unpublished observations. predominate at all pH. A 6-coordinate form of Arabidopsis thaliana deoxy-trHb is short-lived and reverts to a 5-coordinate species (13Watts R.A. Hunt P.W. Hvitved A.N. Hargrove M.S. Peacock W.J. Dennis E.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10119-10124Google Scholar). In the known trHb three-dimensional structures the proximal His-F8 imidazole ring is markedly staggered relative to the heme pyrrole N atoms (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar, 14Yeh D.C. Thorsteinsson M.V. Bevan D.R. Potts M. La Mar G.N. Biochemistry. 2000; 39: 1389-1399Google Scholar). This together with the high value of His-F8 NE2-Fe stretching frequencies (220–232 cm−1) (10Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Google Scholar, 11Das T.K. Weber R.E. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Van Hauwaert M.L. Moens L. Rousseau D.L. Biochemistry. 2000; 39: 14330-14340Google Scholar,15Yeh S.R. Couture M. Ouellet Y. Guertin M. Rousseau D.L. J. Biol. Chem. 2000; 275: 1679-1684Google Scholar) 3Although M. tuberculosis trHbN was originally proposed to be a cooperative dimer with n = 2 (12Couture M. Yeh S-R. Wittenberg B.A. Wittenberg J.B. Ouellet Y. Rousseau D.L. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11223-11228Google Scholar), ongoing investigations indicate that oxygenation of this trHb may not be cooperative (M. Guertin, unpublished observations). indicates an unstrained proximal His, whose coordination to the heme iron facilitates a heme in-plane location of the iron atom that in turn supports fast oxygen association and electron donation to the bound distal ligand (16Bolognesi M. Bordo D. Rizzi M. Tarricone C. Ascenzi P. Prog..Biophys. Mol. Biol. 1997; 68: 29-68Google Scholar). Moreover, the O-O stretching frequencies measured for oxygenatedC. eugametos, Synechocystis sp., and M. tuberculosis trHbO (1136, 1133, and 1140 cm−1, respectively) (17Das T.K. Couture M. Ouellet Y. Guertin M. Rousseau D.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 479-484Google Scholar)2 are consistent with a ferric superoxide character of the heme-ligand pair. The heme distal site of trHb is characterized by the nearly invariant Tyr-B10, the main residue providing direct hydrogen bonding to the heme-bound ligand (as observed in aquo-Met P. caudatum trHb, cyano-Met C. eugametos trHb, andM. tuberculosis oxy-trHbN (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar, 11Das T.K. Weber R.E. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Van Hauwaert M.L. Moens L. Rousseau D.L. Biochemistry. 2000; 39: 14330-14340Google Scholar) (see Fig.3). A distal network of H-bonds has been shown to stabilize the ligated O2 in M. tuberculosis trHbN (with Leu at the E7 site) through direct interaction with the Tyr-B10 phenolic oxygen atom and hydrogen bonding of this oxygen atom to Gln-E11 (3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar).3 Analysis of the crystal structures shows that distal site networks based on Tyr-B10 ligand and Tyr-B10-Gln/Thr-E11 hydrogen bonds are conserved in ferric cyano-Met C. eugametos and aquo-Met P. caudatumtrHbs, respectively (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar). Additionally, the latter ferric trHbs display hydrogen bonds between Gln-E7 and the heme ligand, indicating that polar E7 residues are effective in ligand stabilization and that hydrogen bonding between E7 and Tyr-B10 may also contribute to effective structuring of the distal site residues. In this respect, it should be noted that the occurrence of small apolar residues (Ala, Gly) at the E7 site may be compensated by CD1 Phe-224 Tyr/His mutations such as observed in different trHbs of group II (Fig. 1, Supplemental Material). The role played by residues Tyr-B10 and Gln-E7 in ligand stabilization is further stressed by mutational studies (10Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Google Scholar, 12Couture M. Yeh S-R. Wittenberg B.A. Wittenberg J.B. Ouellet Y. Rousseau D.L. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11223-11228Google Scholar, 15Yeh S.R. Couture M. Ouellet Y. Guertin M. Rousseau D.L. J. Biol. Chem. 2000; 275: 1679-1684Google Scholar) and by results of Das et al. (17Das T.K. Couture M. Ouellet Y. Guertin M. Rousseau D.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 479-484Google Scholar) on the simultaneously observed O-O and Fe-O stretching frequencies in C. eugametos andSynechocystis sp. trHbs. It should also be noticed that the trHb hydrogen-bonded network centered on Tyr-B10 is strongly reminiscent of that observed for the very high oxygen affinity Hb from the nematode Ascaris suum (18Yang J. Kloek A.P. Goldberg D.E. Matthews F.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4224-4228Google Scholar, 19Peterson E.S. Huang S. Wang J. Miller L.M. Vidugiris G. Kloek A.P. Goldberg D.E. Chance M.R. Wittenberg J.B. Friedman J.M. Biochemistry. 1997; 36: 13110-13121Google Scholar). In these cases, extraordinarily low oxygen dissociation rates (0.004–0.0014 s−1) result in a high oxygen affinity (0.004–0.005 mm Hg) (9Hvitved A.N. Trent III, J.T. Premer S.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 34714-34721Google Scholar, 10Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Google Scholar, 20Gibson Q.H. Smith M.H. Proc. R. Soc. Lond. B Biol. Sci. 1965; 163: 206-214Google Scholar). The Fe-OO stretching frequency of P. caudatum oxy-trHb detected by resonance Raman spectroscopy indicates strong polar interactions (including hydrogen bonding) between the bound ligand and the nearby Tyr-B10 residue, implying slow dissociation and high ligand affinity (11Das T.K. Weber R.E. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Van Hauwaert M.L. Moens L. Rousseau D.L. Biochemistry. 2000; 39: 14330-14340Google Scholar). In addition, the two Fe-CO stretching frequencies observed in the resonance Raman spectra of both M. tuberculosis trHbN-CO and Ascaris Hb-CO derivatives indicate two conformers expected to display slow and rapid ligand dissociation rates, respectively (15Yeh S.R. Couture M. Ouellet Y. Guertin M. Rousseau D.L. J. Biol. Chem. 2000; 275: 1679-1684Google Scholar, 21Das T.K. Friedman J.M. Kloek A.P. Goldberg D.E. Rousseau D.L. Biochemistry. 2000; 39: 837-842Google Scholar). The actual dissociation of bound O2 or CO is attributed to a fast equilibrium between two conformers, with the ligand off-rate being determined either by the rate of conformer interconversion or by ligand dissociation from the conformer with the higher rate. Because of the orientation of the CD-D region, the E-helix of trHb falls close to the distal face of the heme. Crowding by distal residues and their interactions with the heme block access to the distal site cavity through the classical E7 residue gate, typically achieved in vertebrate Hbs by His-E7 (22Bolognesi M. Cannillo E. Ascenzi P. Giacometti G.M. Merli A. Brunori M. J. Mol. Biol. 1982; 158: 305-315Google Scholar, 23Scott E.E. Gibson Q.H. Biochemistry. 1997; 36: 11909-11917Google Scholar, 24Das T.K. Couture M. Guertin M. Rousseau D.L. J. Phys. Chem. B. 2000; 104: 10750-10756Google Scholar, 25Scott E.E. Gibson Q.H. Olson J.S. J. Biol. Chem. 2001; 276: 5177-5188Google Scholar). Remarkably, however, a different route for ligand diffusion to/from the heme appears to be coded in trHb structures as a cavity network or tunnel through the protein matrix. InC. eugametos trHb and M. tuberculosis trHbN, the tunnel is composed of two roughly orthogonal branches converging at the heme distal site from two distinct protein surface access sites. On one hand, a 20-Å long tunnel branch connects the globin region nestled between the AB and GH corners to the heme distal site. On the other, a path of about 8 Å connects an opening in the protein structure between G- and H-helices to the heme. The tunnel branches display inner diameters in the 5–7 Å range for a ligand-accessible volume of 330–360 Å3 (1Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar). Residues lining the tunnel branches are hydrophobic and are substantially conserved throughout the trHb family, suggesting that the tunnel plays a functional role and is suited for small nonpolar ligand diffusion or storage. A study of ligand rebinding following photolysis of CO in C. eugametosor P. caudatumtrHbs 4U. Samuni, D. Dansker, A. Ray, L. Moens, M. Guertin, and J. F. Friedman, manuscript in preparation. suggests that the tunnel/cavity network in these proteins may indeed act as a CO store whose filling strikingly affects ligand rebinding kinetics. Protein cavities, accessible to xenon atoms in Mb, have been shown to act dynamically as CO secondary docking sites. This has led to the suggestion that protein cavities modulate ligand dynamics and reactivity (Refs. 26Brunori M. Gibson Q.H. EMBO Rep. 2001; 2: 674-679Google Scholar and 27Frauenfelder H. McMahon B.H. Austin R.H. Chu K. Groves J.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2370-2374Google Scholar, and references therein). On the other hand, protein matrix tunnels connect the surface to active sites in Ni-Fe hydrogenases (28Montet Y. Amara P. Volbeda A. Vernede X. Hatchikian E.C. Field M.J. Frey M. Fontecilla-Camps J.C. Nat. Struct. Biol. 1997; 4: 523-526Google Scholar), in methane monooxygenase hydroxylase (29Whittington D.A. Rosenzweig A.C. Frederick C.A. Lippard S.J. Biochemistry. 2001; 40: 3476-3482Google Scholar), and in carbon monoxide dehydrogenase (30Dobbek H. Svetlitchnyi V. Gremer L. Huber H. Meyer O. Science. 2001; 293: 1281-1285Google Scholar). Tunnels serve for internal substrate translocation in some enzymes (31Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Google Scholar). Different residues may modulate ligand diffusion processes along the trHb tunnel. For instance, Phe-E15 of M. tuberculosis trHbN is observed in two distinct conformations in the crystal structure and may act as a gate controlling ligand diffusion along the main tunnel branch (3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Google Scholar). The functional roles of trHbs are little known and may be various. The trHb of the unicellular green alga C. eugametos is induced in response to active photosynthesis and is localized, in part, along the chloroplast thylakoid membranes (32Couture M. Chamberland H. St.-Pierre B. Lafontaine J. Guertin M. Mol. Gen. Genet. 1994; 243: 185-197Google Scholar). The soluble trHb of the cyanobacterium Nostoc commune is localized on the cytoplasmic face of the cell membrane and is expressed only under anaerobic conditions (33Hill D.R. Belbin T.J. Thorsteinsson M.V. Bassam D. Brass S. Ernst A. Boger P. Paerl H. Mulligan M.E. Potts M. J. Bacteriol. 1996; 178: 6587-6598Google Scholar, 34Thorsteinsson M.V. Bevan D.R. Potts M. Biochemistry. 1999; 38: 2117-2126Google Scholar). In addition, the gene encoding this trHb is coexpressed with genes of the nitrogen fixation complex (34Thorsteinsson M.V. Bevan D.R. Potts M. Biochemistry. 1999; 38: 2117-2126Google Scholar). Oxygen supply to the mitochondria of the protozoan Paramecium is impeded by levels of CO sufficient to block trHb but not cytochrome oxidase (35Wittenberg J.B. Mangum C.P. Advances in Comparative and Environmental Physiology: Oxygen Carriers in Blood and Tissues. 13. Springer-Verlag New York Inc., New York1991: 59-85Google Scholar). There is a great deal of evidence that NO generated by nitric-oxide synthase II in macrophages controls the development of M. tuberculosis infection in mouse and man and restricts the bacteria to a latent state (36Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Google Scholar). However, tuberculous infection is in a dynamic balance that teeters for years in a competition between host immunity and M. tuberculosis growth, indicating the presence of an endogenous mechanism for NO resistance in the tubercle bacillus. That the oxygenated form of trHbN may be involved in the protection of the bacilli against NO produced in the granuloma during latency is supported by the observation that Mycobacterium bovis BCG cells that no longer express trHbN are severely impaired in their ability to metabolize NO in vitro. 5M. Guertin, unpublished results. Oxy-trHbN could scavenge NO in vitro by promoting dioxygenation as observed in human oxy-Hb, oxy-Mb, and Escherichia coliflavohemoglobin, in which NO is converted to nitrate by reaction with the oxygenated heme (37Eich R.F. Li T. Lemon D.D. Doherty D.H. Curry S.R. Aitken J.F. Mathews Smith G.N. Olson J.S. Biochemistry. 1996; Scholar, P.R. Proc. Natl. Acad. Sci. U. S. A. Scholar, S. M. T. Biochemistry. 2001; 40: Scholar). The indicate that the dramatically 2-on-2 version of the globin fold observed in trHbs may functions distinct from O2 or In the combination of a distal site with the presence of an protein matrix tunnel at internal ligand diffusion different from based on the E7 distal gate in observed in are as ligand secondary docking sites, the trHb protein matrix tunnel may not only for heme but also for of O2 Notably, in and the tunnel in trHbs may their and relative to the globin are The a of the globin with on Hb structure and on functions within the Hb