A TyrCD1/TrpG8 hydrogen bond network and a TyrB10—TyrCD1 covalent link shape the heme distal site of Mycobacterium tuberculosis hemoglobin O

Abstract

Truncated hemoglobins (Hbs) are small hemoproteins, identified in microorganisms and in some plants, forming a separate cluster within the Hb superfamily. Two distantly related truncated Hbs, trHbN and trHbO, are expressed at different developmental stages in Mycobacterium tuberculosis. Sequence analysis shows that the two proteins share 18% amino acid identities and belong to different groups within the truncated Hb cluster. Although a specific defense role against nitrosative stress has been ascribed to trHbN (expressed during the Mycobacterium stationary phase), no clear functions have been recognized for trHbO, which is expressed throughout the Mycobacterium growth phase. The 2.1-Å crystal structure of M. tuberculosis cyano-met trHbO shows that the protein assembles in a compact dodecamer. Six of the dodecamer subunits are characterized by a double conformation for their CD regions and, most notably, by a covalent bond linking the phenolic O atom of TyrB10 to the aromatic ring of TyrCD1, in the heme distal cavity. All 12 subunits display a cyanide ion bound to the heme Fe atom, stabilized by a tight hydrogen-bonded network based on the (globin very rare) TyrCD1 and TrpG8 residues. The small apolar AlaE7 residue leaves room for ligand access to the heme distal site through the conventional ''E7 path,'' as proposed for myoglobin. Different from trHbN, where a 20-Å protein matrix tunnel is held to sustain ligand diffusion to an otherwise inaccessible heme distal site, the topologically related region in trHbO hosts two protein matrix cavities.T runcated hemoglobins (trHbs) are a class of small oxygenbinding hemoproteins, dispersed in eubacteria, cyanobacteria, protozoa, and plants, recently recognized as a separate cluster within the hemoglobin (Hb) superfamily. On the basis of amino acid sequence analysis, three phylogenetic groups (groups I, II, and III) have been identified within the trHb family; some organisms contain genes from more than one group, suggesting different functions for trHbs belonging to the diverse groups (1). Crystal structures of three group I trHbs (2, 3) revealed that trHbs are clearly not just another variation on the motif of vertebrate myoglobin (Mb) and Hb. Neither are they similar to nonvertebrate Hbs, including the heme-containing domain of flavohemoglobins, nor to the plant symbiotic and nonsymbiotic Hbs (4-10). Major structural differences associated with known trHbs are an unprecedented 2-on-2 ␣-helical sandwich fold, resulting from striking editing of the classical 3-on-3 globin ␣-helical sandwich, and an extended hydrophobic tunnel͞cavity network linking the solvent space and the distal heme pocket (1-3). Much smaller and topologically unrelated cavities, known by their ability to incorporate Xe atoms, have been found in Mb and interpreted as temporary ligand-docking sites (11,12). In trHbs, the positioning and size of the hydrophobic tunnel suggest important roles in controlling ligand access to the heme, in ligand storage, and͞or accumulation (1-3).An additional major diffe...

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

“…In fact, a hydrogen bonding network involving distal Tyr and Trp residues stabilizes the heme-bound ligand in Mycobacterium tuberculosis truncated hemoglobin O (trHbO) [45,46]. Furthermore, Tyr145 stabilizes the heme-bound NO in soluble guanylyl cyclase (sGC) by hydrogen bonding [47].…”

Purification and Spectroscopic Characterization of Ctb, a Group III Truncated Hemoglobin Implicated in Oxygen Metabolism in the Food-Borne Pathogen Campylobacter jejuni

“…The most striking feature is the CD1 residue, which is not Phe as in almost all globins studied to date, but Tyr-36 (35). Furthermore, in the dodecameric crystalline state, the B10 Tyr is covalently bonded to the CD1 Tyr in six of the subunits, whilst in the remaining six the residue side chains are in intimate contact but without the covalent linkage (15). This unique CD1/B10 pair creates a rigid distal environment for heme-bound ligands.…”

Protein structure in the truncated (2/2) hemoglobin family

“…GlnE11, TrpG8, and ThrE7 complete the distal site polar cage, with GlnE11 side chain and the TrpG8 indolic nitrogen atom hydrogen bonded to the heme ligand (14). The occurrence of TyrCD1, however, is sufficient to drastically modify the hydrogen bonding distal network in Mt-2/2HbO, where TyrCD1, not TyrB10, is the residue hydrogen bonding to the heme diatomic ligand (13). Further stabilizing polar interactions are provided in Mt-2/2HbO by TrpG8, whose indole NE1 atom is hydrogen bonded to the heme-bound ligand and to TyrCD1 OH; notably, residue E11 is Leu.…”

“…Among the family-conserved structural features we notice a drastically shortened A-helix (fully deleted in group III), the absence of a D-helix, an extended polypeptide segment (pre-F), followed by a short F-helix (one helix turn in groups I and III) supporting the heme proximal HisF8 residue (8,13,17) (Fig. 1).…”

“…(Ss-2/2HbN) (10 -12), four group II HbOs from M. tuberculosis (Mt-2/ 2HbO) (13), from Bacillus subtilis (Bs-2/2HbO) (14), from Thermobifida fusca (15), from Geobacillus stearothermophilus (16), and one group III HbP from C. jejuni (Cj-2/ 2HbP) (17).…”