Nitrosylation mechanisms of Mycobacterium tuberculosis and Campylobacter jejuni truncated hemoglobins N, O, and P

Abstract

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tuberculosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.

Figure 1. Consensus phylogenetic tree of major globin groups from the three kingdoms of life.

This phylogenetic tree was based on the alignment of 150 sequences representing the following groups of globins: 10 plant non symbiotic 3/3 Hbs, 5 plant symbiotic 3/3 Hbs, 15 bacterial 3/3 globin-coupled sensors (GCSs), 4 3/3 protoglobins (Pgbs), 9 bacterial 2/2 Hb1s, 19 bacterial 2/2 Hb2s, 10 bacterial 2/2 Hb3s, 2 Chlamydomonas reinhardtii 2/2 Hbs, 2 ciliate 2/2 Hbs, 3 plant 2/2 Hbs, Thalassiosira pseudonana 2/2 Hb, 20 bacterial 3/3 flavohemoglobins (FHbs), 19 bacterial 3/3 single-domain Hbs, 9 eukaryote 3/3 FHbs, 1 diplomonad Giardia lamblia 3/3 Hb, and 1 mycetozoan Dictyostelium discoideum 3/3 Hb, Cyanidioschyzon merolae and Thalassiosira pseudonana 3/3 single-domain globins, and 3 vertebrate (i.e., human, bird, and fish) 3/3 neuroglobins (Ngbs), cytoglobins (Cygbs), α- and β-globins and myoglobins (Mbs), and 2 urochordate 3/3 Hbs. Modified from (Copyright (2005) National Academy of Sciences, U S A).

Figure 2. Three-dimensional structure of Mt-trHbN, Mt-trHbO, and Cj-trHbP.

(Top) Ribbon views of Mt-trHbN, Mt-trHbO, and Cj-trHbP, including the heme-Fe group (red) and the protein matrix cavity/tunnel systems (blue mash). (Bottom) The heme-Fe pocket of Mt-trHbN, Mt-trHbO, and Cj-trHbP. The heme group is colored in red. The heme ligand (a cyanide ion in all the three structures) and the side chains of residues in the B10, CD1, E7 and F8 topological positions are highlighted. Atomic coordinates were taken from the PDB entries 1S56 (Mt-trHbN), 1NGK (Mt-trHbO), and 2IG3 (Cj-trHbP). All pictures have been drawn with the Swiss-PdbViewer .

Figure 3. Mt-trHbN(II) nitrosylation at 20.0°C.

(A) Difference absorbance spectrum of Mt-trHbN(II) minus Mt-trHbN(II)-NO, at pH 7.0. (B) Difference absorbance spectrum of Mt-trHbN(II) minus Mt-trHbN(II)-NO, at pH 9.0. (C) Normalized averaged time courses of Mt-trHbN(II) nitrosylation, at pH 7.0. The NO concentration was 5.0×10⁻⁶ M (trace a) and 1.2×10⁻⁵ M (trace b). The time course analysis according to Eqn. (1) allowed the determination of the following values of k = 9.2×10¹ s⁻¹ (trace a) and 2.8×10² s⁻¹ (trace b). (D) Normalized averaged time courses of Mt-trHbN(II) nitrosylation, at pH 9.0. The NO concentration was 5.0×10⁻⁶ M (trace a) and 1.2×10⁻⁵ M (trace b). The time course analysis according to Eqn. (1) allowed the determination of the following values of k = 7.4×10¹ s⁻¹ (trace a) and 1.9×10² s⁻¹ (trace b). (E) Dependence of the pseudo-first-order rate-constant k for Mt-trHbN(II) nitrosylation on the NO concentration, at pH 7.0. The analysis of data according to Eqn. (2) allowed the determination of k _on = (2.1±0.3)×10⁷ M⁻¹ s⁻¹. (F) Dependence of the pseudo-first-order rate-constant k for Mt-trHbN(II) nitrosylation on the NO concentration, at pH 9.0. The analysis of data according to Eqn. (2) allowed the determination of k _on = (1.6±0.3)×10⁷ M⁻¹ s⁻¹. The Mt-trHbN(II) concentration was 1.5×10⁻⁶ M. The NO concentration was 1.0×10⁻⁴ M (panels A and B). Where not shown, the standard deviation is smaller than the symbol. For details, see text.

Figure 4. Nitrite-mediated nitrosylation of Mt-trHbN(II), at 20.0°C.

(A) Difference absorbance spectrum of Mt-trHbN(II) minus Mt-trHbN(II)-NO, at pH 7.4. (B) Normalized averaged time courses of nitrite-mediated nitrosylation of Mt-trHbN(II), at pH 7.4. The nitrite concentration was 2.5×10⁻³ M (trace a) and 1.0×10⁻² M (trace b). The time course analysis according to Eqn. (3) allowed the determination of the following values of h = 4.0×10⁻² s⁻¹ (trace a) and 1.6×10⁻¹ s⁻¹ (trace b). (C) Dependence of h on [NO₂ ⁻] for nitrite-mediated nitrosylation of Mt-trHbN(II), at pH 7.4. The continuous line was generated from Eqn. (4) with h _on = (1.6±0.2)×10¹ M⁻¹ s⁻¹. (D) pH-Dependence of h _on for the nitrite-mediated nitrosylation of Mt-trHbN(II). The slope of the continuous line was −1.00±0.01. The Mt-trHbN(II) concentration was 1.5×10⁻⁶ M. Where not shown, standard deviation is smaller than the symbol. For details, see text.

Figure 5. Reductive nitrosylation of Mt-trHbN(III), at 20.0°C.

(A) Difference absorbance spectra of Mt-trHbN(III) minus Mt-trHbN(III)-NO and of Mt-trHbN(III)-NO minus Mt-trHbN(II)-NO (open and fillrd circles, respectively), at pH 9.0. (B) Normalized averaged time courses of Mt-trHbN(III) reductive nitrosylation, at pH 9.0. The NO concentration was 2.5×10⁻⁵ M (trace a) and 2.0×10⁻⁴ M (trace b). The time course analysis according to Eqns (5a)–(5c) allowed the determination of the following values of parameters α, l, and b: trace a - α = 0.61, l = 6.0 s⁻¹, and b = 2.5×10⁻³ s⁻¹; and trace b - α = 0.89, l = 2.6×10¹ s⁻¹, and b = 2.4×10⁻³ s⁻¹. (C) Dependence of l on [NO] for Mt-trHbN(III) reductive nitrosylation, at pH 9.0. The continuous line was generated from Eqn. (6) with l _on = (1.4±0.2)×10⁵ M⁻¹ s⁻¹ and l _off = 1.6±0.2 s⁻¹. (D) Dependence of α on [NO] for Mt-trHbN(III) reductive nitrosylation, at pH 9.0. The continuous line was generated from Eqn. (7) with L = (1.6±0.2)×10⁻⁵ M. (E) Dependence of b on [NO] for Mt-trHbN(III) reductive nitrosylation, at pH 9.0. The average b value is 2.5×10⁻³ s⁻¹ (dashed line). (F) Dependence of b on [OH⁻] for Mt-trHbN(III) reductive nitrosylation. The continuous line was generated from Eqn. (8) with b _OH− = (1.7±0.2)×10² M⁻¹ s⁻¹ and b _H2O = (6.4±0.7)×10⁻⁴ s⁻¹. The Mt-trHbN(III) concentration was 1.5×10⁻⁶ M. Where not shown, standard deviation is smaller than the symbol. For details, see text.