Figure 1.
Representation of the long and short branches of the tunnel system.
The graphical display is based on the X-ray crystallographic structure of Mtb HbN (PDB entry 1IDR), and the access routes of O2 and NO in the dual-path ligand-modulated mechanism proposed for this protein are indicated. The gating residue PheE15 (residue 62) is shown in the two conformations found in the X-ray structure as sticks.
Figure 2.
Spectral properties of PheE15Ala mutant of HbN.
(A) Optical absorption spectra of oxygenated (solid line) and sodium dithionite reduced species of mutant HbN, recorded in 50 mM Tris.Cl (pH 7.5). (B) CO-difference spectrum of PheE15Ala mutant of HbN. Spectral profile of other PheE15 gate mutants (PheE15Tyr, PheE15Trp and PheE15Trp) appeared similar and matched with the wild type spectrum reported earlier [1].
Table 1.
Oxygen binding and CO association kinetics of PheE15 gate mutants of HbN of M. tuberculosis.
Table 2.
NO-dioxygenase activity of PheE15 gate mutants of HbN.
Figure 3.
NO oxidation profile of PheE15 gate mutants of HbN.
Titration of oxygenated HbN protein (20 µM) was done by adding 15 µM NO sequentially and recording spectra after each addition. Wild type HbN displayed fully oxidized spectra after 12 additions (A), whereas mutants PheE15Tyr (B), PheE15Ile (C), PheE15Trp (D) and PheE15Ala (E) displayed very slow oxidation of the protein and could not be fully oxidized even after 20 additions of NO. The first and last additions are labeled as 1 and 2, respectively.
Figure 4.
Representation of rmsd and rmsf profiles for PheE15 gate mutants of HbN.
(Left) Rmsd (Å) of the protein backbone determined using the X-ray structure (1IDR; subunit A) as reference. The rmsd of the whole protein is shown in green, whereas the rmsd of the residues in the protein core (excluding those in the pre-A segment; residues 1–15) is shown in blue. (Right) Representation of the rmsf (Å) of residues side chains in the protein. The plots correspond to the mutants (A) PheE15Ala, (B) PheE15Ile and (C) PheE15Tyr. The location of the mutated residue Phe(62)E15 is indicated in the plots by an arrow (helix E encompass residues 51–66).
Table 3.
Global similarity index determined by comparison of the motions of the protein backbone in the oxygenated form of wild type HbN and the PheE15 mutants.
Figure 5.
Representation of the accesible volume in wt HbN and its mutants.
The accessible volume determined from MDpocket analysis is achieved for a density isocontour of 6.7 in the case of wt protein (A). The use of the same isocontour shows discontinuities in the accessible volume of the tunnel long branch for the different mutants. The disruption is located around the position of the gate in the case of PheE15Ile (C) and PheE15Tyr (D) mutants. For the PheE15Ala species (B) the major disruption involves the region close to the channel entry. Continuous progression of the accessible volume is achieved when the isocontour value is reduced to 5.4 for PheE15Ile and PheE15Tyr, and to 3.4 for PheE15Ala. In the plots the protein backbone corresponds to the energy-minimized structure obtained by averaging the snapshots sampled in the last 0.1 ns for each trajectory.
Figure 6.
Free energy profile determined for NO migration along the long tunnel branch.
MSMD calculations were performed to determine the energetics of ligand migration through the tunnel long branch for Phe15Ile (green) and PheE15Tyr (blue). The profiles are compared with those determined for ligand migration in both open (solid line) and closed (dashed line) states of oxygenated wt HbN. The free energy is given in kcal/mol, and the distance of the ligand from the heme iron is given in angstroms. For the sake of clarity, error bars are not displayed.
Figure 7.
Representative trajectories followed by free NO ligand through the protein matrix.
MD simulation of free NO located at random positions from the protein were followed to investigate the pathways leading to the heme cavity for oxygenated forms of (A) PheE15Ala, (B) PheE15Ie and (C) PheE15Tyr. The distinct pathways are indicating by showing the position of NO (represented as sticks) along the trajectory using different colors: long branch (blue), short branch (brown), EH (green), and other (yellow). Note that helix G is displayed as highly transparent cartoon for the sake of clarity.
Table 4.
Analysis of the migration pathways followed by a free NO in a simulation box containing the solvated proteins PheE15Ala, PheE15Ile and PheE15Tyr.