Limits...
Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Boron I, Bustamante JP, Davidge KS, Singh S, Bowman LA, Tinajero-Trejo M, Carballal S, Radi R, Poole RK, Dikshit K, Estrin DA, Marti MA, Boechi L - F1000Res (2015)

Bottom Line: In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W.These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site.We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EGA, Argentina.

ABSTRACT
Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

No MeSH data available.


Related in: MedlinePlus

•NO decomposition by Mt-trHbN.(A)•NO decay was monitored amperometrically in the absence (red trace) and the presence (black trace) of Mt-trHbN added at the apex of the signal response to 2 μM ProliNONOate. Data are representative of 3 technical repeats. (B) Mean rates of•NO decay in the presence of wild type Mt-trHbN or site-directed mutants from 3 technical repeats ± S.E.M *P < 0.05, unpairedt-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591903&req=5

f6: •NO decomposition by Mt-trHbN.(A)•NO decay was monitored amperometrically in the absence (red trace) and the presence (black trace) of Mt-trHbN added at the apex of the signal response to 2 μM ProliNONOate. Data are representative of 3 technical repeats. (B) Mean rates of•NO decay in the presence of wild type Mt-trHbN or site-directed mutants from 3 technical repeats ± S.E.M *P < 0.05, unpairedt-test.

Mentions: Mt-trHbN has previously been described as a dioxygenase, capable of O2-dependent•NO consumption36,37. Consequently,•NO decomposition by purified Mt-trHbN and the VG8F, VG8W mutants was determined in a reaction mixture containing buffer, NADPH andE. coli FdR, to enable cyclic restoration of heme iron to the oxyferrous state.Figure 6A shows that in the absence of protein (red trace) decay of the•NO signal was monophasic until•NO was exhausted. The decay of NO in the presence of Mt-trHbN (black trace) was biphasic, with an almost linear initial rapid rate in decay, which was used to compare the various Mt-trHbN derivatives, followed by a slower rate in decay. This suggests that•NO is not being turned over in a cyclic manner, but is simply binding available heme.•NO consumption results show that the VG8F and VG8W mutans have a statistically significant reduced•NO binding capacity compared to HbN (Figure 6B).


Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Boron I, Bustamante JP, Davidge KS, Singh S, Bowman LA, Tinajero-Trejo M, Carballal S, Radi R, Poole RK, Dikshit K, Estrin DA, Marti MA, Boechi L - F1000Res (2015)

•NO decomposition by Mt-trHbN.(A)•NO decay was monitored amperometrically in the absence (red trace) and the presence (black trace) of Mt-trHbN added at the apex of the signal response to 2 μM ProliNONOate. Data are representative of 3 technical repeats. (B) Mean rates of•NO decay in the presence of wild type Mt-trHbN or site-directed mutants from 3 technical repeats ± S.E.M *P < 0.05, unpairedt-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591903&req=5

f6: •NO decomposition by Mt-trHbN.(A)•NO decay was monitored amperometrically in the absence (red trace) and the presence (black trace) of Mt-trHbN added at the apex of the signal response to 2 μM ProliNONOate. Data are representative of 3 technical repeats. (B) Mean rates of•NO decay in the presence of wild type Mt-trHbN or site-directed mutants from 3 technical repeats ± S.E.M *P < 0.05, unpairedt-test.
Mentions: Mt-trHbN has previously been described as a dioxygenase, capable of O2-dependent•NO consumption36,37. Consequently,•NO decomposition by purified Mt-trHbN and the VG8F, VG8W mutants was determined in a reaction mixture containing buffer, NADPH andE. coli FdR, to enable cyclic restoration of heme iron to the oxyferrous state.Figure 6A shows that in the absence of protein (red trace) decay of the•NO signal was monophasic until•NO was exhausted. The decay of NO in the presence of Mt-trHbN (black trace) was biphasic, with an almost linear initial rapid rate in decay, which was used to compare the various Mt-trHbN derivatives, followed by a slower rate in decay. This suggests that•NO is not being turned over in a cyclic manner, but is simply binding available heme.•NO consumption results show that the VG8F and VG8W mutans have a statistically significant reduced•NO binding capacity compared to HbN (Figure 6B).

Bottom Line: In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W.These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site.We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EGA, Argentina.

ABSTRACT
Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

No MeSH data available.


Related in: MedlinePlus