Deciphering the MDeciphering the Molecular Basis of the olecular Basis of the NO detoxification mechanism in NO detoxification mechanism in
Truncated Hemoglobin N Truncated Hemoglobin N
F. Javier LuqueF. Javier Luque
Departament of Physical Chemistry and Departament of Physical Chemistry and Institute de BiomedicineInstitute de Biomedicine
FacultadFacultad de Farmacia de FarmaciaUniversity of BarcelonaUniversity of Barcelona
Modeling Interactions in Biomolecules III
Prague, Sept. 2007
During infection, nitric oxide (NO) formed in macrophages contribute to restrict bacteria in latency
However, the toxic effects of NO can be minimized by resistance mechanisms; this is the role played by the truncated hemoglogin N, which converts NO to the harmless nitrate anion.
Mycobacterium Tuberculosis (Electron micrograph)
Tuberculosis (TB) remains a threat to the health and well-being of people around the world.
Among infectious diseases, it is the second leading killer of adults in the world (2
million TB-related deaths each year + latently persist in 1 billion people).
−+→++ 32 NO)III(FeNOO)II(Fe
Truncated Hemoglobin N (Mycobacterium tuberculosis)
PDB entry 1IDR
Myoglobin (sperm whale)
PDB entry 1A6M
A
B
E
G
C
H
Enzyme O2 binding to ferrous trHbN NO oxidation by oxy trHbN
kon (M-1 s-1) koff (s-1) K (M) k (M-1 s-1)
trHbN 2.5 x 107 2.0 x 10-1 8.0 x 109 7.5 x 108
NO conversion to nitrate by oxy-trHbN is faster than O2
binding to the deoxy protein, i.e. the detoxification efficiency is determined by the ability to capture O2 and NO.
Biochemical data
Enzyme O2 binding to ferrous trHbN NO oxidation by oxy trHbN
kon (M-1 s-1) koff (s-1) K (M) k (M-1 s-1)
trHbN 2.5 x 107 2.0 x 10-1 8.0 x 109 7.5 x 108
NO conversion to nitrate by oxy-trHbN is faster than O2
binding to the deoxy protein, i.e. the detoxification efficiency is determined by the ability to capture O2 and NO.
How does the enzyme control ligand access to the heme site?
Biochemical data
Truncated Hemoglobin N (Mycobacterium tuberculosis)
PDB entry 1IDR
A
B
E
G
C
H
Long branch (∼ 20Å)B: Ile19, Ala24, Ile25, Val28, Val29, Phe32E: Phe62, Ala63, Leu66
Short branch (∼ 10Å)G: Ala95, Leu98H: Leu116, Ile119
Phe62
Phe32
Leu98
helix Bhelix E
Phe62 acts as the gate of the tunnel long branch by adopting two different conformations in oxy trHbN
Glu70
Does the pre-helix A play a regulatory role?
Glu70
Arg10
Arg10
CMIP energy isocontour
Closed State Axis of the long branch of the tunnel
Phe62 acts as a gate in the long branch of the tunnel
Phe62(helix E)
Phe32(helix B)
Tyr33(helix B)
CMIP energy isocontour
Open State
Phe62(helix E)
Axis of the long branch of the tunnel
Entry to main
channel
Heme
Fre
e e
ne
rgy
(kc
al/m
ol)
Distance Fe-N(NO) (Å)
Free energy profile for NO diffusion along the main channel(steered MD simulations)
Closedstate
Openstate
How does the protein control the opening/closing of the gate (Phe62)?
Heme
Phe61
Gln58
Tyr33
Essential Movements Deoxy trHbN
Shift in the H-bond
Gln58 in all-trans conformation
(E helix)
(B helix)
Essential MovementsOxy trHbN Deoxy trHbN
Shift in the H-bond
Gln58 in all-trans conformation
Frozen H-bond
Torsional flexibility in Gln58 side chain, which adopts an staggered conformation
(E helix)
(B helix)
Heme
Phe62
Gln58
Tyr33
Heme-O2
Phe62
Gln58 Tyr33
Helix C
Helix HHeme group
Essential dynamics analysis
Helix G
Deoxy trHbN
Heme group
Essential dynamics analysisOxy trHbN
Helix B
Helix E
Fre
e E
nerg
y (k
cal/m
ol)
Dihedral angle (Phe62)
PMF for torsional change in Phe62
Openstate
Closedstate
Oxy trHbN
Deoxy trHbN
What is the diffusion pathway followed by O2 to get the heme cavity?
Access to the heme group occurs through the secondary tunnel
Phe91(helix G)
Closedstate
Openstate
Free energy profile for ligand diffusion along the secondary channel(steered MD simulations)
Fre
e en
ergy
(kc
al/m
ol)
Distance Fe-N(NO) (Å)
Entry tosecondary channel
Heme
Deoxyform
Oxy form
The Tyr33-Gln58 pair is essential for trHbN because it contributes:
1) to modulate binding affinity of O2 to the heme,
2) to assist anchoring of the incoming NO reactant, and
3) to act as a molecular switch that regulates access of NO through the long branch of the tunnel
Are viable mutants such as Tyr33→Phe or Gln58→Ala?
The Tyr33-Gln58 pair is essential for trHbN because it contributes:
1) to modulate binding affinity of O2 to the heme,
2) to assist anchoring of the incoming NO reactant, and
3) to act as a molecular switch that regulates access of NO through the long branch of the tunnel
NO consumption
a) cells with wt protein (WT)
b) cells where the trHbN gene was inactivated (∆ HbN)
c) cells where the trHbN gene was inactivated, but later restored through a plasmid (∆ HbN: HbN)
d) cells where the trHbN gene was inactivated, but later the Tyr33→Phe mutant was restored through a plasmid (∆ HbN: YB10F)
Can the Tyr->Phe mutant inactivate the enzyme
by affecting the catalytic efficiency or
by altering the dynamical behavior of the protein?
Gas phase Water Protein (w.t.) Tyr33->Phe mutant
Fe(III) [OONO-] -> Fe(IV)=O + NO2
Fe(IV)=O + NO2 ->
Fe(III)[NO3-]
Fe(II)-O2 + NO ->
Fe(III) [OONO-]
Ene
rgy
(kca
l/m
ol)
d(O2-NNO; Å)E
nerg
y (k
cal/m
ol)
Ene
rgy
(kca
l/mol
)
d(O1-O2; Å)d(O1-NNO; Å)
∆E
(kcal/mol)
Fe(II)-O2 + NO ->
Fe(III) [OONO-]
Fe(III) [OONO-] ->
Fe(IV)=O + NO2
Fe(IV)=O + NO2 ->
Fe(III)[NO3-]
Tyr33 -23.0 -8.1 -18.0
Tyr33->Phe -21.5 -7.7 -19.3
The conversion to nitrate is a favorable process, where the protein environment (in particular Tyr33) does not play a significant contribution to the catalysis of the chemical reaction
Reactant
ProductReactant Reactant
Product Product
Tyr33→PheGln58→Ala
closed
Phe62
open
Phe62
Torsional flexibility of Phe62 for theoxy state of the mutants
CMIP energy contour
Tyr33->Phe Gln58->Ala
wt-protein
Essential Dynamics
C
H
G
loop-F
Tyr-Phe mutant
C
H
G
loop-F
Gln-Ala mutant
Oxy state
2
11
)(1 B
j
n
i
Ai
n
jAB n
ν•ν=γ ∑∑==
γ AB Tyr→Phe Gln→Ala
WT 0.54 0.44
Tyr→Phe 0.49
Main local conformations at the heme cavity
Tyr-Phe mutant Gln-Ala mutant
wild type
wild type
Distribution of interesidue distances between Gln58 and Phe62
wt mutant
Tyr-Phe mutant
ConclusionTrHbN has evolved to develop a mechanism for selective ligand diffusion to ensure survival under NO stress conditions
According to this mechanism, the diffusion of NO and O2 takes place selectively through different channels, and entry of NO is not permitted until the heme group has already captured O2, thus enabling trHbN to accomplish its dioxygenase role.
Is the product, nitrate anion, released efficiently to the bulk solvent?
The driving force for the release of nitrate anion is the large hydration free energy due to the presence of the negative charge,
but
1) must it be released through highly hydrophobic, narrow channels?
2) how the (heme)Fe-O(nitrate) is broken in an efficient way?
The formation of the nitrate anion promotes a relevant structural rearrangement in the heme active site
Isodensity contour of water molecules along the simulation of the trHbN with heme-bound nitrate anion
Water molecules contribute to weaken the Fe-O bond
Model LACV3P* basis
∆ E d
D 1.1 2.07
W 0.6 2.24
DL 0.9 2.15
WL 3.1 2.32
PR 7.2
Model WL
QM subsystem
D: heme + nitrate + distal HisDL: + TyrB10 + GlnE11W: heme + nitrate + distal His + 3 watersWL: + TyrB10 + GlnE11
∆E (kcal/mol): High/low spin energy gap d (Å): Fe-O(nitrate) bond distance
Simulations show that release of the unbound nitrate to the bulk solvent is fast
The release involves an eggression pathway distinct from the two hydrophobic tunnels, and is apparently guided by Thr49.
Release of nitrate anion to the bulk solvent involves a new eggression pathway different from the hydrophobic tunnels used for the entry of O2 and NO
Besides Phe62 (gate), and the Tyr33-Gln58 (O2-activated switching mechanism), Thr49 appears to be important in order to ensure fast release of the product.
Mutants will be valuable to assess trHbN as a potential therapeutical target.
Thr49 Gln58
Tyr33
Phe62
University of BarcelonaA. Bidon-Chanal
A. MorrealeM. OrozcoJ. L. Gelpí
University of Buenos AiresD. EstrínA. CrespoM. Martí
University of MilanM. Bolognesi
M. Milani
Acknowledgments
Barcelona Supercomputer Center(Marenostrum supercomputer)
Spanish Ministerio Educación y Ciencia
Barcelona Supercomputer CenterV. Guallar
Thank you !