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 !