Sophie Bernad

Relating the Diffusion of Small Ligands in Human Neuroglobin to Its Structural and Mechanical Properties

Neuroglobin (Ngb), a recently discovered member of the globin family, is overexpressed in the brain tissues over oxygen deprivation. Unlike more classical globins, such as myoglobin and hemoglobin, it is characterized by a hexacoordinated heme, and its physiological role is still unknown, despite the numerous investigations made on the protein in recent years. Another important specific feature of human Ngb is the presence of two cysteine residues (Cys46 and Cys55), which are known to form an intramolecular disulfide bridge. Since previous work on human Ngb reported that its ligand binding properties could be controlled by the coordination state of the Fe(2+) atom (in the heme moiety) and the redox state of the thiol groups, we choose to develop a simulation approach combining coarse-grain Brownian dynamics and all-atom molecular dynamics and metadynamics. We have studied the diffusion of small ligands (CO, NO, and O(2)) in the globin internal cavity network for various states of human Ngb. Our results show how the structural and mechanical properties of the protein can be related to the ligand migration pathway, which can be extensively modified when changing the thiols redox state and the irons coordination state. We suggest that ligand binding is favored in the pentacoordinated species bearing an internal disulfide bridge.

Frontier residues lining globin internal cavities present specific mechanical properties.

The internal cavity matrix of globins plays a key role in their biological function. Previous studies have already highlighted the plasticity of this inner network, which can fluctuate with the proteins breathing motion, and the importance of a few key residues for the regulation of ligand diffusion within the protein. In this Article, we combine all-atom molecular dynamics and coarse-grain Brownian dynamics to establish a complete mechanical landscape for six different globins chain (myoglobin, neuroglobin, cytoglobin, truncated hemoglobin, and chains α and β of hemoglobin). We show that the rigidity profiles of these proteins can fluctuate along time, and how a limited set of residues present specific mechanical properties that are related to their position at the frontier between internal cavities. Eventually, we postulate the existence of conserved positions within the globin fold, which form a mechanical nucleus located at the center of the cavity network, and whose constituent residues are essential for controlling ligand migration in globins.

Probing the Role of the Internal Disulfide Bond in Regulating Conformational Dynamics in Neuroglobin

In this report, we demonstrate that the internal disulfide bridge in human neuroglobin modulates structural changes associated with ligand photo-dissociation from the heme active site. This is evident from time-resolved photothermal studies of CO photo-dissociation, which reveal a 13.4+/-0.9 mL mol(-1) volume expansion upon ligand photo-release from human neuroglobin, whereas the CO dissociation from rat neuroglobin leads to a significantly smaller volume change (DeltaV=4.6+/-0.3 mL mol(-1)). Reduction of the internal disulfide bond in human neuroglobin leads to conformational changes (reflected by DeltaV) nearly identical to those observed for rat Ngb. Our data favor the hypothesis that the disulfide bond between Cys46 and Cys55 modulates the functioning of human neuroglobin.

Reduction of the internal disulfide bond between Cys 38 and 83 switches the ligand migration pathway in cytoglobin

Despite the similar tertiary structure between cytoglobin (Cygb) and myoglobin, several structural features indicate a distinct mechanism of Cygb interactions with exogenous ligands. Here we present a spectroscopic investigation of the dynamics and thermodynamics of structural changes associated with the exogenous ligand migration between the solvent and the heme active site in Cygb with reduced and oxidized Cys 38 and Cys 83 side-chains (Cygb(ox) and Cygb(red), respectively). Photo-acoustic and transient absorption data show that disulfide bond formation alters the ligand migration pathway(s) as evident from the distinct geminate quantum yields (Φgem=0.35 for Cygb(ox) and Φgem=0.63 for Cygb(red)) and rate constants for bimolecular CO rebinding. Moreover, ligand escape from the protein matrix is fast (<40ns) and coupled with an enthalpy change of 18±2kcalmol(-1) in Cygb(red), whereas the disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4kcalmol(-1). These results demonstrate that the disulfide bond connecting helix E and helix B modulates the conformational dynamics in Cygb including the size and energy barrier between the internal hydrophobic sites. Based on the comparison of the thermodynamic profiles for CO photo-dissociation from Cygb, myoglobin, and neuroglobin we propose that in Cygb(red) the photo-dissociated ligand escapes through the hydrophobic tunnel, whereas the CO preferably migrates through the His64 gate in Cygb(ox) suggesting that Cygbs physiological role may vary in response to intracellular redox conditions.

Conformational Dynamics in Human Neuroglobin: Effect of His64, Val68, and Cys120 on Ligand Migration

Neuroglobin belongs to the family of hexacoordinate hemoglobins and has been implicated in the protection of neuronal tissue under hypoxic and ischemic conditions. Here we present transient absorption and photoacoustic calorimetry studies of CO photodissociation and bimolecular rebinding to neuroglobin focusing on the ligand migration process and the role of distal pocket residues (His64 and Val68) and two Cys residues (Cys55 and Cys120). Our results indicate that His64 has a minor impact on the migration of CO between the distal heme pocket and protein exterior, whereas the Val68 side chain regulates the transition of the photodissociated ligand between the distal pocket and internal hydrophobic cavities, which is evident from the increased geminate quantum yield in this mutated protein (Φ(gem) = 0.32 for WT and His64Gln, and Φ(gem) = 0.85 for Val68Phe). The interface between helix G and the A-B loop provides an escape pathway for the photodissociated ligand, which is evident from a decrease in the reaction enthalpy for the transition between the CO-bound hNgb and five-coordinate hNgb in the Cys120Ser mutant (ΔH = -3 ± 4 kcal mol(-1)) compared to that of the WT protein (ΔH = 20 ± 4 kcal mol(-1)). The extensive electrostatic/hydrogen binding network that includes heme propionate groups, Lys67, His64, and Tyr44 not only restricts the heme binding but also modulates the energetics of binding of CO to the five-coordinate hNgb as substitution of His64 with Gln leads to an endothermic association of CO with the five-coordinate hNgb (ΔH = 6 ± 3 kcal mol(-1)).

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q(A) and Q(B). These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P(+)Q(A)(-) state (P is the primary electron donor) and temperature dependence of the rate of P(+)Q(A)(-) charge recombination that are in good agreement show that in the two RC variants, both Q(A)(-) and Q(B)(-) are destabilized by about the same free energy amount: respectively approximately 100 +/- 5 meV and 90 +/- 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q(A)(-) and Q(B)(-). This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.

A novel cryo-reduction method to investigate the molecular mechanism of nitric oxide synthases.

Nitric oxide synthases (NOSs) are hemoproteins responsible for the biosynthesis of NO in mammals. They catalyze two successive oxidation reactions. The mechanism of oxygen activation is based on the transfer of two electrons and two protons. Despite structural analogies with cytochromes P450, the molecular mechanism of NOS remains yet to be elucidated. Because of extremely high reaction rates, conventional kinetics methods failed to trap and characterize the major reaction intermediates. Cryo-reduction methods offer a possibility to circumvent this technological lock, by triggering oxygen activation at cryogenic temperatures by using water radiolysis. However, this method is not adapted to the NOS mechanism because of the high instability of the initial Fe(II)O2 complex (extremely fast autoxidation and/or reaction with the cofactor H4B). This imposed a protocol with a stable Fe(II)O2 complex (observed only for one NOS-like protein) and that excludes any redox role for H4B. A relevant approach to the NOS mechanism would use H4B to provide the (second) electron involved in oxygen activation; water radiolysis would thus provide the first electron (heme reduction). In this context, we report here an investigation of the first electron transfer by this alternative approach, i.e., the reduction of native NOS by water radiolysis. We combined EPR and resonance Raman spectroscopies to analyze NOS reduction for a combination of different substrates, cofactor, and oxygen concentrations, and for different NOS isoforms. Our results show that cryo-reduction of native NOS is achieved for all conditions that are relevant to the investigation of the NOS mechanism.

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.

Corrigendum to: Chemical Evidence for the Dawn of Life on Earth

The dating of the dawn of life on Earth is a difficult task, requiring an accumulation of evidences from many different research fields. Here we shall summarize findings from the molecular scale (proteins) to cells and photosynthesis-related-fossils (stromatolites from the early and the late Archaean Eon), which indicate that life emerged on Earth 4.2–3.8 Ga (i.e. 4.2–3.8 × 109 years) ago. Among the data supporting this age, the isotopic and palaeontological fingerprints of photosynthesis provide some of the strongest evidence. The reason for this is that photosynthesis, carried out in particular by cyanobacteria, was responsible for massive changes to the Earths environment, i.e. the oxygenation of the Earths atmosphere and seawater, and the fixation of carbon from atmospheric CO2 in organic material. The possibility of a very early (>3.8 Ga ago) appearance of complex autotrophic organisms, such as cyanobacteria, is a major change in our view of lifes origins.

Impact of A90P, F106L and H64V mutations on neuroglobin stability and ligand binding kinetics

Human neuroglobin (Ngb) is a hexacoordinated globin which binds some small ligands. Its function is still not well-established, even though Ngb seems to be implicated in the protection against neurodegenerative diseases. It has been shown by molecular dynamics and crystallography that ligand binding could occur thanks to a haem sliding mechanism specific to Ngb. In this paper, we studied some regions which could participate in this mechanism. We used UV–visible spectroscopy, CD and NMR to have a look on the protein structure and NMR and stopped-flow to study the ligand binding properties of the proteins. In the haem environment we mutated the distal histidine H64, the alanine A90 which is on the proximal F helix and the phenylalanine F106 which is close to the haem. We showed that both H64V and A90P variants, which affect the haem coordination, seemed to be important to haem and protein secondary structure stabilities whereas F106L mutation did not affect those properties. Then we confirmed that the cyanide binding kinetics were isomer dependent on wild-type Ngb and A90P and F106L variants. H64V Ngb variant had a behavior similar to wild-type Mb or Hb with a loss of the haem kinetic differentiation. Moreover, our results suggested that one haem isomer was more sensitive to A90P and F106L mutations. Those results brought some evidence that the haem sliding mechanism could occur for the cyanide binding and could be haem isomer dependent. The isomer forms may play distinct roles for the potential function of Ngb in vivo.