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Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Oliveira AS, Damas JM, Baptista AM, Soares CM - PLoS Comput. Biol. (2014)

Bottom Line: These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient.In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC.Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

View Article: PubMed Central - PubMed

Affiliation: ITQB - Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

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O2 free energy landscape obtained from the ILS calculations and lowest free energy pathways obtained from the ILS energy landscape.A-O2 free energy landscape obtained from the ILS calculations. Two isosurface contours are shown, with free energy levels at -5 (blue inner contour) and 5 (cyan contour) kJ·mol−1. B- Comparison of the O2 free energy landscape obtained from the ILS calculations (blue maps) and high-affinity regions from the O2 explicit MD simulations (red maps). The probability density contours at 0.00015 Å−3 are represented as a red isosurface. C- O2 lowest free energy pathways obtained from the ILS energy landscape. The spheres represent the local free energy minima while the tubes connecting the minima represent the pathways. The size of the spheres representing the local energy minima scales linearly along the displayed free energy range (small spheres indicates high free energy while large spheres indicate low free energy). The diameter of the pathways scales linearly with the free energy and thus with the oxygen affinity (thinner radius represent high free energies and low oxygen affinity). The free energy at the minima and along the pathways follows the same color scale. The channel 1 coincides with the channel inferred from the X-ray data [7], [13] whereas the channel 2 and channel 3 are alternative routes to reach the BNC.
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pcbi-1004010-g003: O2 free energy landscape obtained from the ILS calculations and lowest free energy pathways obtained from the ILS energy landscape.A-O2 free energy landscape obtained from the ILS calculations. Two isosurface contours are shown, with free energy levels at -5 (blue inner contour) and 5 (cyan contour) kJ·mol−1. B- Comparison of the O2 free energy landscape obtained from the ILS calculations (blue maps) and high-affinity regions from the O2 explicit MD simulations (red maps). The probability density contours at 0.00015 Å−3 are represented as a red isosurface. C- O2 lowest free energy pathways obtained from the ILS energy landscape. The spheres represent the local free energy minima while the tubes connecting the minima represent the pathways. The size of the spheres representing the local energy minima scales linearly along the displayed free energy range (small spheres indicates high free energy while large spheres indicate low free energy). The diameter of the pathways scales linearly with the free energy and thus with the oxygen affinity (thinner radius represent high free energies and low oxygen affinity). The free energy at the minima and along the pathways follows the same color scale. The channel 1 coincides with the channel inferred from the X-ray data [7], [13] whereas the channel 2 and channel 3 are alternative routes to reach the BNC.

Mentions: From the 3D affinity map for Ccox obtained from the ILS calculations (Fig. 3A), we can see that Ccox possesses a complex free energy landscape with several possible O2 binding cavities, most of them located at the protein surface and that do not directly connect to the BNC. Moreover, the calculated affinity map from ILS not only correlates well with some of the high probability regions found in the explicit O2 simulations (see Fig. 3B) but also provides a more complete description of the free energy landscape for O2 inside Ccox due to the sampling of lower affinity zones, not sufficiently sampled by the normal MD simulations. Indeed, the ILS approach allowed us to identified three major routes (named channel 1, channel 2 and channel 3) with high probability of O2 occupancy, interconnecting the protein surface to the BNC. Two of the channels (channel 1 and channel 2) approach the BNC from the subunit I side whereas the third one (channel 3) approaches the BNC from the subunit II side (Fig. 3A). Interestingly all three channels start in the membrane spanning region where the O2 concentration is higher than in aqueous phase, which makes physical sense.


Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Oliveira AS, Damas JM, Baptista AM, Soares CM - PLoS Comput. Biol. (2014)

O2 free energy landscape obtained from the ILS calculations and lowest free energy pathways obtained from the ILS energy landscape.A-O2 free energy landscape obtained from the ILS calculations. Two isosurface contours are shown, with free energy levels at -5 (blue inner contour) and 5 (cyan contour) kJ·mol−1. B- Comparison of the O2 free energy landscape obtained from the ILS calculations (blue maps) and high-affinity regions from the O2 explicit MD simulations (red maps). The probability density contours at 0.00015 Å−3 are represented as a red isosurface. C- O2 lowest free energy pathways obtained from the ILS energy landscape. The spheres represent the local free energy minima while the tubes connecting the minima represent the pathways. The size of the spheres representing the local energy minima scales linearly along the displayed free energy range (small spheres indicates high free energy while large spheres indicate low free energy). The diameter of the pathways scales linearly with the free energy and thus with the oxygen affinity (thinner radius represent high free energies and low oxygen affinity). The free energy at the minima and along the pathways follows the same color scale. The channel 1 coincides with the channel inferred from the X-ray data [7], [13] whereas the channel 2 and channel 3 are alternative routes to reach the BNC.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256069&req=5

pcbi-1004010-g003: O2 free energy landscape obtained from the ILS calculations and lowest free energy pathways obtained from the ILS energy landscape.A-O2 free energy landscape obtained from the ILS calculations. Two isosurface contours are shown, with free energy levels at -5 (blue inner contour) and 5 (cyan contour) kJ·mol−1. B- Comparison of the O2 free energy landscape obtained from the ILS calculations (blue maps) and high-affinity regions from the O2 explicit MD simulations (red maps). The probability density contours at 0.00015 Å−3 are represented as a red isosurface. C- O2 lowest free energy pathways obtained from the ILS energy landscape. The spheres represent the local free energy minima while the tubes connecting the minima represent the pathways. The size of the spheres representing the local energy minima scales linearly along the displayed free energy range (small spheres indicates high free energy while large spheres indicate low free energy). The diameter of the pathways scales linearly with the free energy and thus with the oxygen affinity (thinner radius represent high free energies and low oxygen affinity). The free energy at the minima and along the pathways follows the same color scale. The channel 1 coincides with the channel inferred from the X-ray data [7], [13] whereas the channel 2 and channel 3 are alternative routes to reach the BNC.
Mentions: From the 3D affinity map for Ccox obtained from the ILS calculations (Fig. 3A), we can see that Ccox possesses a complex free energy landscape with several possible O2 binding cavities, most of them located at the protein surface and that do not directly connect to the BNC. Moreover, the calculated affinity map from ILS not only correlates well with some of the high probability regions found in the explicit O2 simulations (see Fig. 3B) but also provides a more complete description of the free energy landscape for O2 inside Ccox due to the sampling of lower affinity zones, not sufficiently sampled by the normal MD simulations. Indeed, the ILS approach allowed us to identified three major routes (named channel 1, channel 2 and channel 3) with high probability of O2 occupancy, interconnecting the protein surface to the BNC. Two of the channels (channel 1 and channel 2) approach the BNC from the subunit I side whereas the third one (channel 3) approaches the BNC from the subunit II side (Fig. 3A). Interestingly all three channels start in the membrane spanning region where the O2 concentration is higher than in aqueous phase, which makes physical sense.

Bottom Line: These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient.In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC.Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

View Article: PubMed Central - PubMed

Affiliation: ITQB - Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

Show MeSH
Related in: MedlinePlus