Posted comment on ´Light Effect on Water Viscosity: Implication for ATP Biosynthesis` by A P Sommer, M K Haddad and H.J Fecht published in Scientific Reports 5, Article number: 12029 doi: 10.1038/srep12029 8th July 2015
Sommer, Haddad and Fecht describe in their article their investigations into water viscosity on hydrophilic (aluminium, zinc, copper and gold) and hydrophobic (polystyrene) surfaces and the effect of 670nm laser light. They related their results to the effect of water viscosity on mitochondrial ATPase activity. Using a nanoindenter, Sommer, Haddad and Fecht investigated the load required to penetrate 100 nanometers into the surface of specific model substrates with or without 670nm laser light irradiation. In the case of single crystal gold, they found that at a relative humidity threshold of 67% , irradiation with the laser light reduced the load required to penetrate the hydrophilic sample by 72%. The effect disappeared when the humidity threshold was decreased to 48% indicating that the water at the tip was too small to build up a substantial viscous layer. No effect with the laser light was seen with the hydrophobic surface.
The observation was explained with relation to water viscosity at surfaces. Nanoscopic interfacial water layers confined in subnanometer gaps exist on hydrophilic surfaces and these are responsible for the reduction of the viscous friction at the tip by the laser light. The authors equate their observations to the efficiency of biological rotary nanometers such as ATPase where viscosity and torque of the rotary arm are linked. The enzymes are also tuned to biologically tolerated intensities of red to NIR laser light since a reduction of viscosity by the laser light will result in an increase in enzyme efficiency reflected by a rise in ATP production. The example given is mitochondrial ATPase which increases ATP synthesis when irradiated in vitro with 632.8nm red laser light. This increase is explained by the increased proton-motive force resulting from the reduced interfacial viscosity within and/or around the enzyme complex in comparison to the previous assumption that the water in the mitochondria around the enzyme had the properties of bulk water.
The authors also linked interfacial viscosity to the negative effect of reactive oxygen species on ATP synthesis. Reactive oxygen species (ROS) induced oxidative stress was shown to cause depletion of ATP levels in mitochondria which the authors ascribe to an increase in the hydrophilic nature of the intra-mitochondrial space due to the oxygen. This leads to an increase in viscous friction and a reduced performance of the rotary motor and decrease in ATP production.
This article is interesting because it highlights not only the differences between laboratory experiments and the real biological world, but also the differences between reactions investigated at the larger scales and those at the nanometer scale. The article investigates a physical property in the laboratory and correlates the results to a particular biological enzyme which might be affected if that physical principle applies to the biological world. The physical principle is that water viscosity affects penetration depth of a tip into hydrophilic or hydrophobic surfaces and the biological enzyme to which this principle is applied is the mitochondrial ATPsynthase. The authors conclude that environmental changes on the nanometer scale can affect the enzyme`s conformational changes that lead to alterations in the level of activity.
The ATPsynthase is an important mitochondrial enzyme responsible for the synthesis of ATP, but also in its reverse reaction, ATP hydrolysis back to ADP and Pi. Its structure is highly complex and it spans the membrane bilayers and therefore, it consists of a hydrophobic unit (the Fo) and a predominantly hydrophilic unit (the F1). Its main function in the mitochondria is the synthesis of ATP and this is carried out by the catalytic beta subunits that form part of the F1 unit. However, release of the synthesized ATP requires the translocation of protons (in the form of H+) from the external environment through the bilayer via the Fo subunit which forms a rotor arm down through the middle of the F1. The rotation of this rotor arm brings about the correct conformation of the F1 subunits so that the synthesised ATP can be released. The high concentration of protons on the external membrane is brought about by the chain of events that form oxidative phosphorylation and the transfer of the protons across the membrane proton gradient is the transmembrane proton-motive force.
The authors claim that local variations in water viscosity might affect the dynamics and efficiency of this motor molecule near the hydrophilic F1 and in the contact zone between the F1 and Fo. This might not be observed by normal imaging techniques because they are usually carried out in a vacuum. The authors also believe that water in the mitochondria may have the properties of interfacial water since nanoscopic water layers in sub-nanometer gaps are bound to the hydrophilic surfaces instead of having the properties of bulk water as assumed. This may mean that viscosity values are orders of magnitude larger than those expected and hence, enzyme performance is affected.
Several factors may cloud this water viscosity issue and enzyme performance. The enzyme structure itself of the mitochondrial ATPsynthase means that for the Fo subunit, the only contact with the cytoplasm on the exterior side of the membrane has a high pH due to the high concentration of H+ needed to drive the proton translocation and ATP release. This means that any water molecules are likely to be in the clathrate form and unlikely to be bound to the hydrophilic subunit. On the matrix side, the Fo subunits are shrouded by the alpha, beta, y and e subunits so that there is unlikely to be any contact with water molecules from the matrix environment and water formed from the ATP synthesis reaction is likely to also be in the clathrate form. Water molecules could be in nanoscopic pockets, but it would not be a reason for an effect on the enzyme`s overall efficiency.
The authors also demonstrated the effect of NIR laser light on water viscosity and suggested that the increased ATPsynthase activity and increased ATP turnover observed with NIR laser light was due to a decrease in water viscosity in the structure of the nanoscopic interfacial water layers brought about by the irradiation. Again, the argument about the unlikelihood of water viscosity playing a role in enzyme performance due to structure can be put forward and is supported by work by Kujawa et al. 2004 who showed an effect of NIR laser light on red blood cell ATPase activity was due to a change in erythrocyte membranes and lipid bilayer fluidity. Others have also provided explanations as to why this increase in ATP turnover can be observed. Amat et al. 2006 suggested that the effects of NIR laser light could be due to:
1) the production of an oscillating low frequency electric field that causes dipole transitions; the active centre of the enzyme (the Fo) experiencing an internal resonant type field that is trapped inside the structure. This field produces strong electrical interactions and conformational changes which could lead to the release of ATP by the rotor arm rotation;
2) the formation of a new unstable form of ATP that is more easily hydrolysed by the ATPase enzyme due to the last phosphate bond of the ATP having different interconvertible resonance isoforms of similar, but not equal energy. Hence, the ATP, ADP, phosphate bonds could be more easily released from the Fo unit in the ATPsynthase case;
3) an increase in calcium ion concentration by increasing release from the endoplasmic stores by the light induced increase in membrane potential and hence, an increase in enzyme activity. ATP synthase also relies on membrane potential and therefore, any effect on it will have an effect on its activity; or a change in the ratio of ATP to ADP. Amat et al. 2006 reported that in the case of HeLa cells there is an increase in ATP synthesis 25mins after irradiation stops. They suggested that this delay was due to the delayed synthesis of ADP (exists stored in the cell by binding to other molecules but becomes free when needed, but the greatest source is from breakdown of ATP). Irradiation with the NIR laser light leads to irradiated ATP reacting with the enzyme and substrates, increasing the free ADP concentration and decreasing the ATP/ADP ratio. Hence, a shift in ATP synthesis is observed.
Hence, there are other alternative explanations to the increase in ATP synthesis observed with NIR laser light and these should be considered. In the second part of their investigation into water viscosity effect on ATP synthase efficiency, the authors looked at how reactive oxygen species (ROS) causes depletion of ATP levels in the mitochondria. The authors used the premise that bursts of ROS will enhance the hydrophilic nature of the intra-mitochondrial space due to the oxygen, and thereby increase the viscous friction between surfaces moving relative to each other. Hence, performance of the rotary motor would be reduced, which explains the drop in ATP production. Amat et al. 2006 also investigated this and explained the observations by chromophores (in this case the neighbouring electron transport chain enzyme cytochrome c oxidase) absorbing the NIR light which leads to electronic excitation and a transference of the energy to a nearby molecule that is in triplet ground state eg oxygen. This leads to the production of ROS, in this case, hydrogen peroxide which can increase the concentration of calcium ions and as seen above, increase ATP production. In this case, water is produced as a result of hydrogen peroxide breakdown, just like in ATP production, hence the effects on enzyme performance should be the same.
Therefore, although the authors have used a laboratory experiment to make deductions about a biological mechanism there other alternative explanations for the effect observed. Hence, the matter of why ATP synthesis is increased with irradiation with NIR laser light is not yet resolved and further investigations should be made.
Since we´re talking about the topic ……..
….Amat et al. 2006 showed the same effect on ATP synthesis with membranes exposed to electric pulses as with NIR laser light. Therefore, can we assume if the same experiments described here were then repeated with pulsed electric fields the same penetration effect would be seen with the hydrophilic surfaces?
…would a similar laboratory experiment using cloned alpha3beta3y subunits immobilized on a nickel-ion coated glass sheet give the same results?
…..mitochondria and chloroplasts share evolutionary paths so can we assume that if the same experiments were carried out with ATPsynthase of chloroplasts, the same increase in ATP production would be seen with NIR laser light irradiation?