Photosynthesis apparatus was assembled in an artificial cell

Anna Kaznadzei, N+1

Italian scientists have created a photosynthetic apparatus in an artificial cell. The reaction centers are correctly oriented in the membrane of giant vesicles and are able to effectively create a proton gradient. An article describing the system is published in PNAS – Biophysics and Computational Biology (Altamura et al., Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocols).

Photosynthesis is a reaction in which the energy of light is converted into the energy of chemical bonds. This allows, for example, plants to convert carbon dioxide and water into organic compounds and oxygen with the help of light quanta. This is how plants feed, and due to this process, a huge number of other organisms on Earth survive: firstly, they eat these "living crystals" that create themselves from light and air, and secondly, they breathe oxygen that plants produce. The photosynthetic apparatus of plants is located in the membranes of special organelles – chloroplasts. During the operation of chloroplasts, a flow of protons is created through the membrane, and a proton gradient occurs, thanks to which cells store energy by synthesizing high-energy ATP molecules. Some bacteria are also capable of photosynthesis. Their photosynthetic apparatus is located in cytoplasmic membranes, and a proton gradient is created between the cytoplasm and the external environment.

The researchers worked with the photosynthetic apparatus of purple bacteria. Most of these bacteria are anaerobic, and their photosynthesis is oxygen–free - instead of oxygen, sulfur, for example, is formed at the end of the reaction. The reaction center (RC) in their photosynthetic apparatus is arranged in such a way that when a quantum of light is absorbed, one quinone molecule is restored, which requires two protons from the cytoplasm. The resulting proton gradient allows them to synthesize ATP.

Confocal microscopy of reaction centers (colored red)
in the protocells (the inner cavity contains the green dye calcein).
Here and below are the drawings from the PNAS article.

To create an artificial system, scientists used only the main transmembrane protein of the reaction center of the bacterium Rhodobacter sphaeroides, without auxiliary molecules. This protein consists of two highly hydrophobic subunits and one hydrophilic. The simplified reaction center was embedded in various lipid membranes, including flat bilipid layers. One of the significant problems of such experiments is the orientation of proteins in membranes: when embedded, they often turn out to be oriented in different directions, and therefore they cannot create the correct proton gradient. In the new work, scientists managed to create a real protocell – a giant lipid vesicle – with RC embedded in the membrane, ninety percent of which are oriented in the right direction.

The principle of micelle assembly with selective orientation of reaction centers.

To obtain gigantic vesicles, the droplet transfer method was applied. Initially, the protein was isolated from the bacterial membrane, dissolved in water and obtained a homogeneous micellar substance. It contained fully active reaction centers surrounded and protected from water by detergent molecules. Then it was emulsified in a mixture of phosphatidylcholine and phosphatidylglycerin fats and placed on the surface of water, creating a two-phase system, which was then centrifuged and giant lipid vesicles with RC embedded in them were obtained. Hydrophobic interactions in contact with the lipid-water emulsion caused the reaction centers to unfold in the same direction, since the hydrophilic subunit of the protein tended to water, and the hydrophobic ones tended to move away from it and thus fell into the lipid bilayer. The resulting vesicles had a diameter of about 20 microns, were stable and had a fairly high protein density – they contained ~1 reaction center per 2,200 lipid molecules, which is about a third of the corresponding density in the membranes of photosynthetic bacteria.

Further analysis showed that photosynthetic proteins in such vesicles are active. In order to verify this, the vesicles were illuminated with short flashes of light and the recombination reaction rate was measured. In addition, the addition of water–soluble cytochrome to the solution as an electronic donor showed that 90 percent of the reaction centers were indeed oriented towards the external environment - the signal indicating recombination practically disappeared, since cytochrome was effectively oxidized, which would not have happened if they were oriented in the other direction. After that, an analogue of ubiquinone, decylubiquinone, was added to the solution, which, when the flashes resumed, began to work as an electron acceptor, turning into decylhydrobiquinone due to electron donors – cytochromes. Over time, recombination reappeared as electron donors ran out. In the future, it was also possible to measure that the rate of proton movement through the membrane: it was approximately one proton per minute for each reaction center. The effectiveness of the work depends, apparently, on a number of physical factors, including the size of the vesicles and the composition of the lipid membrane.

Earlier we have already told how a bionic sheet was created, also capable of simulating photosynthesis, but with the help of the work of living bacteria (and another one without bacteria, based on fluorescent solar concentrators – VM). In the same project, the developed method will expand the work with artificial vesicles, scientists believe. The next task is to embed other protein complexes in them in the correct orientation, including ATP synthase, which previously turned out to be turned only into the external environment, and not inside the vesicle, as needed. The joint work of RC and ATP synthase will allow vesicles to store light energy in the bonds of ATP molecules, which brings researchers closer to designing energetically autonomous artificial cells.

Portal "Eternal youth" http://vechnayamolodost.ru  23.03.2017