The evolution of respiratory chain complex I from smaller functional modules

Sammanfattning: Popular Abstract in English The cell is the basic unit in all organisms. The molecular machinery in cells converts the energy in sunlight or food into a form of energy that the cell can use for physiological processes in the organism. In humans, this molecular machinery is located in the mitochondria; therefore they are often called the power plants of the cell. The molecular machines are actually enzymes, proteins that can catalyze the chemical reactions. In this case, the enzymes are quite complex, and really work like small machines, that sit embedded in a membrane. The substrates from food are oxidized and electrons are released at the active sites, then the electrical current through the enzyme drives proton pumping across the membrane. This process resemble when you charge a battery. In the next step, that proton gradient is used to drive ATP synthesis. ATP (adenosine-tri-phosphate) is the general energy currency of the cell that is used when for instance a muscle is moved. In this thesis, we have focused on one part of the molecular machinery, an enzyme called complex I. This machine is generally built up by 14 different proteins. They can be divided into three parts, the first part contain the electron donor site and also an electron transfer wire. The second part contains the actual engine where the process of electron transfer and proton pumping are coupled. These first and second parts are located towards from the membrane. The last part contains parts for moving the protons across the membrane thus they are sitting in the membrane. Each of the 14 proteins in complex I can be regarded as functional building blocks that are also found in smaller enzymes. During evolution, these similar election transfer and proton pumping related parts comes together and give birth to complex I. There are even smaller variants of complex I in nature reflecting that the building of the full size machine is an ongoing process today. One of these variants does not contain the electron donor wire but contains the other two parts of complex I. In this thesis, we showed that the smaller complex I was the ancestor of present day complex I. As mentioned before, parts for proton pumping are located in the membrane part. Three proteins in this part are named NuoL, NuoM and NuoN, are similar to a particular type of transporter proteins called Mrp antiporters. Generally antiporters exchange one proton with one sodium ion across a membrane. Mrp antiporters can be of MrpA and MrpD type. You can compare the similarity between two proteins by looking at their amino acid sequence. When the sequence is similar, we can say the proteins are homologous. The NuoL protein was more similar to MrpA and NuoM and NuoN were more similar to the MrpD antiporters. In this work, we investigated the possible functions of the NuoL, NuoM and NuoN proteins of complex I by comparing their function to those of the real antiporter proteins such as MrpA and MrpD. We made a model system where we used bacteria that use Mrp antiporter to survive salt (sodium chloride, table salt) in their growth media. Removing of MrpA or MrpD genes resulted in that the bacteria did not to grow in a media containing salt (sodium chloride, table salt). When the MrpA and MrpD encoding genes could be put back into the bacteria on a plasmid, resulted the bacteria were able to grow with salt in the growth medium again. But it only worked, when the correct genes had been returned to corresponding strains. As a further step, we incorporated the genes encoding the complex I proteins NuoL, NuoM and NuoN in the same (as used above) bacteria. We discovered that NuoL could rescue the bacteria lacking MrpA, so that they could grow in the presence of sodium again. Likewise, NuoN could rescue the MrpD removed bacterial strain. This showed that the amino acid sequence similarity reflected actual functional similarity in these proteins. Hence, we could conclude that NuoL has similar function to MrpA and NuoM and NuoN have similar function to MrpD.