The evolution of respiratory chain complex I from smaller functional modules
(2011)- Abstract
- NADH:quinone oxidoreductase, or complex I, is a large membrane bound enzyme in the respiratory chain of living organisms, that has evolved from the combination of smaller functional building blocks. The enzyme has a conserved core structure, comprising 14 protein subunits: Seven subunits protrude from the membrane and contain a flavin and eight iron-sulfur clusters. The remaining seven subunits in the membrane domain are lacking prosthetic groups and thus have no color or other spectral features. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery.
In this work a novel type of cytochrome c fusion protein was created, to... (More) - NADH:quinone oxidoreductase, or complex I, is a large membrane bound enzyme in the respiratory chain of living organisms, that has evolved from the combination of smaller functional building blocks. The enzyme has a conserved core structure, comprising 14 protein subunits: Seven subunits protrude from the membrane and contain a flavin and eight iron-sulfur clusters. The remaining seven subunits in the membrane domain are lacking prosthetic groups and thus have no color or other spectral features. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery.
In this work a novel type of cytochrome c fusion protein was created, to facilitate studies of these subunits. The fusion proteins remained functional, and could be quantified spectroscopically or detected using anti-cyt c antibodies. Using whole genome sequences, the distribution of different types of complex I in the tree of life was surveyed. A compact, 11-subunit complex I was found both in the archeal and the eubacterial kingdoms, whereas the full size complex I was only found in some eubacterial phyla.
Bacillus subtilis strains genetically deleted for their antiporter subunits MrpA or MrpD were used to study the function of the homologous ion transporter proteins in complex I and its evolutionary progenitors. By comparing the complementation capacity of the Na+ and pH sensitive growth phenotypes, the function of the individual subunits could be deduced. It was concluded that MrpA and MrpD are single ion transporters that exhibit antiporter activity when working together. NuoN could only rescue the ΔMrpD strain whereas NuoL primarily improved the growth of the ΔMrpA strain. Thus, complex I most likely contain one Na+ transporter and two H+ transporters. The corresponding subunits from an 11-subunit complex I showed essentially the same ion specificity as the full size complex I, whereas the hydrogenase transporter subunit corroborated the theoretical prediction that these proteins are degenerate, less specific forms. (Less) - Abstract (Swedish)
- 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... (More) - 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1776768
- author
- Moparthi, Vamsi LU
- supervisor
- opponent
-
- Gennis, Robert B, Department of Chemistry, University of Illinois, A320 Chemical & Life Sciences Lab, 600 South Mathews Avenue, Urbana IL 61801, USA
- organization
- publishing date
- 2011
- type
- Thesis
- publication status
- published
- subject
- keywords
- Hyf, Hyc, Membrane bound hydrogenases, NADH:quinone oxidoreductase, Mrp antiporter, 16S rRNA
- defense location
- KC sal B
- defense date
- 2011-02-25 10:15:00
- language
- English
- LU publication?
- yes
- id
- e8ae8b58-8b5f-47f8-bfe9-1f669cd1493f (old id 1776768)
- date added to LUP
- 2016-04-04 14:02:55
- date last changed
- 2018-11-21 21:17:57
@phdthesis{e8ae8b58-8b5f-47f8-bfe9-1f669cd1493f,
abstract = {{NADH:quinone oxidoreductase, or complex I, is a large membrane bound enzyme in the respiratory chain of living organisms, that has evolved from the combination of smaller functional building blocks. The enzyme has a conserved core structure, comprising 14 protein subunits: Seven subunits protrude from the membrane and contain a flavin and eight iron-sulfur clusters. The remaining seven subunits in the membrane domain are lacking prosthetic groups and thus have no color or other spectral features. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery. <br/><br>
<br/><br>
In this work a novel type of cytochrome c fusion protein was created, to facilitate studies of these subunits. The fusion proteins remained functional, and could be quantified spectroscopically or detected using anti-cyt c antibodies. Using whole genome sequences, the distribution of different types of complex I in the tree of life was surveyed. A compact, 11-subunit complex I was found both in the archeal and the eubacterial kingdoms, whereas the full size complex I was only found in some eubacterial phyla. <br/><br>
<br/><br>
Bacillus subtilis strains genetically deleted for their antiporter subunits MrpA or MrpD were used to study the function of the homologous ion transporter proteins in complex I and its evolutionary progenitors. By comparing the complementation capacity of the Na+ and pH sensitive growth phenotypes, the function of the individual subunits could be deduced. It was concluded that MrpA and MrpD are single ion transporters that exhibit antiporter activity when working together. NuoN could only rescue the ΔMrpD strain whereas NuoL primarily improved the growth of the ΔMrpA strain. Thus, complex I most likely contain one Na+ transporter and two H+ transporters. The corresponding subunits from an 11-subunit complex I showed essentially the same ion specificity as the full size complex I, whereas the hydrogenase transporter subunit corroborated the theoretical prediction that these proteins are degenerate, less specific forms.}},
author = {{Moparthi, Vamsi}},
keywords = {{Hyf; Hyc; Membrane bound hydrogenases; NADH:quinone oxidoreductase; Mrp antiporter; 16S rRNA}},
language = {{eng}},
school = {{Lund University}},
title = {{The evolution of respiratory chain complex I from smaller functional modules}},
year = {{2011}},
}