Production and Characterization of Mrp AntiporterComplex - The Evolutionary Progenitor of Complex I
(2013) KEMT30 20122Department of Chemistry
- Abstract
- NADH:upiquinone oxidoreductase, or complex I, is the first and largest enzyme in the respiratory chain. It is the last known enzyme in the chain due to its complexity of being a very large complex comprising of a hydrophilic and a hydrophobic domains of about 46 subunits in total in the eukaryote enzyme. Another reason of its complexity is that the hydrophobic domain does not have any redox centers or prosthetic groups making this part in particular difficult to study.
The transfer of two electrons from NADH molecule to FMN in the hydrophilic domain of complex I and then through the Fe/S clusters up to quinone is coupled to proton translocation by the hydrophobic domain of the complex. Recently, the structure of the whole complex was... (More) - NADH:upiquinone oxidoreductase, or complex I, is the first and largest enzyme in the respiratory chain. It is the last known enzyme in the chain due to its complexity of being a very large complex comprising of a hydrophilic and a hydrophobic domains of about 46 subunits in total in the eukaryote enzyme. Another reason of its complexity is that the hydrophobic domain does not have any redox centers or prosthetic groups making this part in particular difficult to study.
The transfer of two electrons from NADH molecule to FMN in the hydrophilic domain of complex I and then through the Fe/S clusters up to quinone is coupled to proton translocation by the hydrophobic domain of the complex. Recently, the structure of the whole complex was solved. However, the coupling mechanism of the electron transfer and the proton translocation is still unknown.
Understanding complex I mechanism is of great importance as it is related to production of reactive oxygen species (ROS). The production of such component leeds to mutations in the mitochondrial DNA and thus causing serious health disorders.
The hydrophobic domain subunits show a high sequence similarity to a Na+/H+-antiporter called Mrp-complex. NuoL, NuoM and NuoN, NuoK and NuoJ subunits in complex I are homologoues to MrpA, MrpD, MrpC and last part of MrpA subunits in Mrp-complex respectively. Such high sequence similarity between the two complexes indicates a functional similarity too, leading to a conclusion that complex I might pump sodium in exchange to protons. The structural similarity of the two complexes would be the strongest link between complex I and Mrp-complex. However, the structure of Mrp-complex has not been solved yet, thus solving the structure of the Mrp-antiporter and understanding its mechanism will solve the mystery of complex I coupling mechanism.
This project is aiming to crystalize the Mrp-complex from Bacillus subtilis. Mrp-subcomplex composed of four subunits MrpA, MrpB, MrpC and MrpD has been successfully constructed so that a his-tagged cytochrome c550 domain was added to the C-terminal end of MrpD subunit, which would facilitate purification and quantification of the complex. MrpABCDcytH complex was successfully produced in E. coli, purified and analyzed for further experiments. (Less) - Popular Abstract
- All living organisms need energy to survive, because it is required for all processes such as respiration, growth, thinking, muscle movement etc. Food is decomposed through a series of chemical reactions and energy is released in the form of electrons. The released electrons are carried by electron carrier molecules such as NADH. These molecules are the major source of electrons that are donated to the main energy production machinery - the respiratory chain.
Complex I, or NADH:upiquinone oxidoreductase, is the first and largest protein in the chain. Electrons from NADH are delivered to complex I and the transfer of electrons through the complex leeds to proton translocation to the periplasm (outside the membrane) by its smaller proteins... (More) - All living organisms need energy to survive, because it is required for all processes such as respiration, growth, thinking, muscle movement etc. Food is decomposed through a series of chemical reactions and energy is released in the form of electrons. The released electrons are carried by electron carrier molecules such as NADH. These molecules are the major source of electrons that are donated to the main energy production machinery - the respiratory chain.
Complex I, or NADH:upiquinone oxidoreductase, is the first and largest protein in the chain. Electrons from NADH are delivered to complex I and the transfer of electrons through the complex leeds to proton translocation to the periplasm (outside the membrane) by its smaller proteins located in the membrane. Other complex proteins in the chain translocate protons too through electron transfer in the chain except complex II. Finally, these protons are used by the ATP synthase complex where they are pumped to the inside of the membrane, aiding the energy synthesis.
The mechanism by which the electron transfer is coupled to proton translocation in complex I is still unknown. The interest in understanding the coupling mechanism is due to the fact that it is linked to many serious health disorders. Studies have shown that complex I subunits (smaller proteins) that are located in the membrane are homologous (similar to) Mrp-complex subunits. Mrp complex is an antiporter protein that pumps sodium outside the cell in exchange for protons. NuoL, NuoM, NuoN, NuoK and NuoJ subunits in complex I are homologous to MrpA, MrpD, MrpC and last part of MrpA subunits in Mrp-complex, respectively. As the two complexes have a high degree of similarity, this gives an indication that both are probably having similar modes of action.
Up to date, there is no structure of Mrp-complex avaliable. In this project, an attempt to solve the Mrp structure has been made. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/4646159
- author
- Al-Yahyaai, Hanan LU
- supervisor
- organization
- course
- KEMT30 20122
- year
- 2013
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- proteinvetenskap, protein science
- language
- English
- id
- 4646159
- date added to LUP
- 2014-09-12 16:21:26
- date last changed
- 2014-09-12 16:21:26
@misc{4646159,
abstract = {{NADH:upiquinone oxidoreductase, or complex I, is the first and largest enzyme in the respiratory chain. It is the last known enzyme in the chain due to its complexity of being a very large complex comprising of a hydrophilic and a hydrophobic domains of about 46 subunits in total in the eukaryote enzyme. Another reason of its complexity is that the hydrophobic domain does not have any redox centers or prosthetic groups making this part in particular difficult to study.
The transfer of two electrons from NADH molecule to FMN in the hydrophilic domain of complex I and then through the Fe/S clusters up to quinone is coupled to proton translocation by the hydrophobic domain of the complex. Recently, the structure of the whole complex was solved. However, the coupling mechanism of the electron transfer and the proton translocation is still unknown.
Understanding complex I mechanism is of great importance as it is related to production of reactive oxygen species (ROS). The production of such component leeds to mutations in the mitochondrial DNA and thus causing serious health disorders.
The hydrophobic domain subunits show a high sequence similarity to a Na+/H+-antiporter called Mrp-complex. NuoL, NuoM and NuoN, NuoK and NuoJ subunits in complex I are homologoues to MrpA, MrpD, MrpC and last part of MrpA subunits in Mrp-complex respectively. Such high sequence similarity between the two complexes indicates a functional similarity too, leading to a conclusion that complex I might pump sodium in exchange to protons. The structural similarity of the two complexes would be the strongest link between complex I and Mrp-complex. However, the structure of Mrp-complex has not been solved yet, thus solving the structure of the Mrp-antiporter and understanding its mechanism will solve the mystery of complex I coupling mechanism.
This project is aiming to crystalize the Mrp-complex from Bacillus subtilis. Mrp-subcomplex composed of four subunits MrpA, MrpB, MrpC and MrpD has been successfully constructed so that a his-tagged cytochrome c550 domain was added to the C-terminal end of MrpD subunit, which would facilitate purification and quantification of the complex. MrpABCDcytH complex was successfully produced in E. coli, purified and analyzed for further experiments.}},
author = {{Al-Yahyaai, Hanan}},
language = {{eng}},
note = {{Student Paper}},
title = {{Production and Characterization of Mrp AntiporterComplex - The Evolutionary Progenitor of Complex I}},
year = {{2013}},
}