Lecture 5 - H+ translocation by the Respiratory Chain Flashcards
What is the function of mitochondria in the respiratory chain?
Mitochondrial function - Responsible for generating ATP via the electron transport chain. Proton pumping complexes I, II, III and IV are integral parts of the electron transport chain pump protons across the inner mitochondrial membrane which generates an electrochemical gradient which powers ATP synthesis.
What is the malate shuttle?
Malate Shuttle - NADH produced during glycolysis transfers electrons into the mitochondria. NADH is converted to malate which is able to cross the mitochondrial membrane. The malate is then oxidised to regenerate NADH inside the mitochondria.
What is the electron transport chain?
A series of protein complexes located in the inner mitochondrial membrane which transfer electrons from donors such as NADH and FADH2 to acceptors such as oxygen in a controlled manner releasing energy in the process. The electrons move from a higher energy state to a lower energy state releasing energy which is harnessed to pump protons across the inner mitochondrial membrane, this creates an electrochemical gradient.
- Electrons flow from one redox centre to the next which has a more positive redox potential
- The redox potential difference between the redox couples NADH and O2 is 1.2V equivalent to a release of 231kJ/mol of Gibbs free energy
How is electron transport and proton pumping measured?
Proton pumping can be measured by monitoring changes in pH oxygen is necessary for this to take place as it is the terminal acceptor and is therefore required for the flow of electrons.
To understand the specific roles of individual complexes in the electron transport chain inhibitors can be used to isolate the activity of each complex.
How are H+/e- stoichiometries determined?
Refers to the number of protons pumped across the membrane for every 2e- that pass through the electron transport chain. It is important for evaluating the efficiency of proton pumping by different complexes.
Complex I (NADH dehydrogenase)
Crucial in the transfer of electrons from NADH to ubiquinone in the inner mitochondrial membrane
Structure:
Complex I is a large protein complex consisting of 41 subunits, 7 of these are integral to the membrane and responsible for the transport of electrons and protons.
Redox Centres:
Complex I contains several important redox centres important in electron transfer and pumping
* NAD+ binding site
* 7 Fe-s Centres
* FMN (Flavin adenine mononucleotide)
* Tightly bound UQ (ubiquinone)
Proton Pumping:
Complex I pumps four protons across the mitochondrial membrane for every two electrons that pass through the complex. (H+/2e- = 4)
Mechanism for proton pumping
In Complex I electrons move from the N (mitochondrial matrix) side to the P (Intermembrane space) side. This movement isn’t solely due to redox potential but also the fixed positions of the redox centres within the complex. As the electrons move across the membrane they create an opportunity for the uptake of protons from the N side and release on the P side.
Initially it was thought that the bound ubiquinone in complex I was responsible for releasing protons from N to P but understanding has evolved. Most recent investigations link the reduction of the UQ to conformational changes in the complex protein which open and close H+ channels allowing translocation.
The mechanism involves the electrostatic transfer of a negative charge from the ubiquinone to the protein complex via specific residues, including glutamate and lysine. This transfer induces horizontal movement of subunits integral to the membrane. The movement of charge causes the proton channels to open.
Complex II (Succinate Dehydrogenase)
Responsible for transferring the electrons from succinate oxidation to fumarate to ubiquinone which is free in the mitochondrial membrane. Complex II doesn’t directly pump protons.
Complex III (Cytochrome Bc1)
Structure:
Composed of three subunits all Fe containing and integral
* Rieske Protein - 2Fe/2S centre
* Cytochrome c1 - Haem group
* Cytochrome b - Haem groups bL (Low redox potential) and bH (High redox potential)
Haem groups are iron-containing redox centres, they have different affinities for electrons based on their position within complex III
Proton pumping: H+/2e- = 4
Mechanism:
The structure of complex III is designed to facilitate vectorial (directional) electron movement . The position of redox centres in relation to the position in the membrane allows for the precise transfer of electrons.
The Q Cycle
The Q cycle involves two phases:
Phase I
1. Ubiquinol binds to the Qp site (on P side)
2. It is oxidised to ubiquinone
3. Releases two protons to the P side
The two excess electrons have different pathways. One electron is donated to the Rieske centre, and then it moves to cytochrome c1 where it eventually is donated to cytochrome C which carries the electron to complex IV. The second excess electron is donated to the heme bL and then on the N side to the heme bH resulting in the pumping of protons from the N side to the P side.
Phase II
1. A second Ubiquinol binds to the Qp site
2. It is oxidised to ubiquinone
3. Two protons are released to the P side
Like in phase I the electrons are recycled: one is donated to cytochrome C and the other to ubiquinone causing its partial reduction at the Qn site on the P side. The ubiquinol on the N-side is fully reduced and binds two protons from the N side before being released into the membrane adding to the pool of ubiquinol
Overall as a result of the Q cycle two molecules of ubiquinol are oxidised while one ubiquinone is reduced. The net effect is of one ubiquinol being oxidised, four protons being pumped from the N side to the P side and two electrons being moved to Complex IV.
The ability to split and recycle electrons from the oxidation of UQH2 is key to doubling the H+ pumped per UQH2 oxidized by Complex III. The membrane potential ensures energy is retained even as electrons are transferred from one side of the membrane to another (bL to bH)
Complex IV (Cytochrome c oxidase)
The final complex in the electron transport chain. It is where molecular oxygen accepts electrons and is reduced to form water.
Structure:
Complex IV has 13 subunits but for the bacterium Paracoccus, it has only four subunits which are believed to be essential for electron transport
Redox centres:
* Haem a, a3 and CuB in subunit 1
* CuA in subunit 2
* Subunits 3 and 4 have no redox centres
Proton pumping: H+/2e- = 2
Electron and proton transport
* 4 molecules of Cytochrome c sequentially deliver 4 electrons to complex IV. The electrons are used to convert oxygen to water
* As electrons are transferred four protons from the N side of the membrane are used in converting oxygen into water
As electrons are transferred four protons are pumped from the N side to the P side contributing to the electrochemical gradient
