Flashcards in Cytochrome C oxidase Deck (75):
What is cytochrome C oxidase otherwise known as?
What is the full name of cytochrome C oxidase?
Ferrocytochrome c/oxygen oxidoreductase
Is the reaction catalysed by cytochrome C oxidase endergonic or exogonic?
Exogonic - it releases ~500 meV of energy
Why are 4 cytochromes needed to produce water from oxygen?
Because cytochromes are 1 electron systems
How does cytochrome c oxidase prevent energy loss?
uses energy to generate a proton gradient which is then used to drive ATP synthesis
Where is cytochrome c oxidase found?
spans the inner mitochondrial membrane
Do both half reactions occur on the same side of the membrane?
No - therefore the reactions occur in different environments
What is the effect of the two half reactions occurring on opposite sides of the membrane?
The pH difference causes a shift in the midpoint potential.
Why is cytochrome c oxidase not at its midpoint potential in functional conditions when experimentally measured?
Oxygen is in surplus and shifts the equilibrium to the right, and more water is produced. This means the functional potential for oxygen is lower than the midpoint potential.
Why is the energy released by cytochrome c ~550 meV?
cytochrome oxidation - 300meV
oxygen reduction - 700 meV
Therefore 500 meV released (700-300)
Where has ATPase been shown to be located in the mitochondria?
In the bends of the cristae in mitochondria
Where are protons pumped by cytochrome c oxidase?
From the matrix to the intermembrane space
Describe the structure of cytochrome c oxidase.
Dimeric multisubunit enzyme
Give the main subunits present in cytochrome c oxidase.
Subunit I and subunit II - there are also other regulatory
Where are the genes for subunit I and subunit II encoded in mammals?
In mitochondrial DNA
Where are other regulatory subunits encoded?
In nuclear DNA
What is the role of the nuclear encoded subunits?
To regulate the activity of the mitochondrial encoded subunits. May stabilise dimerisation of the enzyme.
Give an example of a regulatory subunit in cytochrome c oxidase.
COXIV - binds ATP to inhibit the enzyme when sufficient levels of ATP have been produced.
Describe the structure of subunit I.
60 kDa. 12 TM helices form 3 semicircular arcs with 4 TM helices per arc (view from above). Forms 3 pores within the arcs.
Name the cofactors present in subunit I.
heme a3, CuB and heme a
Describe pore A in subunit I.
contains no cofactors but includes the D channel
Describe pore B in subunit I.
contains haem a3, CuB and includes the K channel
Describe pore C in subunit I.
contains haem a
What is the purpose of the calcium and manganese ions found in cytochrome c oxidase?
Structural role - not directly involved in the reaction
Describe the structure of haem groups.
What is the major difference between haem a and haem c?
haem c has a sulfur group which allows it to be covalently bound to proteins
How are most haem groups attached to their proteins?
via their long tail regions
Describe iron in haem a?
Iron has 6 ligands and no open binding site.
How is haem a coordinated to pore C in subunit I?
By 2 imidazole groups in histidine side chains.
Describe the iron in haem a3?
Iron has 5 ligands with an open binding site - allows reaction with substrates
How is haem a3 coordinated to pore B in subunit I?
By 1 imidazole group in a histidine side chain
Why is the iron in haem a3 estimated to have a high functional potential?
Water is not excluded and can get into the open binding site to form the ferryl-like state.
Where is CuB found and how is it coordinated?
CuB is found in pore B of subunit I and is coordinated by 3 imidazole groups in His side chains, where one His240 is cross-linked to Tyr244
Which cofactor is CuB very spatially near to?
Haem a3 - they are considered as one binding site
Describe CuA in subunit II.
A dimer of two copper ions found at a feed in site in subunit II
Which subunit isn't present in quinone oxidases?
Describe the structure of subunit II.
Contains CuA and Cytc. Consists of 2 TM helices.
Why are the first 3 redox pairs all at a similar midpoint potential?
The cofactors are close together and are highly oxidising. This means that the electron transfer can occur quickly.
Why is there a big energy drop following haem a?
This is the energy used to pump protons across the membrane, and is the step which reduces the active site cofactors (in the redox cycle).
Describe the CytC oxidation.
1 electron oxidation
Describe the oxygen reduction.
4 electron oxidation
Which cofactors are involved in the redox cycle in the active site?
haem a3, CuB and Tyr*
Give the order of reduction of the active site cofactors.
Why is the Tyr radical reduced first?
It is the most highly oxidising species in the active site so gains its electron first.
Where does oxygen bind and what does this form?
To the open ligand binding site on haem a3, forming the highly unstable oxyferrous species
Give the fully reduced form of the active site.
Fe2+, Cu+, OH-Tyr
When does the 4 electron reduction of oxygen occur and why does it occur in one step?
Occurs immediately after oxygen binding. Occurs in one step to avoid generation of ROS intermediates.
What is an advantage of avoiding ROS intermediates?
Keeps the reaction more balanced as it does not go through the uneven energy increases/decreases between the ROS species. The steps of equal energy means that a similar mechanism can be used for each reaction step.
What has happened once the P state has been formed?
4 electron reduction of oxygen has occurred. The oxygen has been split to give two deprotonated waters.
What is the purpose of the rest of the redox cycle?
Uses electrons from 4CytC to reduce the oxidised cofactors in the active site and to reform the fully reduced form. Each step uses an electron from CytC and results in proton uptake.
Why does the proton from Tyr-OH remain near to Fe3+-OH in the O/E states?
To accommodate electron addition to the ferryl state
How does the whole system remain neutral throughout the reaction cycle?
Chemical protons enter the matrix to compensate for the charges in the active site.
Why are the protons taken from the matrix considered chemical protons?
They end up on the water that is formed
How much energy is needed to move one proton across the membrane?
Why does water formation generate 200 meV?
Chemical protons from the matrix meet electrons from the intermembrane space at the catalytic site, this is the equivalent of moving one electron the whole way across the membrane, against the proton gradient. This generates 200 meV.
How many protons are translocated in each step?
1 chemical proton which will end up on the water and 1 pumped proton which contributes to the proton motive force.
How do protons cross the membrane?
Via D and K channels which go from the matrix to the active site.
How much of the energy produced is conserved in the electrochemical and proton gradients?
400 meV out of the 500 meV produced
What does the excess driving force allow?
For reactions to still take place in extreme conditions.
What is the excess driving force called?
What is a likely method for proton pumping through the D and K channels?
It is likely that one channel takes a proton to the active site and that the other channel takes a proton across the membrane.
What does the charge neutrality model focus on and what does this suggest?
This model focusses on the D channel, suggesting that the protons don't have to go through separate channels.
What amino acid is necessary for charge separation in the charge neutrality model?
Where is the proton that will be pumped held during the reaction and why?
Moves past the active site to be held in the proton trap site to allow stabilisation of the electron brought in from cytochrome c.
How is the pumped proton released from the proton loading site?
As the rest of the reaction cycle occurs, the proton in the trap site is no longer stabilised by the electron (gets used) and the proton is released into the intermembrane space.
What triggers the proton to move into the proton loading site?
Electron transfer from haem a to the active site
Give a possible role of the K channel.
Likely to be involved in loading the enzyme with protons during earlier stages of the catalytic cycle whilst the D channel moves both chemical and pumped protons.
How might oxygen binding to the active site affect the D and K channels?
Oxygen binding may act as a signal to switch from using the K channel to using the D channel.
Where are disease causing mutations most likely to occur?
In the nuclear encoded subunits.
Give 3 examples of diseases caused by nuclear mutations of CytC oxidase subunits.
Leigh-Syndrome (SURF-1 mutation), neonatal failure (SCO1 mutation), cardioencephalomyopathy (SCO2 mutation)
What can mutations in the COXI subunit I cause?
Leber optic atrophy, anaemia, hearing loss and colorectal cancer.
What can mutations in the COXII subunit cause?
Leber optic atrophy, exercise intolerance, encephalopathy
What is Leber optic atrophy?
A mitochondrially inherited disease. Results in degeneration of retinal ganglion cells, causing acute vision loss.
Give the major differences between quinone oxidases and cytochrome c oxidase.
No subunit II, ubiquinol binding site in subunit I, haem b instead of haem a, haem o3 instead of haem a3, releases one extra proton per electron in comparison with cytochrome c oxidase