Mitochondrial function, inhibition and disease Flashcards
What is complex 1?
The first enzyme in ETC.
How does complex 1 act?
As the rate limiting step for ATP synthesis.
How is complex 1 called?
NADH-coenzyme Q oxo reductase.
Or NADH dehydrogenase.
What are the characteristics of complex 1?
It is the least well understood of all ETC complexes.
It is the most difficult to obtain a crystal structure for it.
How many subunits make complex 1??
46 different subunits.
What much molecular mass of 1 giga Dalton does complex 1 have?
A huge molecular mass.
Where is the complex encoded?
Some in the mitochondrial genome.
The rest by nuclear genome.
Which part of the complex 1 is encoded in the inner mitochondrial membrane?
The hydrophobic part.
What part of complex 1 stays out into the lumen of the mitochondria and it is encoded by nuclear genome?
The hydrophilic part.
How many subunits out of 46 are important in complex 1 for its catalytic and redox activity?
14 of 46 subunits.
What do the rest of 46 subunits of complex 1 that are not important for its redox and catalytic activity do?
They modulate its activity.
What is the function of enzyme complex 1?
It catalyses NADH oxidation.
From where does NADH oxidation comes from?
From Krebs cycle reactions.
Where do Krebs cycle reactions for NADH oxidation occur?
In the mitochondrial lumen.
By what factor do Krebs cycle reactions for NADH oxidation occur?
By ubiquinone.
What is ubiquinone?
A lipid.
Soluble.
Where does ubiquinone sits?
In the inner membrane.
What does NADH oxidation do?
It takes 2 electrons/NADH –> pumps 4 protons –> redox energy released.
What does redox energy released from NADH oxidation allow?
The pumping of protons from the lumen of the mitochondria across the inner membrane against their concentration gradient.
How many modules does complex 1 have?
3 modules.
What are the three modules of complex 1?
- Electron input module / dehydrogenase module (N module).
- Electron output module / hydrogenase module (Q module).
- Proton translocation module (P module).
What is the function of N module of complex 1?
Accepts electrons from NADH.
What is the function of Q module of complex 1?
Delivers electrons to ubiquinone.
What is the function of P module of complex 1?
Pumps protons across inner membrane.
Which two of the three modules of complex 1 are parts of the matrix arm of the complex?
N and Q modules.
Where does the P module of complex 1 lie?
Within the portion in the membrane.
What molecule binds to complex 1 firstly?
NADH.
What does NADH do once it binds to complex 1?
It transfers 2 electrons to the flavin mononucleotide / FMN prosthetic group of complex 1.
What does the transform of 2 electrons from NADH to the complex 1 do?
It recycles NAD+ –> produces H+ –> creates FMNH2 in the complex.
Where are the electrons from NADH transferred then?
They are transferred through FMN via series of iron-sulphur (Fe-S) clusters –> travel up hydrophilic arm of complex.
What does the redox state of the protein induces?
A conformational change –> alters dissociation constant of side chains –> causes 4 H+ –> pumped from mitochondrial matrix –> across intermembrane space.
What happens to the electrons released from redox reaction in complex 1?
They are handed to ubiquinone.
How is ubiquinone called alternatively?
Co-enzyme Q.
Why is ubiquinone called alternative Co-enzyme Q?
Because it is indicated by a Q in the hydrophobic part of the enzyme.
How many electrons from redox reaction in complex 1 does ubiquinone accept?
2 electrons.
To what is ubiquinone reduced after it accepts 2 electrons from NADH in redox reaction in complex 1?
It is reduced to ubiquinol (CoQH2).
What happens to H+ released from NADH oxidation in complex 1 in the begining?
They are trapped in the intermembrane space until they can move back.
Through what can the H+ released from NADH oxidation in the beginning of complex 1 move back?
Through UCP1 –> leak across naturally slowly.
Or through ATP synthase.
What does complex 1 produce in respiration?
Free radicals.
What are the free radicals?
They are highly reactive compounds.
Why are the free radicals highly reactive compounds?
Because they have unpaired electrons.
How are free radicals useful?
In small quantities –> useful cell signals.
What can a build up of free radicals cause?
Cell dysfunction.
Why a build up of free radicals causes cell dysfunction?
Because they damage macromolecules with double bonds.
Where does the damage of macromolecules with double bonds leads to?
To inflammation.
Calcium influx.
Cell death if uncontrolled.
What happens if oxygen delivery is blocked?
The final electron acceptor is in short supply –> ROS levels build up quickly –> damage.
What can produce ROS?
Complex 1.
Why does complex 1 produces ROS?
Because electrons ‘leak’ out –> interact with oxygen –> later complexes work slower.
Where does the production of ROS by complex 1 is important?
In cell signalling.
In apoptosis.
Programmed cell death.
How many complex 1 inhibitors exist?
60.
As what are some of the 60 complex 1 inhibitors developed?
As agents used in research.
Why do they use some of complex 1 inhibitors in research?
To explore mitochondrial function.
Where else can some other complex 1 inhibitors used?
In medicine.
What is the most common use of complex 1 inhibitors?
As pesticides.
In how many pesticides group do complex 1 inhibitors fall into and based on what criteria?
3 groups.
Based on how they work.
What are the 3 groups that complex 1 inhibitors fall into as pesticides?
- Quinone antagonists = acetogenins.
- Semiquinone antagonists = rotenone.
- Quinol antagonists = myxothiazol.
What is the function of Quinone antagonists as complex 1 inhibitors?
They act at the entry of the hydrophobic site.
What is the function of Semiquinone antagonists as complex 1 inhibitors?
They act in the intermediate steps by disrupting the electron transfer between terminal FeS cluster and ubiquinone.
Unknown specific action site.
What is the function of Quinol antagonists as complex 1 inhibitors?
They prevent formation and release of product.
Are the inhibitors that prevent NADH interaction with the enzyme of complex 1 specific?
No.
Where do the inhibitors of complex 1 act on to prevent NADH interaction with the enzyme?
They act on all other enzymes that rely on NADH.
Which of the 3 complex 1 inhibitors is the most commonly used?
Rotenone.
What are Piericidin and Myxobacterial antibiotics?
Acetogenins.
Useful anticancer drugs.
Act on complex 1.
Where do Tranquiliser barbiturate amytal, some neuroleptic drugs and neurotoxins, capsaicin from hot chilli peppers act?
On complex 1.
What does the antidiabetic drug Metformin inhibit?
Complex 1.
Where is the function of the antidiabetic drug Metformin that inhibits complex 1 important?
For its function.
Which is the strongest inhibitor of complex 1?
Bullatacin.
Which organisms are sensitive to complex 1 inhibition?
Insects.
Fish.
Why do Rotenone and similar compounds used as pesticides?
Because insects and fish mitochondria are sensitive to complex 1 inhibition.
What happens to NADH if complex 1 is inhibited?
NADH is not oxidised.
What happens when NADH is not oxidised?
There is no supply of NAD+.
What happens when NAD+ supply does not occur?
Krebs cycle activity is reduced –> Oxygen consumption slows.
What happens when Krebs cycle activity is reduced?
H+ pumping slows down.
What happens when H+ pumping slows down?
Membrane gradient is reduced.
What happens when the membrane gradient is reduced?
protonmotive force is weaker –> the ATP production is slowed down.
What effects can ATP reduction have?
Global effects on cell.
Why does the ATP reduction have global effects on cell?
Because all enzymes are affected.
What is the most notable effect caused by ATP reduction?
Reduced ion pumping –> K+ leaves cells –> Ca2+ floods in.
When is the complex 1 inactivated?
During ischaemia reperfusion.
What happens during ischaemia reperfusion?
Stroke.
Or heart attacks.
–> Tissue damage.
What happens to complex 1 in absence of oxygen?
It loses FMN co factor.
Becomes inactive.
In what disease is complex 1 dysfunction implicated?
In Parkinson’s disease.
What do complex 1 inhibitors cause?
Cell death.
Changes.
Parkinson’s disease in neurones in cell culture.
What diseases are associated with complex 1 dysfunction?
Several neurological diseases.
Type 2 diabetes.
Cardiac disease.
Various cancers.
Where do neurons and pancreatic isle beta cells rely heavily on?
On mitochondrial ATP.
NAD -linked pathways.
What does the reliance of neurons and pancreatic islet beta cells on mitochondrial ATP and NAD-linked pathways makes them?
Very vulnerable.
What is the characteristic of cancer cells in culture?
They have low mitochondrial density.
What does the low mitochondrial density of cancer cells in culture make them?
More vulnerable to complex 1 inhibition than other cells.
What are the motor skill symptoms of Parkinson’s disease?
Bradykinesia. Vocal symptoms. Rigidity and postural inability. Tremors. Walking or gait difficulties. Dystonia.
What are the nonmotor skill symptoms in Parkinson’s disease?
Mental/behavioural issues. Sense of smell. Sweating and melanoma. Gastrointestinal issues. Pain.
How is complex 2 called?
Succinate reductase.
Succinate ubiquinone oxidoreductase.
What does complex 2 contain?
Succinate dehydrogenase.
What is succinate dehydrogenase?
An enzyme in Krebs cycle.
How many protein subunits occur in complex 2 ?
4
Which are the 4 subunits in complex 2?
- Succinate dehydrogenase (SDHA).
- Succinate dehydrogenase iron-sulphur subunit (SDHB).
- Succinate dehydrogenase complex subunit C (SDHC).
- Succinate dehydrogenase complex subunit S (SDHD).
What do SDHA + SDHB subunits of complex 2 do?
They project into the lumen.
What are the subunits C +D of complex 2?
They are membrane bound.
By what genes is complex 2 encoded?
By only nuclear genes.
What does succinate dehydrogenase catalyse?
Succinate oxidation to –> fumarate.
Where does oxidation of succinate to fumarate occur?
In the lumen of mitochondria.
By what subunit of complex 2 does the succinate oxidation to fumarate performed?
By SDHA.
What does subunit SDHA of complex 2 in the process of oxidation of succinate to fumarate?
It reduces FADH –> FADH2.
Transfers electrons through SDHB via iron sulphur complexes.
How is the process of SDHA transferring electrons through SDHB via iron sulphur complexes called?
Electron tunnelling.
Why does SDHA transfers electrons through SDHB via iron sulphur complexes?
To oxidise ubiquinone.
Where does ubiquinone oxidation occur?
In the inner mitochondrial membrane.
What happens in ubiquinone oxidation process?
Q –> QH2.
By which subunits of complex 2 does Q converted into QH2 in the oxidation process of ubiquinone?
By SDHC + SDHC.
What do SDHD + C complex 2 subunits contain?
A haem group.
As what does the haem group of complex 2 SDHD + C subunits act?
As electron sink.
What is the haem group of complex 1 SDHD + C subunits?
A protection mechanism.
What is the function of haem group in complex 2 SDHD + C subunits?
Stops excess electrons that react with molecular oxygen and form free radicals.
Which is the only membrane bound enzyme in Krebs cycle?
Succinate dehydrogenase.
Where are the other Krebs cycle enzymes found?
In the mitochondrial lumen.
Not membrane bound.
What does the unique structure of complex 2 mean?
It can act as a regulator of metabolism.
Why does complex 2 can act as a regulator of metabolism?
Because it is involved in Krebs cycle and ETC.
When does the activity of succinate dehydrogenase drops very low?
Below a membrane potential of -60mV to -80mV.
What happens to the activity of succinate dehydrogenase above -80mV?
It has a very high activity.
What can the high activity of succinate dehydrogenase control?
Krebs cycle during hypoxia.
What happens to ubiquinone during hypoxia in Krebs cycle?
It is reduced.
How is the complex 2 characterised in ETC?
Unique.
Why is complex 2 unique in ETC?
Because it does not pump protons.
What does complex 2 do in association with complex 1?
It runs in parallel to complex 1.
Why complex 2 cannot contribute to protonmotive force?
Because it does not pump protons.
What does complex 2 have?
A prosthetic group.
What is the prosthetic group of complex 2?
Subunit E.
What does subunit E in complex 2 do?
It helps FAD bind to subunit A.
How many complex 2 inhibitors exist?
2.
Which are the complex 2 inhibitors?
- Malonate + Krebs cycle intermediates.
2. Carboxin.
What does Malonate and Krebs cycle intermediates do as inhibitors of complex 2?
They prevent succinate binding.
Prevent electron build up.
Prevent formation of superoxide radical.
What does Carboxin do as an inhibitor of complex 2?
It prevents ubiquinone binding.
As what are ubiquinone binding inhibitors often used?
As fungicides.
What do some fungi have to fungicides?
Developed resistance.
What happens when complex 2 is inhibited?
Proton motive force is not affected.
Free radical production increases.
Low oxygen sensing pathways are switched on.
What does complex 2 generate when complexes 1 and 3 are inhibited?
Large ROS amount.
What does complex 2 normally generate?
Protection against ROS.
What is the importance of complex 2?
In making sure quinone pool in the inner membrane is reduced.
As what does complex 2 act?
As an antioxidant.
Why does complex 2 act as an antioxidant?
Because free radicals steal its electrons –> protect macromolecules from damage by ROS.
What can ROS do and cause damage if complex 2 does not act as an antioxidant?
Steal electrons from lipid and nucleic acids.
What are the consequences of complex 2 inactivation?
ROS increases.
DNA mutations increase.
Cell cycle progression increases.
Why does cell cycle progression increases when complex 2 is inactivated?
Because p53 protein levels drop.
What does the increase in succinate signals causes to the cell?
Low Oxygen levels.
What is the consequence of low oxygen levels in cell?
Hypoxia factor is made.
What do Hypoxia and complex 2 inactivation cause?
Tumours to arise and survive.
What are the diseases associated with complex 2 characterised?
Rare.
Why are the diseases associated with complex 2 rare?
Because complex 2 enzyme is vital.
Any disorders are usually lethal at embryo stage.
Are there any complex 2 disorders?
Yes.
What do the complex 2 disorders produce?
A wide range of symptoms.
What is one of the complex 2 disorders?
Leigh disease.
What is the complex 2 Leigh disease?
A degenerative neurometabolic disorder.
What does Leigh disease of complex 2 involves?
Loss of previously acquired motor skills. Loss of appetite. Vomiting. Irritability. Seizures.
When do the symptoms of Leigh disease of complex 2 onset?
In infants.
3 months old.
2 years old.
When can Leigh disease of complex 2 begin?
Much later in life.
What are the factors that cause Leigh disease of complex 2?
Arginine changes to –> tryptophan.
Where does arginine change to tryptophan and causes Leigh disease of complex 2?
At position 544 in SDHA subunit.
What does the change of arginine to tryptophan at 544 position in SDHA produces and then causes Leigh disease of complex 2?
It slows SDH activity.
Where do mutations of iron-sulphur complexes / cytochrome b subunits involved?
In electron transfer.
What do mutations of iron-sulphur complexes / cytochrome b subunits in electron transfer cause?
Familial neural crest-derived tumours in head / neck.
Where are neural crest-derived tumours in neck and head from mutations in complexes/ cytochrome b subunits in electron transfer found?
In highly vascular organs = carotid body.
What does make the highly vascular organs more reliant on glycolysis for ATP production?
The lack of efficiency of ETC.
Where else do SDHD mutations implicated?
In the development of Huntington’s disease.
What is Huntington’s disease?
A hereditary neurodegenerative disease.
How is complex 2 important in cancer biology?
As a tumour suppressant.
In oxygen sensing.
Where else is complex 2 dysfunction involved?
In development of diabetes through ROS and succinate production - insulin secretion pancreas link.
What are the early symptoms of Huntington’s disease?
Moodiness. Fidgeting. Cognitive issues. Personality shifts. Depression. Trouble focusing. Muscle twitching.
What happens during ischaemia?
Succinate accumulates due to reversal of succinate dehydrogenase (SDH) activity.
As what does accumulated succinate act?
As a store of electrons that drive reverse electron transport (RET) + reactive oxygen species (ROS) production.
When does accumulated succinate act act as a store of electrons?
When it is rapidly re-oxidised on perfusion.
When do SDH reversal and succinate accumulation inhibited?
During ischaemia.
By which substance do SDH reversal and succinate accumulation inhibited during ischaemia?
By malonate.
What is malonate?
A competitive inhibitor of SDH.
What happens on reperfusion?
Accumulated succinate –> rapidly re-oxidised.
By which factor is accumulated succinate re-oxidised on perfusion?
By SDH.
Where does re-oxidation of accumulate succinate by SDH result?
To reverse electron transport (RET).
Superoxide (O2) production from flavin mononucleotide site of complex 1 (C1).
What else does competitive inhibitor of SDH = malonate, inhibits?
Rapid re-oxidation of succinate by SDH on perfusion.
What is the function of malonate in the inhibition of succinate by SDH on perfusion?
It prevents RET.
Prevents ROS production.
What can be used to treat or reduce ischaemia reperfusion injury?
Malonate esters.
Wat are the causes of ischaemia?
Stroke.
Heart attack.
When do stroke and heart attack occur during ischaemia?
During ischaemia reperfusion injury.
Surgery.
Transplantation.
Hypovolemic shock.
Which tissues are the most vulnerable to ischaemia cuses?
Tissues with lots of mitochondria = brain, heart, kidney.
As what is complex 3 known?
As cytochrome C oxidoreductase.
Co enzyme Q cytochrome c oxidoreductase.
Cytochrome bc1 complex.
By how many subunits is complex 3 made?
11 subunits.
What are the 3 subunits of the total 11 subunits in complex 3?
They are respiratory subunits.
Where are the 3 respiratory subunits of complex 3 involved?
In redox.
In proton pumping.
How many proteins occur in the process of redox and proton pumping of the 3 subunits of complex 3?
2 core proteins.
6 small accessory proteins.
By what are the proteins involved in the 3 subunits of complex 3 encoded?
By mitochondrial and nuclear genes.
What else does complex 3 have?
4 co factors.
For what are the 4 co factors of complex 3 required?
For the activity of complex 3.
Which are the 4 co factors of complex 3?
- Cytochrome c1.
- Cytochrome b 562.
- Cytochrome b 566.
- Iron sulphur complex.
With what does the complex 3 couple?
With the inner mitochondrial membrane.
What does the reaction of complex 3 called?
Ubiquinone .
Q cycle.
What does the reaction of complex 3 involve?
The reduction of cytochrome c.
Oxidation of cytochrome c.
Oxidation of coenzyme Q.
What happens in the ubiquinone reaction of complex 3?
4H+ –> pumped in –> inner membrane space.
How many protons are taken up from the matrix in the reaction of complex 3?
Only 2 H+.
What does the fact that : matrix only takes up 2 H+ in the reaction of complex 3?
The inter membrane space –> becomes –> more positive relative to matrix.
What happens to the 2 electrons after they are taken up by the matrix in the reaction of complex 3?
They are transferred from ubiquinol –> to ubiquinone.
How are the 2 electrons transferred from ubiquinol to ubiquinone in the reaction of complex 3 after they are taken up by matrix?
By the two cytochrome c co factors.
How do the cytochrome c co factors help the 2 electrons to transferred from ubiquinol to ubiquinone?
Cytochrome b –> binds –> ubiquinone (QH2) in membrane + ubiquinol (Q).
Where are cytochrome b and ubiquinone + ubiquinol bind?
Within complex 3.
At Qo and Qi sites.
What do iron sulphur complex and bL haem group do?
They pull 2 electros from the Qo site.
They hand one electron to –> cytochrome c1.
They hand the other electron to –> the other haem group (bH).
What does cytochrome c1 in complex 3 do?
It hands electrons to –> cytochrome c.
What does the other electron in complex 3 do?
It tracks down through complex 3.
It hands electron to –> ubiquinone at Qi.
How many does the process of electron that tracks down from complex 3 and hands it to ubiquinone Qi occur?
Twice.
What happens in the process where the electron tracks down from complex 3 and hands it to ubiquinone Qi?
It pulls 4 H+ to –> inner membrane space from ubiquinone oxidation.
It removes 2 H+ from matrix –> use in reducing ubiquinol.
What is the key think that happens in complex 3?
Complex 3 –> pumps H+ as it hands on electrons.
Proton movement = unequal.
Intermembrane space becomes more positive than matrix.
Where does the more positive inter membrane space than matrix help?
To create and maintain proton motive force for ATP synthesis.
Which is one of the main inhibitors of complex 3 used in laboratory settings?
A compound called: Antimycin A.
What is Antimycin A?
A secondary metabolite of Streptomyces bacteria.
What does Antimycin A inhibit?
The transfer of electrons from heme bH to oxidized Q.
Where does Antimycin A act?
At Qi site towards matrix side of complex 3.
What is Atovaquone?
An anti malarial.
What does Atovaquone inhibit?
The transfer of electrons from QH2 to iron sulphur complex.
Where does Atovaquone inhibition process for electron occur?
At Qo site.
Under what name is Atovaquone drug sold?
Under brand name: Mepron.
For what is Atovaquone drug used?
To treat other parasitic diseases: Pneumocystis pneumonia.
Toxoplasmosis.
Babesia.
What is pneumocystis pneumonia?
A type of pneumonia common in AIDS and kidney transplant patients.
What is toxoplasmosis?
A disease caused by a parasite common in cats.
A zoonotic.
What is babesia?
A tick borne parasite that infects livestock.
A zoonotic.
What does ‘zoonotic’ mean?
It can be transferred from animals to humans.
What are some other compounds that acts in a similar way by binding at Qo site?
Myxothiazol.
Stigmatellin.
What is myxothiazol?
An antifungal agent.
What is stigmatellin?
An antibiotic produced by myxobacteria.
What happens when complex 3 is inhibited?
Proton motive force –> collapses.
Cell can longer make ATP to meet its needs.
Why does the cell can no longer make ATP to meet its need after proton motive force collapses when complex 3 is inhibited?
Because there is no longer a head of protons able to rotate ATP synthase molecule.
What occurs naturally from complex 3 when more electrons arrive than reducing ubiquinol?
Electron leakage.
How are the electrons from complex 3 react when more electrons arrive?
Prematurely.
With molecular oxygen.
They produce superoxide free radical.
What happens when ROS levels build up?
They can cause diseases driven by macromolecular damage and inflammation.
What are ROS thought to be responsible for?
Aging.
What does inhibition of complex 3 by antimycin A can cause?
The haem group –> ‘locked’ in reduced state –> re-oxidised.
What does the lock of haem group to re-oxidised mean?
Electrons can’t be handed on from Qo.
They are transferred to oxygen instead.
What can complex 3 inhibition cause?
ROS production at much higher levels than occurs naturally.
What is the result if complex 3 is not functional?
Lethal.
What are people with genetic abnormalities in complex 3?
Very rare.
What are the characteristic of Biornstard syndrome?
Hearing loss.
Brittle hair.
How many people are affected by Biornstard syndrome globally?
50 cases.
For what is GRACILE syndrome stands for?
Growth Retardation, Aminoaciduria, Cholestasis, Iron overload, Lactic acidosis and Early death.
How many people are globally affected by GRACILE syndrome?
30 cases.
What are the causes of Biornstrad and GRACILE syndrome?
ROS build up + oxidative damage.
ATP lack.
From what are milder conditions caused by?
Exercise intolerance.
What happens in exercise intolerance?
Resting metabolism can be supported.
ATP cannot be generated fast enough.
How is complex 4 called?
Cytochrome c oxidase (COX).
Why is complex 4 called cytochrome c oxidase?
Because it oxidises cytochrome c handed on from complex 3.
What is complex 4?
The final complex of ETC.
By what is the reaction finished in ETC by complex 4?
By using H+ + molecular O2 : in mitochondrial matrix –> producing water.
Where is complex bound?
In the inner mitochondrial membrane.
By how many subunits is complex 4 made?
By 14 subunits.
By what else is complex 4 made except from subunits?
Metal cofactors.
Two haems.
Two cytochromes.
Two copper clusters.
Where are the 3 of the complex 4 subunits encoded?
In the mitochondrial DNA.
By what are the 11 complex 4 subunits encoded?
By nuclear genes.
Where are two haem molecules of complex 4 found?
In subunit 1.
Where do the two haem molecules in complex 4 help?
In electron transport to subunit 2.
What happens in subunit 2 of complex 4?
2 copper molecules receive and pass on electrons.
How many electron does complex 4 receives from each of four cytochrome c molecules that have gained electrons from complex 3?
1 electron from each.
What does complex 4 do with the electrons that receives from the 4 cytochrome c molecules?
It transfers them via 2 haem + 2 copper groups to –> 1 oxygen molecule.
It combines the oxygen with 4 H+ from mitochondrial lumen –> produce 2 water molecules.
What does complex 4 pumps across membrane to inter membrane space?
4 H+.
What the pumping of 4 H+ in the inter membrane space from complex 4 increases?
The electrochemical difference across membrane.
Membrane potential initiated by complex 1 and complex 3 actions.
What does the adding to proton motive force by complex 4 help to maintain?
ATP synthesis.
How can the adding to proton motive force by complex 4 maintain ATP synthesis?
By ensuring there is a head of protons in intermembrane space.
Where is superoxide radical generated at complex 1?
In mitochondrial lumen.
Where else is superoxide radical generated in mitochondrial lumen?
At complex 2.
Where is superoxide released at complex 3?
On either side of the membrane.
By which factor is superoxide released at complex 3?
By leakage of electrons from ubiquinone.
Why is complex 4 not such an important site of electron leakage?
Because it is already handing electrons to molecular oxygen.
The leaks tend to happen before.
Where can complex 4 inhibitors act?
On complex 4.
When can inhibitors of complex 4 act on it?
When it is at one of the three different states that affect its conformation.
What are the three states that affect the conformation of complex 4?
- Fully oxidized (pulsed).
- Partially reduced.
- Fully reduced.
When does the fully pulsed state of complex 4 has highest activity?
When both haem a3 and one of the copper groups are oxidized.
When does partially reduced state of complex 4 occur?
When receiving 2 electrons from cytochrome c has a shape that allows oxygen to bind.
When does the fully reduced state occur?
When the enzyme is inactive.
What do different inhibitors have for the different states of complex 4?
Different affinity.
Where do cyanide, azide, and carbon monoxide act?
On complex 4?
What inhibitors are cyanide, azide and carbon monoxide?
Non competitive inhibitors.
Where do cyanide, azide and carbon monoxide bind on the complex 4?
Tightly on its active site.
What happens to the oxygen levels when cyanide, azide and carbon monoxide inhibitors present on complex 4?
More oxygen is needed.
Why more oxygen is needed when cyanide, azide and carbon monoxide inhibitors occur in complex 4?
To maintain cellular respiration.
What are nitric oxide and hydrogen sulphide on complex 4?
Allosteric regulators of COX.
Where does cyanide sit?
Between haem and copper groups.
What is the function of cyanide in complex 4?
It prevents haem and copper groups from participating in redox reaction.
They can not receive electrons from cytochrome c.
Where does cyanide inhibitor bind in complex 4?
It binds with high affinity in pulsed and partially reduced states.
What are the consequences of cyanide binding on complex 4?
It produces chemical asphyxia.
Histotoxic hypoxia.
What is histotoxic hypoxia?
A situation where oxygen is available but can’t be used.
What can the swallow of a small quantity of solid cyanide or a cyanide solution of 200 mg or exposure to airborne cyanide of 270 ppm do to us?
Kill us in minutes.
Why do spies have cyanide pills to take when captures to avoid interrrogation?
Because it can kill us in minutes.
Are there any ante-dotes to cyanide?
Yes.
Need to be taken quickly.
Why do ante-dotes of cyanide need to be taken quickly?
To rapidly produce a lot of ferric iron (FE3+) –> displace cyanide from cytochrome.
Why ante-doted displace cyanide from cytochrome?
Because cyanide has a higher affinity for the ferric iron.
What is the old method of displacing cyanide from cytochrome?
Inhalation of amyl nitrite.
Swallowing sodium nitrite + sodium thiosulphate.
What will nitrites do?
Oxidise haemoglobin.
Produce methaemoglobin.
Where does methaemoglobin bind?
To cyanide.
What does the binding of methaemoglobin to cyanide comfort?
The COX enzyme binding.
What is happening to the cyanide after it binds to methaemoglobin?
It is converted to an easily excretable form.
Removed by kidneys.
Peed out in urine.
What does the more modern version of displacing cyanide from cytochrome avoid?
Messing with haemoglobin.
What does haemoglobin do?
It creates oxygen delivery problems.
What does the modern method of displacing cyanide from cytochrome uses instead of haemoglobin in order to not create oxygen delivery problems?
It uses hydroxocobalamin.
Which is the cobalt used in the modern method of displacing cyanide from cytochrome?
The metal that moves cyanide.
What can high nitric oxide concentration displace and comfort?
It can displace cyanide.
It can comfort cyanide’s effects.
Are nitric oxide concentration levels high enough endogenously?
No.
What does nitric oxide do?
It binds reversibly to metal groups.
Becomes reduced to –> nitrite.
What can nitric oxide facilitate at low levels?
Oxygen delivery into deeper tissues.
How can nitric oxide deliver oxygen into deeper tissues ate lower levels?
By slowing down the rate of activity in complex 4.
What happens when the rate of activity in complex 4 is slowed down by nitric oxide?
Oxygen is not used up as fast.
Oxygen can diffuse further into tissue.
What are carbon monoxide and hydrogen sulphide as inhibitors?
Non competitive.
What do carbon monoxide and hydrogen sulphide produce?
Histotoxic hypoxia.
Where does carbon monoxide bind?
To haemoglobin.
What does carbon monoxide do once it binds to haemoglobin?
It disrupts its supply.
Disrupts oxygen use.
Why is it really important to get boilers serviced, based on the carbon monoxide disruption of haemoglobin supply and oxygen use?
Because no one asphyxiates from fumes from incomplete combustion.
What can ATP inhibit in complex 4?
The enzyme.
Why does ATP inhibit the complex 4 enzyme?
To reduce the activity of electron transport chain.
How does ATP act when it reduces the activity of electron transport chain because it inhibits the enzyme of complex 4?
As a negative feedback form.
What does complete complex 4 inhibition reduces?
The proton motive force.
Why is ROS generation not associated with complex 4?
Because ROS hands electrons to oxygen anyway.
All the ‘leaks’ in the system happen before this step.
Which tissues have high metabolic rates?
Brain.
Heart.
Kidney.
What are the tissues that have high metabolic rates most susceptible to?
Complex 4 inhibition or dysfunction.
What are the defects in complex 4?
Fatal.
What are the genetic abnormalities in complex 4?
Rare.
Where do disorders of complex 4 manifest?
In early childhood.
What do the disorders of complex 4 affect most severely?
The tissues with the highest metabolic rates.
With what are the disorders of complex 4 associated?
With severe impairments.
Where are the most often impairments in complex 4 diseases?
In genes.
What do the genes do with the impairments in complex 4 diseases?
They control the meeting of complex.
Which are the diseases of complex 4?
Leigh syndrome. Cardiomyopathy. leukodystrophy. anaemia. deafness.
From where does deafness occur in complex 4 diseases?
From impaired neural function.
Where do patients experience lactic acidosis in complex 4 diseases?
Because, oxidative phosphorylation is impaired.
Pyruvate cannot be departed to mitochondria very quickly.
What are the symptoms of Leigh disease?
Damaged eye sight = Retinitis Pigmentosa. Lactic acidosis. Hypoglycaemia. Aciduria. Deafness. Muscle tone lack. Motor function loss. Cognitive abilities. Brain development impairment. Speed failure. Recurrent infections.
Why is ‘hypoglycaemia’ a symptom of Leigh syndrome?
Because people with this syndrome rely heavily on glycolysis for ATP synthesis.
What do the symptoms of Leigh syndrome highlight?
How the central normal electron transfer and ATP generation underpin health.
What is ATP synthase?
One of the most ancient and highly conserved enzymes.
Why is ATP synthase ancient and highly conserved?
Because its function is vital for life.
Where do ATP hydrolysis and synthesis occur?
On three catalytic sites in F1 sector.
Where does proton transport occur?
Through membrane-embedded Fo section.
By how many subunits is Fo made?
8.
Which are the subunits of Fo?
c-ring.
b subunits.
Where is Fo section embedded?
In the membrane.
By what subunits is F1 made?
α
β
γ
δ
Where are proteins trapped as they cannot pass back through the inner mitochondrial membrane?
In the intermembrane space.
Why can protons no pass back through the inner mitochondrial membrane on their own?
Because, the core of bilayer membrane is too hydrophobic for ions to get through large amounts.
What do protons need to pass back through the inner mitochondrial membrane?
Help.
In the form of ATP synthase.
Complex 5.
What is the formation of ATP from ADP?
Energetically favourable.
Why is the formation of ATP from ADP energetically favourable?
Because, it requires energy input to add on phosphate group to ADP.
What does ATP synthase do to drive the synthesis of ATP from ADP?
It traps the energy from the proton gradient.
By bow many subunits does ATP synthesis consist?
2.
Which are the 2 subunits of ATP synthesis?
Fo.
F1.
What can F1 do?
Rotate in relation to Fo.
What does Fo anchor?
In the membrane.
What does the ability of F1 to rotate mean?
That it is a molecular machine/turbine.
Though where do protons move?
Through a channel in Fo.
Where do protons through Fo channel bind to?
To a ring on Fo.
What does the proton binding to a ring on Fo cause?
Fo to rotate a notch.
From where can protons exit after they bind to a ring on Fo and cause it to rotate a notch?
They exit from Fo into the lumen.
What is the exit of protons from Fo into the lumen like?
Like a one way revolving door.
Where are the protons then transferred after they exit from Fo into the lumen?
To the F1 subunit.
By which factor are the protons transferred to F1 subunit?
By a central stalk that connects Fo + F1 subunits.
What is the shape of the stalk that connects the Fo+F1 subunits of ATP synthase?
A cam shaft.
What happens when the stalk that connects Fo+F1 subunits of ATP synthase rotate?
It squashes F1 subunit.
Causes conformational change in F1.
By how many dimers is F1 made?
3.
Where are the 3 dimers of F1 arranged?
In a ring.
What happens in ATP synthesis total process then?
ADP +Pi bind in gap between Fo+F1 subunits of ATP synthase. Stalk rotates. Fo+F1 squash together. ADP+Pi squash together. ADP+Pi fuse. ADP+Pi form ATP.
By which factors is the second rotation of the stalk driven?
By H+ movement through Fo.
What does the other stalk rotation let happen?
Dimers pop apart again.
They release new formed ATP.
Allow ADP+Pi to bind again.
What does keep the 2 dimers together in ATP synthase?
A flexible peripheral stalk.
What the flexible peripheral stalk that holds the 2 dimers of ATP synthase allows?
The 2 dimers to flex with each rotation of the internal stalk.
What gives the inner mitochondrial membrane its characteristic folds called cristae?
ATP synthase dimerization.
What is the meaning of ATP synthase dimerization?
Proton gradient is focussed near ATP synthase –> makes the process highly efficient.
What are the different classes of ATP synthase inhibitors?
Peptide inhibitors. Polyphenolic phytochemicals. Polyketides. Organotin compounds. Polyenic α-pyrone derivatives. Cationic inhibitors. Substrate analogues. Amino acid modifiers.
Which antibiotics inhibit ATP synthase?
Efrapeptins.
Aurovertins.
Oligomycins.
What do ‘efapeptins’ and ‘aurovertins’ inhibit?
ATP synthesis.
ATP hydrolysis.
Where does ‘efrapeptins’ bind?
To ATP synthase at a site that extends from rotor.
Across central cavity of enzyme.
In the specific β-subunit catalytic site.
What does the binding of ‘efrapeptins’ into β-subunit catalytic site of the enzyme prevent?
Closure of β subunit during rotation.
Where do molecules of ‘aurovertin’ bind?
To the cleft between the nucleotide-binding and C-terminal domains of 2 β subunit domains.
How does ‘oligomycin’ inhibit ATP synthase?
By binding in Fo sector.
Blocking proton conduction.
Why is Fo called Fo?
Because, it binds oligomycin.
What do the ATP synthesis inhibitors uncouple?
Oxidation from phosphorylation.
What does the uncoupling of oxidation from phosphorylation mean?
Electron transfer works normally.
Protonmotive force remains intact.
Oxygen consumption occurs.
ROS generation occurs but is not used for ATP synthesis.
What is ‘Rotenone’ ?
The most well known and used lab inhibitor of complex 1.
What does ‘Carboxin’ inhibit?
Complex 2.
What do ‘Antimycin A’ and ‘ Strobilurin’ impact?
Complex 3.
By which factors is complex 4 inhibited?
By compounds like ‘ cyanide’ , ‘ phosphine’ , ‘nitric oxide’, ‘hydrogen sulphide’, ‘carbon monoxide’, ‘azide’.
What does ATP exert?
Negative feedback on complex 4.
By which factors are ATP synthase or complex 5 inhibited?
By ‘oligomycin’, ‘carbodiimide’, ‘diafenthurion’.
What are the diseases of ATP synthase?
Rare.
Why are the diseases of ATP synthase rare?
Because, ATP synthesis is vital to multicellular life.
With what are the human neuromuscular disorders associated with?
Defects in ATP synthase.
How many known mutations are in mitochondrial genes?
58.
How many subunits encode mitochondrial genes?
8.
α of ATP synthase.
How many distinct genetic mitochondrial dysfunction syndromes occur?
150.
By which factor are genetic mitochondrial dysfunction syndromes caused by?
Reduced oxidative phosphorylation.
How many human births are affected by genetic mitochondrial dysfunction syndromes?
1 in 5000.
What are the typical symptoms of genetic mitochondrial dysfunction syndromes?
Visual/hearing defects. Encephalopathies. Cardiomyopathies. Myopathies. Diabetes. Dangerous liver and kidney function.
To what are the mitochondrial genes sensitive?
To mutations.
Why are mitochondrial genes sensitive to mutations?
Because, they are close to the sites of ROS production.
Mitochondrial genome has less developed repair systems than nuclear genome.
What is FILA?
Fatal Infantile Lactic Acidosis.
What is LHON?
Leber Hereditary Optic Neuropathy.
What is MELAS?
Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episodes.
What is GRACILE?
Growth retardation Aminoaciduria Cholestasis Iron overload Lactic acidosis and Early death.
What is MILS?
Maternally Inherited Leigh Syndrome.
What is MLASA?
Mitochondrial myopathy Lactic Acidosis Sideroblastic Anemia.
What do complex 1 mutations cause?
Leigh Syndrome. Leucoencepahlopathy. FILA. LHON. MELAS.
What do complex 2 mutations cause?
Leucoencephalopathy.
Hereditary tumours of neural crest.
What do complex 3 mutations cause?
Leucoencephalopathy.
GRACILE.
Bjoornstad syndromes.
What do complex 4 mutations cause?
Leigh syndrome.
Neonatal hepatopathy.
Cardiomyopathy.
What do complex 5 mutations cause?
Adult onset Neuropathy. Ataxia. Retinitis pigmentosa. MILS. MLASA. Adult onset Cerebral ataxia. Motor neurone syndrome.
Where does dysfunction of different complexes lead?
To hypoglycaemia.
Lactic acidosis.
ROS damage.
Limited tissue function.
By which factors are the complexes lead to sympotms?
By lack of efficient ATP generating capacity.
Lack of generation of proton motive force.
Inability to use force to drive ATP synthase.
Where is the treatment of patients with complex mutations focuses?
On correcting acidosis or hypoglycaemia.
What can the treatment of complex mutation’s symptoms not redress?
The issues of limited ATP synthesis.
High ROS production.
What can be a way of correcting the problems of complex mutations?
Gene editing techniques.
What is the disadvantage of using gene editing techniques to correct the problems of complex mutations?
It has a long way to go before it can be used clinically.
