sakai-mitochondrial shuttles, ETC and oxidative phosphorylation Flashcards
Describe the malate-aspartate shuttle. How do you form malate in cytosol, and how do you generate NADH in the mitochondrial matrix?
The malate-aspartate shuttle describes the formation of malate from oxaloacetate in cytosol using cytosolic NADH and forming NAD+. This provides NAD+ in cytosol in order to continue glycolysis.
NADH cannot pass by itself through the inner mitochondrial membrane, and the transport of malate instead into the mitochondria allows NADH generation from malate inside of the mitochondria.
The generated mitochondrial oxaloacetate is used to form aspartate by transamination and aspartate is transported into the cytosol. This aspartate can be used to form oxaloacetate in cytosol and malate can be formed again and be transported into mitochondria.
In short: malate is transported into the mitochondria, aspartate is transported out of mitochondria. The NADH in cytosol is used to form malate which generates NAD+ in cytosol for glycolysis. Inside of mitochondria, NADH can be formed from malate by mitochondrial malate DH. NADH can then be oxidized in complex I of the ETC, leading to 3 (2.5) ATP in oxidative phosphorylation.
In the glycerophosphate shuttle, do you also use NADH in cytosol and generate it inside of mitochondria? Explain.
In the glycerophosphate shuttle, NADH and DHAP are used to form glycerol 3-phosphate and NAD+. Glycerol 3-P is not transported into the mitochondria.
[Fig 6.15 Lippincotts is a bit misleading, in A the inner mitochondrial membrane is colored in blue, in B it is the mitochondrial matrix that is shown in blue]
Glycerol 3-P is substrate for the mitochondrial glycerophosphate dehydrogenase, which is bound to the inner mitochondrial membrane, contains FAD and forms dihydroxyacetone phosphate (DHAP) and FADH2.
FADH2 can enter the ETC at CoQ leading to 2 (1.5) ATP in oxidative phosphorylation.
Why is it important to regenerate cytoplasmic NAD+?
Glycolysis needs NAD+ in the step catalyzed by glyceraldehyde 3-P dehydrogenase.
When the malate-aspartate shuttle or the glycerophosphate shuttle is used, then the NAD+ is regenerated and aerobic glycolysis can be performed. In addition, FADH2 or NADH are formed that can be used in the ETC and oxidative phosphorylation.
Describe the role of the inner mitochondrial membrane in electron transport and oxidative phosphorylation. Which phospholipid is characteristic for in the inner mitochondrial membrane and allows ETC and oxidative phosphorylation?
The inner mitochondrial membrane binds the components of the ETC, it is protein-rich.
This membrane is normally impermeable for protons and allows the generation of a proton gradient by active pumping in complex I, III and IV.
Cardiolipin is essential for the functioning of the ETC and oxidative phosphorylation.
[Cardiolipin, diphosphatidylglycerol, contains 4 fatty acids, mainly linoleic acid]
Describe the different types of electron carriers used in the ETC.
NADH and also the enzyme succinate dehydrogenase (FADH2) donate each two electrons to the lipid CoQ which forms CoQH2 after uptake of protons from the matrix.
[CoQ is also the entry point of electrons from the glycerophosphate shuttle and from flavoproteins linked to -oxidation of fatty acids. ]
[This is the part of the ETC where an electron can be lost and superoxide may be formed. Molecular oxygen and one electron forms superoxide which is an anion and radical]
The ETC changes then by using cytochromes to perform the electron transport, one electron at a time. The electron binds to the heme groups of cytochromes. The ferric iron (3+) changes to ferrous iron (2+) after uptake of one electron. The electron leaves then to the next cytochrome.
Only at complex IV, cytochrome c oxidase, the electrons are meant to interact with molecular oxygen and water is formed.
[please compare: the heme in cytochromes of the ETC are meant to change from ferric to ferrous to ferric and so on, as they transport electrons.
The heme groups however are not meant to interact with molecular oxygen unless in complex IV (cyt a3).
On the other hand, the heme in hemoglobin and myoglobin is supposed to stay in the ferrous state and it shall interact with molecular oxygen in hemoglobin and myoglobin].
Describe the ETC entering with NADH and succinate dehydrogenase (FADH2) and finishing with formation of water.
NADH enters at complex I (NADH dehydrogenase, NADH/CoQ reductase) which contains FMN and iron sulfur clusters. Two electrons are transported to CoQ and CoQH2 is formed using protons from the matrix. This electron transport allows the pumping of protons into the inter membrane space and the establishment of a proton gradient.
CoQ (ubiquinone) is a nonpolar lipid with ten isoprenoid units in humans (CoQ10). It is inside of the inner mitochondrial membrane and it can move in the fatty acyl region of phospholipids.
[CoQ synthesis branches out of cholesterol synthesis using farnesyl-PP and isopentenyl-PP. Inhibition of HMG CoA reductase for treatment of hypercholesterolemia also leads to less synthesis of CoQ]
CoQ also accepts two electrons from FADH2 of succinate dehydrogenase (TCA) in complex II. This enzyme has the prosthetic group FAD and that is why this enzyme is the only enzyme of the TCA cycle that is bound to the inner mitochondrial membrane. Complex II does not generate the energy to pump protons.
After CoQH2 is formed, only one electron at a time is transported by cytochromes which allows a safe way to transport electrons without superoxide formation.
The first cytochromes are in complex III which contains cytochrome b and c1. Complex III is a proton pumping complex and also named cytochrome c reductase as the electrons are transferred to cytochrome c.
Cytochrome c is bound in the inner mitochondrial membrane toward the intermembrane space.
From cytochrome c, the electrons are transferred to complex IV which is also known as cytochrome c oxidase. This complex contains copper and the cytochromes a/a3. This is the only complex where the electrons are meant to interact with molecular oxygen and water is formed. Complex IV is the third proton pumping complex of the ETC.
Many drugs were used to investigate the ETC. Some drugs block the electron transport. Name eight drugs or compounds that inhibit the ETC.
The ETC is inhibited by Amytal, Rotenone , Pericidin A, Antimycin A, Sodium azide, cyanide, carbon monoxide and hydrogen sulfide.
Please match the above drugs or compounds with the very specific location of the inhibition in the ETC. Which drugs inhibit directly NADH/CoQ reductase, cyotochrome c reductase and cytochrome c oxidase, respectively?
Amytal and rotenone and pericidin A inhibit NADH/CoQ reductase
Antimycin A inhibits Cytochrome c reductase
Cyanide, carbon monoxide and Sodium azide and hydrogen sulfide inhibit Cytochrome c oxidase
Name 4 synthetic uncouplers of oxidative phosphorylation. The uncouplers are drugs that separate ETC from oxidative phosphorylation. They generate heat.
The uncouplers are 2,4 Dinitrophenol and valinomycin and gramicidin A and also aspirin
at a high dose.
[an overdose of aspirin often leads to fever in the individual]
Name an ATP synthase inhibitor that interferes with the ATP formation. ATP synthase is sometimes named Complex V.
Oligomycin binds to F0 of ATP synthase and blocks the proton channel. The F0 domain is bound in the inner mitochondrial membrane, the F1 domain reaches knob-like into the matrix.
Name 2 inhibitors of ADP-ATP translocase
The ADP-ATP translocase is inhibited by the toxins from plants like atractyloside and bongkrekic acid.
Name the complexes of the ETC that are proton pumping and generate the proton gradient.
Complex I, III and IV are proton pumping and build the proton gradient that leads to higher proton concentration in the inter membrane space than in the mitochondrial matrix.
How can ATP formation be described using the Mitchell’ Chemiosmotic theory?
The generation of a proton gradient by complex I, III and IV allows the usage of the energy of the generated proton gradient when the protons flow back into mitochondria through ATP synthase.
The energy generated by the proton gradient is enough to perform the ATP generation by ATP synthase, however, the ATP formation has to be connected to the ETC transport chain.
Describe the action of ATP synthase. Explain the functions of the F1 and F0 domains
The protons from the inter membrane space can only flow into mitochondria via the F0 domain of ATP synthase. The flow leads to rotation and the F1 domain links ADP and inorganic phosphate to each other and releases the formed ATP.
How is the uncoupling of ETC and oxidative phosphorylation achieved by 2,4-DNP?
2,4-DNP is a lipophilic proton carrier that readily diffuses through the inner mitochondrial membrane. At high proton concentration in the inter membrane space, it picks up a proton.
Then it can move back into the matrix and release the proton inside. With that it “transports” protons in its structure back into the mitochondrial matrix and the proton gradient cannot be efficiently built.
