Lecture 32-35 CAC and oxidative phosphorylation PDF

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Notes on Lecture 32-35 CAC and oxidative phosphorylation...

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Lectures 32-35 Pyruvate decarboxylation 

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Pyruvate (3 carbons) can be reduced to form lactic acid, or be transported to the mitochondrial matrix to be broken down into acetyl CoA (2C). This means that a CO2 is produced. An NADH molecule is also produced. Once inside the mitochondria, a trio of enzymes known as pyruvate dehydrogenase complex catalyse the reaction. Thiamine (Vitamin B1) is part of the pyruvate dehydrogenase complex, therefore a thiamine deficiency results in an inability to turn pyruvate into acetyl CoA. This causes the excess pyruvate to turn into lactic acid, which is responsible for the symptoms of beriberi and Wernicke Korsakoff.

Citric acid cycle     

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Acetyl CoA (2C) enters the citric acid cycle by combining with oxaloacetate (4C) to form citrate aka citric acid. Citrate (6C) is rearranged by the enzyme aconitase to form isocitrate (6C). Important step because this step gets poisoned by 1080 aka sodium fluoroacetate. Isocitrate (6C) loses a carbon, giving off CO2 and producing a NADH to form alphaketoglutarate (5C). Alpha-ketoglutarate loses a carbon, so CO2 is produced and a NADH is also produced. It also combines with Coenzyme A to form succinyl-CoA (4C). Succinyl-CoA (4C) is broken off from CoA and the energy used to create a guanosine triphosphate (GTP), which is pretty much equivalent to an ATP molecule. Example of substrate level phosphorylation. Succinate is produced (4C). Succinate (4C) is oxidised to fumarate (4C) and a FAD molecule is reduced to FADH2. This step is special because it occurs on the inner mitochondrial membrane, as part of the electron transport chain, unlike all the other reactions which occur in the mitochondrial matrix. Similar to the first step of beta-oxidation (oxidation and FADH2 produced). Fumarate (4C) has H2O added to it to become malate (4C). Similar to second step of betaoxidation (addition of water). Malate (4C) is oxidised to form oxaloacetate (4C), which was our starting substrate. NADH is also produced. This is similar to the third step of beta-oxidation (oxidation and production of NADH). 3 NADH, 1 FADH2 and 1 ATP produced. Also 2 CO2 molecules produced.

1080 aka sodium fluoroacetate    

Sodium fluoroacetate is a compound that can poison the citric acid cycle. Fluroacetate can form fluoroacetyl CoA, which can combine with oxaloacetate to form fluorocitrate. Fluorocitrate is a competitive inhibitor of aconitase, which normally catalyses the isomerisation of citrate. This poisons the citric acid cycle and acetyl CoA can’t be broken down into NADH, FADH2 or ATP, which means that energy production is greatly reduced. This leads to insufficient ATP for the cell to function and results in death of the organism.

Amino acid breakdown 

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Amino acids can be broken down for energy. This can occur if we have consumed excess protein, or during periods of starvation. Transamination also allows the body to synthesise amino acids that we need from amino acids that we have an excess of. Amino acids need to have their amino (NH2) group removed in order to be used for energy. The amino group can be removed and released into solution as NH3 aka ammonia (deamination), or it can be transferred onto another molecule (transamination). Deamination involves the amino acid being oxidised and losing it’s amino group. This releases NH3 into solution. The problem with this is that NH3, or ammonia, is a weak base and will become protonated to turn into NH4+, or ammonium, which is toxic in high concentrations in the blood.

Transamination is when the amino group is transferred to an alpha-keto acid. This requires aminotransferase enzymes (also called transaminase enzymes). The enzyme requires pyridoxal phosphate as a coenzyme, which is made from pyridoxine (Vitamin B6).

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Once the amino group has been removed, the carbon skeleton can be used to make acetyl CoA or used as substrates in the citric acid cycle, thus producing energy.

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If the muscles are breaking down amino acids for energy, then the excess nitrogen that is being produced can be transported to the liver and turned into urea, which is non-toxic and can be excreted in the urine.

Oxidative phosphorylation  

Oxidative phosphorylation involves the electron transport chain and ATP synthase. The two are coupled by a proton gradient. This means the energy required for the production of ATP comes from oxidative phosphorylation, which is indirect, as opposed to substrate level phosphorylation.

Electron transport chain   

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The electron transport chain is located on the inner mitochondrial membrane and is comprised of 4 protein complexes. They are called Complex 1, 2, 3 and 4. The electrons get passed on to carriers that have increasing reduction potential. Higher reduction potential means they want electrons more aka they want to be reduced. They receive electrons from NADH and FADH2, the electron carriers, and extract the energy from those electrons to pump protons across the inner mitochondrial membrane into the intermembrane space. This creates a high concentration of protons in the intermembrane space. The electrons are then transferred to O2 to form H2O. This is why we need oxygen, to act as the terminal electron acceptor for the electron transport chain.

NADH    

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NADH drops off two electrons at complex 1, this powers the pumping of 4 protons into the intermembrane space. The electrons are passed onto CoQ, which delivers them to complex 3. CoQ is also known as CoQ10, Coenzyme Q, or ubiquinone. Yes it’s very confusing ☹ Complex 3 then pumps 4 more protons and passes the electrons onto cytochrome c, which is a peripheral protein located on the intermembrane side of the inner mitochondrial membrane. Cytochrome c delivers the electrons to complex 4, which pumps 2 protons. The electrons are passed onto oxygen and water is formed. Path of electrons for NADH – Complex 1 (4 protons), Complex 3 (4 protons), Complex 4 (2 protons), oxygen. This means 10 protons are pumped for each NADH molecule.

FADH2    

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Remember that succinate is oxidised to fumarate in the citric acid cycle. This reaction occurs on Complex 2 of the electron transport chain. During this reaction, 2 electrons are passed onto FAD to form FADH2. Complex 2 does NOT pump any protons. The electrons are passed onto CoQ, which delivers them to complex 3. Complex 3 then pumps 4 protons and passes the electrons onto cytochrome c, which is a peripheral protein located on the intermembrane side of the inner mitochondrial membrane. Cytochrome c delivers the electrons to complex 4, which pumps 2 protons. The electrons are passed onto oxygen and water is formed. Path of electrons for FADH2 – Complex 2, Complex 3 (4 protons), Complex 4 (2 protons), oxygen. This means 6 protons are pumped for each FADH2 molecule.

Rotenone  

Rotenone inhibits CoQ, the electron carrier that delivers electrons from Complex 1 and 2 to Complex 3. If electrons can’t be delivered to Complex 3, then the whole ETC stops and no proton gradient is formed, which means no ATP.

Cyanide     

Cyanide binds to the Fe3+ of cytochrome a3 in complex 4. Cytochrome a3 is also known as cytochrome oxidase. Because the Fe of cytochrome a3 can be in the 2+ or 3+ oxidation state, this means that carbon monoxide can also bind to it when it is in the ferrous (2+) state. Inhibition of cytochrome a3 means the electrons cannot move through the ETC and no ATP is produced. Remember that carbon monoxide binds to Fe2+, while cyanide binds to Fe3+.

ATP synthase    

The pumping of electrons into the intermembrane space creates a electrochemical gradient and a proton motive force (pmf). This means that protons want to flow back into the mitochondrial matrix, however they can only pass through ATP synthase under normal conditions. ATP synthase uses the flow of protons to generate ATP. ATP synthase can be divided into F0 or F1, where F0 is in the inner mitochondrial membrane and F1 is in the matrix.

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It can also be divided into rotor or stator parts. The stator doesn’t move (is stationary), while the rotor parts rotate.

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1 ATP is made for every 4 protons pumped. This means each NADH produces 2.5 ATP since it pumps 10 protons. FADH2 produces 1.5 ATP since it pumps 6 protons.

DNP  

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DNP, or dinitrophenol, can uncouple the electron transport chain from ATP production. It works by picking up protons from the intermembrane space, and then crossing the inner mitochondrial membrane. Thus it reduces the proton gradient, which means less protons pass through ATP synthase and less ATP is produced. It does NOT affect the electron transport chain. The citric acid cycle will be accelerated since there is a shortage of ATP. This results in excess heat production and overdose results in death due to hyperthermia (overheating)....