Kreb’s Cycle and Electron Transport Chain PDF

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Summary

Covers Kreb's cycle in detail, along with the electron transport chain in detail....

Description

Introduction to Life Sciences Lecture 30 Kreb’s Cycle and Electron Transport Chain 11/12/20 Krebs cycle - AKA citric acid or tricarboxylic acid cycle. - Occurs in matrix of mitochondria. - Acetyl group plus oxaloacetate forms citric acid. - NADH, FADH2 capture energy rich electrons which then drive ATP formation. - Two cycles per glucose molecule. - Produces 4 CO2, 2 ATP, 6 NADH and 2 FADH2 per glucose molecule.

Step 1: citrate synthesis - Citrate synthase catalyses the first step in the Krebs cycle. - Acetyl CoA from glycolysis combines with oxaloacetate to form citrate, which releases CoA.

Co A

oxaloacetate acetyl CoA

citryl CoA

citrate

Step 2: isomerase reaction - In the second step in the TCA cycle, aconitase converts citrate to isocitrate. - This is an isomerase reaction.

citrate

cis-aconitate

isocitrate

Step 3: isocitrate dehydrogenase - the next step, mediated by isocitrate dehydrogenase (DH), oxidises isocitrate to -ketoglutarate.

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NAD+ is reduced to NADH, and CO2 released.

isocitrate

oxalosuccinate

-ketoglutarate

Step 4: alpha-Ketoglutarate DH - -ketoglutarate DH catalyses the oxidative addition of CoA. - NAD+ is again used as the oxidising agent, and a further molecule of CO 2 is released.

-ketoglutarate

succinyl CoA

Step 5: Succinyl CoA Synthase - Removal of the CoA moiety to produce succinate provides the energy to phosphorylate GDP. - This is the only tri-phosphate nucleotide to be produced directly by the Krebs cycle. - GTP is counted as an ATP equivalent when calculating the energy yield from glucose oxidation and the Krebs cycle.

succinyl CoA

succinate

Step 6: succinate dehydrogenase - In the next step, succinate is oxidised to fumarate. - For this reaction FAD is used as the electron acceptor, forming FADH2.

succinate

fumarate

-

Fumarase catalyses the hydration of fumarate to malate. The enzyme requires no cofactors.

fumarate

malate

Step 8: malate dehydrogenase - Malate dehydrogenase catalyses the oxidation of malate to form oxaloacetate. - NAD+ is reduced to NADH and an additional hydrogen ion is also released.

malate

oxaloacetate

Overall Krebs cycle - During the Krebs cycle, each molecule of acetyl CoA that is oxidised leads to the formation of 3 NADH + 1 FADH2 + 2 CO2 + 1 GTP (4 pairs of electrons). 2C-CoA

4C

CoA

6C

4C

6C 1C

4C

5C 1C 4C

NADH and FADH2

4C

-

Oxidoreductase enzymes require electron donors (for reduction) or electron acceptors (for oxidation). - The cofactors NAD+/NADH and FAD/FADH2 perform this role: - NAD+ + 2H+ + 2eNADH + H+ + - FAD + 2H + 2e FADH2 - Oxidized forms reduced forms Krebs cycle: control - Whereas glycolysis is controlled by regulating PFK, the Krebs cycle is controlled at several steps. - the strongest regulation applies to the two steps where CO 2 is released (i.e., high G0) - Both ATP and NADH inhibit these reactions, while ADP and calcium ions are activators Krebs cycle summary - Each glucose becomes two molecules of pyruvate... - ...these become two acetyl CoAs plus two CO2s - Acetyl CoA is further broken down to CO2 accompanied by the production of GTP, NADH and FADH 2

The electron transport chain

1 FADH2

TCA cycle

8 e-

3 NADH (8 electrons)

H +

I

II

H +

H +

III

IV

H2O ½O2

Complex I: NADH dehydrogenase

Complex II: succinate dehydrogenase - Part of Krebs cycle (succinate to fumarate). - Succinate to fumarate reaction generates FADH2. - FADH2 oxidized to regenerate FAD. - Released electrons transferred to CoQ via Fe-S clusters. Coenzyme Q (CoQ) - Also called CoQ10, ubiquinone (UbQ) or just “Q”. - Small lipid soluble compound (a hydrophobic quinone). - Diffuses rapidly within IMM (inner mitochondrial membrane)– mobile carrier. - Accept electrons from Fe-S proteins from Complex I and Complex II. - Transfers electrons to Complex III / cytochrome c (“Q cycle”).

Complex III: Cytochrome C reductase

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Both complex III and cytochrome C use heme prosthetic groups as an electron acceptor/donor. Cytochrome C - Peripheral membrane protein loosely bound to IMM. - Binds to Complex III and transfers electrons to Complex IV. - Highly conserved. Complex IV: Cytochrome C oxidase - Four cytochrome C molecules and eight protons required to oxidise one oxygen molecule.

How is energy produced?

Complex V: ATP synthase - Embedded in inner mitochondrial membrane. - Composed of two subunits: F1 ATPase (generates ATP), and F0 coupling factor; a proton channel. - Protons pumped to the cytosolic side of mitochondrial membrane reenter matrix through F0 proton channel. - The passage of protons through the channel drives the rotation of the C-ring of F0. - This sequentially alters conformation of -subunit of F1 domain, driving formation of ATP from ADP & Pi.

ATP yield and P/O ratios

Overview of cellular respiration - Electrons from NADH and FADH 2 are transferred to oxygen along the electron transport chain (respiratory chain). - Components located in the inner mitochondrial membrane (IMM). - Energy released is used to pump protons into intermembrane space to create a proton (i.e., pH) gradient. - Protons flow back through ATP-synthase complex to generate ATP. - Whole pathway tightly coupled. Overall energetics

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The accepting carrier (oxygen) has a higher affinity for electrons than the donating carriers NADH & FADH2. - e.g., for: NADH + ½ O2 + H+  H2O + NAD+. E0’ = 1.14 V means G°’ = -52.6 kcal mol-1. Concept of the metabolic pool - Catabolism : degradative reactions, such as the oxidation of food, and are mostly exergonic- release energy. - Anabolism : overlapping pathways that synthesise complex compounds required for life and are mostly endergonic- require heat. Pyruvate carboxylase and anapleurosis - The Krebs cycle is involved in both catabolic and anabolic processes (amphibolic). - Biosynthetic pathways deplete Krebs cycle components, which must be replaced (anapleurosis). - Pyruvate carboxylase is a key part of this, generating oxaloacetate (OAA) from pyruvate. - In consequence, it is found in all tissues.

Chemiosmotic theory - This is the theory that ATP synthesis is driven by a proton motive force across the mitochondrial membrane. - Proposed by Peter Mitchell who determined: O2 consumption in isolated mitochondria is dependent on the concentration of ADP, respiration continues until all ADP is converted to ATP, uncoupling agents (see next slide) eliminate the dependence of substrate oxidation on ADP. Uncoupling agents - Substances that dissipate the proton gradient will decouple the electron transport chain from ATP synthesis. - Examples include chemicals such as dinitrophenol, which forms proton ionophore, uncoupling proteins e.g., thermogenin (uncoupling protein 1; UCP1), which are found in mitochondria of brown adipose tissue, generate heat (non-shivering thermogenesis), and are found in newborns and hibernating animals. ATP synthase: proof of rotation - ATP synthase is a molecular motor, which runs on protons for fuel. - This has been demonstrated by attaching actin filaments to F1 and “running” the enzyme in reverse, e.g., ATP hydrolysis. 10 revolutions were completed per second, and 30 ATP molecules were consumed. Export of ATP - ATP is synthesised in mitochondria but are mostly used in the cytoplasm. - Therefore, it needs a transport mechanism. - This is achieved by ADP/ATP antiporter and a Pi/H+ co-transporter. - The latter makes it an active process, which depletes the H+ gradient. -

Adenine nucleotide translocase...