Lecture 5: Citric Acid Cycle:

Where is the Citric Acid Cycle Located?

Located in the mitochondrial matrix, pyruvate is transported here and converted into acetyl-CoA and where most of the reactions of CAC occur

• exception: step 6 where succinate dehydrogenase is used is embedded in the inner mitochondrial membrane because it also functions as complex II in ETC

How does Pyruvate convert into Acetyl-CoA:

Pyruvate dehydrogenase complex converts pyruvate into Acetyl-CoA by oxidative decarboxylation (carboxyl group removed and diffused out as CO2)

• Products: Acetyl-CoA + CO2 + NADH (per pyruvate)

Thioester Bond:

a high-energy chemical linkage formed by a carbonyl group attached to a sulfur atom (located in acetyl-CoA)

Citric Acid Cycle:

eight-step enzyme catalysis cycle that creates reduced electron carries in which the two-carbon acetyl group from ACoA combines with four-carbon oxaloacetate to form six-carbon citrate

Intermediate Substrates of CAC

oxaloacetate + CoA, citrate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate

Enzyme of CAC:

citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketogutarate dehydrogenase complex, succinyl-CoA synthetase, succinate dehydrogenase, fumerase, malate dehydrogenase

Oxidative Decarboxylation

oxidation and electron transfer ocur while carbon is being released as CO2 (occurs isocitrate into alpha-ketoglutarate, and alpha-ketoglutarate into succinyl CoA)

When is the thioester bond broken and what does this mean`/

in step 5, when going from succinyl CoA to succinate, the high-energy thioester bond is broken, this means it can be coupled to the reaction of `atp creation

Overall Reaction of CAC:

acetyl CoA + ADP + Pi + 3NAD + FAD + 2H2o → ATP + 3 NADH + 2H+ + FADH2 + 2 CO2 + CoA

Purpose of CAC:

Main energetic purpose is to create NADH and FADH2 as reductive electron carriers so that can donate high-energy electrons to the ETC and chemiosmosis for much larger ATP production

Oxidative Phosphorylation Stages:

ETC: electrons pass through 4 protein complexes in the inner mitochondrial membrane, and the released energy pumps H+ from the matrix into the intermembrane space to form a H gradients

Chemiosmosis: H+ flows back into the marix through ATP synthase, providing the energy for ATP productions

How many hydrogens are pumped per electron pair of NADH and FADH2

NADH: 10 H+ are pumped

FADH2: 6 H+ are pumped (bypasses protein complex 1)

What are the 4 different protein complexes:

NADH Dehydrogenase, Succinate Dehydrogenase, Cytochrome c Oxidoreductase, Cytochrome C Oxidase

Complex I: NADH Dehydrogenase (Function, Proton Pumping, Route)

• Function: Oxidises NADH to NAD+ and transfers two electrons to coenzyme q

• Proton Pumping: transport 4H+ from the matrix to the intermembrane space per electron pair

• Route: Electrons from NADH enter at complex I and bypass complex II

Complex II: Succinate Dehydrogenase: (Function in CAC and ETC, Proton Pumping and Route)

• Function CAC: catalyses the reaction from succinate to fumarate, and reduces FAD to FADH2

• Function ETC: oxidises FADH2 to FAD and transfers the electron to coenzyme q

• Pumping: Complex II does not pump any protons, which explains why FADH2 produces less ATP than NADH

• Route: electrons from FADH2 enter through complex 2 and do not pass through complex 1

Complex III: Cytochrome C Oxidoreductase (Function and Proton Pumping)

Functions: receives electrons from coenzyme Q and transfers them to cytochrome C

Proton Pumping: transports 4 H+ into the intermembrane space per electron pair

Complex IV: Cytochrome C Oxidase (Function, PP, Final Reaction)

• Function: receives electrons from cytochrome c and transfers them to O2

• Proton Pumping: transports 2H+ into the intermembrane space per electron pair

• Final Reaction: four electrons are transferred to O2 and with 4H+ they form 2H2O

Why is Oxygen Essential

because it is the final electron acceptor, and without it the electron flow through the ETC stops

What is Coenzyme Q

conezyme Q is a lipid-soluble mobile electron carrier within the inner mitochondrial membrane

• It carries NADH electrons from complex I to III

• It carries FADH2 electrons from complex II to III

• Other pathways can also donate electrons to coenzyme q, making it a central link between multiple metabolic routed and the ETC

What is Cyctochrome C

small, water-soluble mobile electron carrier on the outer surface of the inner mitochondrial membrane

• It carries electrons from complex III to IV

• It only transports one electron at a time

What is Reduction Potential:

a measure of a molecules tendency to acquire electrons and be reduced

• a more positive reduction potential means it has a greater affinity for electrons and therefore a higher tendency to acquire electrons and be reduced, and has less free-energy

Reduction Potential along the ETC:

along the ETC there is a progressive increase in reduction potential (-0.32V for NADH/NAD to +0.82V for O2/H2O)

• Free energy decreases as electrons move toward carriers with higher reduction potential, yhis released energy is used for proton pumping

What Happens when there is a Disruption of Redox Transfer

Disruption of redox transfer stops the ETC and therefore prevents oxidative phosphorylation

What does Proton Pumping Create

an electrochemical H+ gradient across the inner mitochondrial membrae

• energy is provided to ATP synthesis by a proton circuit consisting of membrane potential and chemical gradient

What is the Proton Motive Force

Membrane Potential + Chemical Gradient = Proton Motive Force

• Membrane Potential: the inter membrane space becomes more positively charged relative to the matrix

• Chemical Gradient: the inter membrane space has a higher concentration of H+ ions making it have a lower pH than the matrix

Chemiosmosis:

the movement of H+ ions down the electrochemical gradient through ATP synthase into the matrix

ATP Synthase, its speed and energy cost

is a membrane-associated rotary enzyme that couples H+ back flow to ATP formation from ADP + Pi

• Can produce more than 100 ATP per second (very fast)

• Energy Cost: Needs 4 H+ ions to return to the matric make 1 ATP

Parts of the ATP synthase

stator, rotor, second channel in stator, internal rod, knob, catalytic sites in knob

Process of ATP Creation through ATP Synthase

1. H+ ions flow down the gradient entering a channel in the stator, which is anchored in the membrane

2. H+ ions enter binding sites within a rotor, changing the shape of each subunit so the rotor spins within the membrane

3. Each H+ ion makes one complete turn before leaving the rotor and passing through a second channel in the stator into the mitochondrial matrix

4. Spinning of the rotor causes the internal rod to also spin. The rod extends into the knob below it, which is held stationary by part of the stator

5. Turning of the rod activates catalytic sites in the knob that produce ATP from ADP and Pi

Total Yield of ATP Per Glucose

• Glycolysis: Produce 2ATP and 2NADH (3 or 5 ATP)

• Pyruvate Oxidation: 2NADH (5 ATP)

• CAC: 2 ATP, 6 NADH (15 ATP), 2 FADH2 (3 ATP)

In Total 30-32 ATP

Why is there a range in amount of ATP 1 glucose can make

Because cytosolic NADH from glycolysis cannot cross the inner mitochondrial membrane, it’s electrons use a shuttle. The shuttle used determines whether each cytosolic NADH yields 1.5 or 2.5 ATP

What are the two types of shuttles and their ATP Yield

Malate-Aspartate Shuttle and Glycerol-3-Phosphate Shuttle

• Malate-Aspartate Shuttle: 2.5 ATP per cytosolic NADH

• Glycerol-3-Phosphate Shuttle: Yields 1.5 ATP per cytosolic NADH

Malate-Aspartate Shuttle: Tissues, Purpose, EC Regenerated

• Tissues: Liver, Heart and Kidney

• Purpose: Transports electrons from cytosolic NADH into the matrix so it can be used in ETC

• EC Regenerated: mitochondrial NADH is regenerated and enter through complex I

Glycerol-3-Phosphate Shuttle: Tissues, Purpose, EC Regenerated

• Tissues: Skeletal Muscle and brain

• Purpose: Transports electrons from cytosolic NADH into the matrix so it can be used in ETC (cannot cross the inner mitochondrial membrane itself)

• EC Regenerated: mitochondrial FADH2 is regenerated and enter through complex II and bypass I

Fatty Acid Breakdown:

Fatty Acids can be broken down into acetyl CoA through beta-oxidation, when then enters the CAC

• a fatty acid first reacts with CoA, to form fatty acyl-CoA catalysed by acyl-CoA synthetase (ATP is converted to ADP + Pi)

What is Beta-Oxidation

consists of four recurring reactions in which oxidative cleavage occurs at the beta-carbon of fatty acyl CoA

• Each round shorten the fatty acyl chain by two carbons and produces acetyl CoA, NADH and FADH2,

• this cycle completes until fatty acid is completely degraded

Why does the CAC rely on oxygen

• Because it is the final electron acceptor, and if it was not there it would stop the ETC

• If it stops the ETC, then there would be too much NADH and FADH2 and no NAD+ and FAD+, meaning agents of the CAC cannot be oxidised causing it to stop

Main Indicators of CAC Regulation

ATP/ADP and NADH/NAD+ ratios because they show the cells energy state

Low Energy: ADP and pyruvate stimulate pathway activity, High Energy: ATP and NADH

Key Control Points of CAC Regulation:

Pyruvate Dehydrogenase Complex:

• inhibited by ATP, acetyl CoA, NADH

• Activated by ADP and pyruvate

Isocitrate Dehydrogenase:

• Inhibited by ATP and NADH

• Activated by ADP

alpha-Ketoglutarate Dehydrogenase:

• Inhibited by ATP, succinyl CoA and NADH

• Activated by ADP

Amphibolic Pathway:

Catabolic: oxidises acetyl-CoA and generates energy in NADH, FADH2 and ATO

Anabolic: CAC intermediates provide starting materials for biosynthesis

• these must be replenished so the cycle can continue operating

Anabolic Role for Intermediates of CAC used for biosynthesis:

Citrate: can contribute to fatty acid and cholesterol synthesis

alpha-Ketoglutarate: contribute to glutamate, other amino acids and purines

Succinyl CoA: contribute to heme synthesis

Oxaloacetate: contribute to asparate, other amino acids, purines and pyramidines

What EC is made at each step

1. H2O (Oxaloacetate + ACoA to Citrate) CoAS-H

2. H2O removed (Citrate to Isocitrate) H2O added

3. NAD+ + (Isocitrate to alpha-ketoglutarate) CO2 + NADH

4. NAD+ + CoA (alpha-glutarate to succinyl CoA) CO2 + NADH

5. ADP + Pi (succinyl CoA to succinate) ATP + CoA

6. FAD (succinate to fumerate) FADH2

7. H2O (fumerate to malate)

8. NAD+ (malate to oxaloacetate) NADH