BICH Exam 2 notes

Pyruvate Dehydrogenase Complex (PDHC)

1. Overview of the TCA Cycle (Krebs Cycle)

• The TCA cycle is a crucial pathway in aerobic respiration.

  • Central pathway for recovering energy from several metabolic pathways

  • Yields ~36 ATPs

• It allows the oxidation of acetyl-CoA (derived from carbohydrates, fats, and proteins) to produce CO₂, NADH₂ and FADH₂.

  • Pyruvate is converted to acetyl-CoA

  • Acetyl-CoA is oxidized to CO₂

• NADH and FADH₂ carry electrons to the electron transport chain for ATP production.

  • Electrons stored as NADH and FADH₂ are delivered to a membrane-associated electron-transport chain to the final electron acceptor O₂. 

  • Electron transfer is coupled to a proton gradient across the membrane to drive the synthesis of ATP in a process known as oxidative phosphorylation.

2. Connection Between Glycolysis and the TCA Cycle

• Glycolysis (occurring in the cytoplasm) breaks glucose into pyruvate, generating 2 ATP per glucose molecule.

• Pyruvate Dehydrogenase Complex (PDHC) converts pyruvate into acetyl-CoA, linking glycolysis to the TCA cycle.

  • Pyruvate must be oxidatively decarboxylated to acetyl-CoA

• The reaction:

Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

3. The Chemical Logic of the TCA Cycle

• The full oxidation of glucose to CO₂ involves 24 electrons.

  • Glycolysis: 4 electrons

  • Pyruvate → Acetyl-CoA (PDHC): 4 electrons

  • TCA Cycle: 16 electrons (8 per acetyl-CoA)

• These electrons are stored as NADH and FADH₂, which drive oxidative phosphorylation.

4. Pyruvate Dehydrogenase Complex (PDHC)

• PDHC is irreversible and a key regulatory step.

  • Gateway to TCA cycle

• It consists of three enzymes:

  • E1: Pyruvate Dehydrogenase

  • E2: Dihydrolipoyl Transacetylase

  • E3: Dihydrolipoyl Dehydrogenase

• Uses five cofactors:

  • TPP (Thiamine pyrophosphate, Vitamin B1)

    • Bound to E₁

    • Decarboxylates pyruvate, yielding a hydroxyethyl-TPP carbanion

  • Lipoic acid

    • Covalently linked to a Lys residue on E₂ (lipoamide)

    • Accepts the hydroxyethyl carbanion from TPP as an acetyl group

  • CoA

    • Substrate for E₂

    • Accepts the acetyl group from lipoamide

    • CoA functions as a carrier of acetyl and other acyl groups

    • Acetyl- CoA is a high-energy compound

    • The ΔG°’ for the hydrolysis of its thioester bond is –31.5 kJ/mol

  • FAD (from riboflavin, Vitamin B2)

    • Bound to E₃ (immobile)

    • Reduced by lipoamide

  • NAD⁺ (from niacin, Vitamin B3)

    • Substrate for E₃ (mobile)

    • Reduced by FADH₂

• Catalyzes 4 sequential reactions to convert pyruvate → acetyl-CoA

  • KNOW THE MECHANISMS FOR EACH REACTION

5. Electron Transfer and ATP Production

• NADH and FADH₂ donate electrons to the electron transport chain.

• Electron transfer creates a proton gradient, driving ATP synthesis.

• Final electron acceptor: O₂.

6. Importance of Cofactors in the PDHC

• TPP (Vitamin B1) is necessary for decarboxylation of pyruvate.

• Lipoic acid helps transfer intermediates within the enzyme complex.

  • Like a swinging arm

• CoA forms high-energy acetyl-CoA.

  • Thioester linkage is a "high energy" bond (~31 kJ/mol)

• FAD and NAD+ act as electron acceptors.

  • FAD → has a standard reduction potential to reduce NAD⁺ (immobile)

  • NAD⁺ → final e^– acceptor and NADH delivers to ETC (mobile)

7. Clinical Relevance: Arsenic Poisoning & Vitamin Deficiencies

• Arsenic poisoning disrupts respiration by inactivating lipoamide, a crucial cofactor in PDHC.

  • Organic arsenic compounds are more toxic to microorganisms than humans so they were used as treatments for syphilis and other bacterial and parasitic diseases

• Vitamin B1 (Thiamine) deficiency causes beriberi, affecting the nervous and cardiovascular systems.

8. Dietary Sources of Essential Cofactors

• Thiamine (B1): Meat, nuts, whole grains.

  • Vitamin B1, is one of eight essential B vitamins

• Lipoic Acid: Spinach, broccoli, organ meats.

  • Made from fatty acid precursor in animals

  • Many foods contain alpha-lipoic acid in very low amounts.

• CoA (B5): Eggs, meat, whole grains.

  • CoA biosynthesis requires cysteine, pantothenate (vitamin B5) and ATP

• FAD (B2): Dairy, eggs, green vegetables.

  • Synthesized from riboflavin (Vitamin B2)

• NAD+ (B3): Meat, fish, fortified cereals.

  • Biosynthesized from tryptophan, but relies on salvage pathways that use niacin (vitamin B3)

• Vitamins help the process your body uses to get or make energy from the food you eat

Conclusion

• PDHC is a crucial link between glycolysis and the TCA cycle.

• The TCA cycle efficiently extracts energy from acetyl-CoA.

• The electron carriers (NADH, FADH₂) power ATP synthesis.

• Proper function depends on essential vitamins and cofactors.

TCA Cycle

1. Big Picture & Review of Pyruvate Dehydrogenase Complex (PDHC)

• Pyruvate Dehydrogenase Complex (PDHC) links glycolysis to the TCA cycle.

• It converts pyruvate to acetyl-CoA, which is essential for entering the TCA cycle.

• The reaction:

Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

• PDHC is irreversible, making it a key regulatory step.

2. Membrane Potential & Transport Thermodynamics

• Pyruvate enters mitochondria via the Pyruvate-Proton Symporter, which relies on the proton gradient.

  • The proton gradient drives pyruvate into the mitochondria; electrically neutral

• The electrochemical potential drives metabolite transport and ATP synthesis.

∆G = RTln ([A]_in/[A]_out) + Z_AF∆Ψ

Chemical Potential + Electrical Potential

• A long-lasting drop or rise of ΔΨ (mV) normal levels may induce unwanted loss of cell viability and be a cause of various pathologies

  • ΔΨ is an important factor in selection of non-functional mitochondria

  • ΔΨ drives inward transport cations (+) and outward transport of anions (-)

  • Heterogeneity (differences) of ΔΨ within a single cell may be a sign of a pathology

• Membrane potential (ΔΨ) is crucial for cellular viability—disruptions can lead to pathologies.

  • The inside of the cell typically has a lower potential than the outside, resulting in a negative membrane potential

    • Inside of cell is more negative than the outside

3. Overview of the TCA Cycle

• Acetyl-CoA (2C) enters the cycle and combines with oxaloacetate (OAA, 4C) to form citrate (6C).

• A dehydration-rearrangement yields isocitrate.

• Two successive decarboxylations produce α-KG (5C) and then succinyl-CoA (4C).

• Multiple rearrangements to regenerate OAA.

• The cycle completes via a series of reactions, regenerating OAA and releasing two CO₂ molecules per turn.

• Energy is extracted in the form of:

  • NADH

  • 1 FADH₂

  • 1 GTP (ATP equivalent)

4. Key Enzymes & Reactions of the TCA Cycle

1. Citrate Synthase

• First reaction: acetyl-CoA (2C) + oxaloacetate (4C) → citrate (6C)

• Highly exergonic (ΔG = -53.9 kJ/mol) → regulated step.

  • Initiates TCA cycle

  • Irreversible (highly spontaneous)

• Citrate synthase is classic CoA chemistry

• Mechanism:

  • Cα of the acetyl group in acetyl-CoA is acidic and can be deprotonated to form a carbanion

  • Nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate

  • Thioester hydrolysis

2. Aconitase

• Converts citrate (tertiary alcohol) to isocitrate (secondary alcohol) via a reversible dehydration-rehydration step.

  • Citrate is a poor candidate for oxidation, so the enzyme aconitase catalyzes an isomerization reaction converting citrate to isocitrate

• Uses an iron-sulfur cluster for stereospecificity.

  • Aconitase removes pro-R hydrogen of the pro-R arm of citrate

  • Hydroxide (-OH) is a poor leaving group

    • The added iron (Fe²⁺) atom coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

• Mechanism:

  • Aconitate intermediate

  • Dehydration: The enzyme, using its [4Fe-4S] cluster, binds citrate and removes a water molecule to form cis-aconitate.

  • Rehydration: It then re-adds water to cis-aconitate at a different position, yielding isocitrate.

3. Isocitrate Dehydrogenase 

• Oxidizes isocitrate (6C) to α-ketoglutarate (5C), producing the first NADH and releasing CO₂.

• Classic NAD⁺ chemistry (hydride removal) followed by a decarboxylation

• A link to the electron transport pathway because it makes NADH

• Mechanism:

  • Oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate

  • β-decarboxylation reaction that expels the central carboxyl group as CO₂

  • OH not a good e^– sink; need to convert the alcohol to a carbonyl before decarboxylating with NAD+ (reducing it into NADH)

4. α-Ketoglutarate Dehydrogenase

• Second oxidative decarboxylation, forming succinyl-CoA (4C).

  • Irreversible and favorable (very exergonic)

α-ketoglutarate + CoA + NAD⁺ → succinyl-CoA + CO₂ + NADH

• Produces NADH and CO₂.

  • Second NADH produced

• Mechanistically similar to PDHC, using TPP, CoA, lipoic acid, NAD⁺, and FAD.

  • E1 = α-ketoglutarate dehydrogenase

  • E2 = dihydrolipoyl transsuccinylase

  • E3 = dihydrolipoyl dehydrogenase

• KNOW MECHANISM (refer to PDHC mechanism and replace pyruvate and Acetyl-CoA with α-ketoglutarate and succinyl-CoA)

5. Succinyl-CoA Synthetase

• Converts succinyl-CoA to succinate (symmetrical).

  • Reversible

• Uses substrate-level phosphorylation to generate GTP (ATP equivalent).

  • The thiol bond (S—C=O) is a high energy bond that drives the substrate level phosphorylation to make GTP

• Since succinate is symmetric, we can no longer distinguish which carbons came from acetyl-CoA (pyruvate)

• Mechanism:

  • Synthesis driven by hydrolysis of a CoA ester

  • Formation of a phosphohistidine to produce succinate

  • A nucleoside triphosphate (GTP) is made

• KNOW MECHANISM

6. Succinate Dehydrogenase (Complex II in the ETC)

• Converts succinate to fumarate, producing FADH₂.

  • Reversible

  • FAD is an immobile electron carrier

    • FADH₂ is used rather than NADH because the standard free energy change is near equilibrium (around 0)

• Uses iron-sulfur clusters.

  • Contains three types of Fe-S centers

    • A 4Fe-4S center, a 3Fe-4S center, and a 2Fe-2S center

  • Aides in the stereospecificity

• Directly linked to the electron transport chain.

  • Connected to ETC Complex II

• Mechanism:

  • Involves hydride removal by FAD and a deprotonation

  • The electrons transferred from succinate to FAD (to form FADH₂) are passed directly to ubiquinone (UQ) in the electron transport pathway

7. Fumarase

• Hydrates fumarate to malate.

  • Reversible

• Mechanisms:

  • Two possible mechanisms

    • Carbonium ion (+)

    • Carbonion (-) - more common

8. Malate Dehydrogenase

• Oxidizes malate to oxaloacetate.

  • Reversible

• Produces the final NADH.

  • NAD⁺-dependent oxidation

• Energetically unfavorable (ΔG°’ = +30 kJ/mol) but driven forward by citrate synthase activity.

  • Keep [OAA] low

• This reaction and the previous two form a reaction triad 

5. Net Reaction of the TCA Cycle

Acetyl-CoA  + 3NAD⁺ + FAD + GDP + P_i + 2H₂O → 2CO₂ + 3NADH + FADH₂ + GTP + CoA + 3H⁺

• Highly exergonic (ΔG°’ = -40 kJ/mol; actual ΔG in cells = -115 kJ/mol).

  • VERY SPONTANEOUS

• The NADH and FADH₂ fuel oxidative phosphorylation.

  • The synthesis of ATP in the Electron Transport Chain (ETC)

6. Electron Transfer & ATP Production

• Each NADH = ~2.5 ATP, Each FADH₂ = ~1.5 ATP.

• The total ATP yield from one glucose molecule (glycolysis + TCA + ETC) ≈ 36 ATP.

7. Key Concepts & Importance

• TCA Cycle is the hub of metabolism: integrates carbohydrate, fat, and protein metabolism.

• NADH & FADH₂ drive oxidative phosphorylation in the electron transport chain.

• Regulation occurs at key steps (citrate synthase, isocitrate DH, α-KG DH).

• Disruptions in cycle enzymes (e.g., α-KG DH or isocitrate DH) can be linked to metabolic disorders.

8. Expected Knowledge

• Know enzyme names, cofactors, reaction mechanisms, and metabolic intermediates.

• Understand carbon tracking from acetyl-CoA through the cycle.

• Recognize reduction potentials and why FAD is used in succinate DH instead of NAD⁺.

Final Thought

The TCA cycle is central to bioenergetics, not only generating ATP but also providing precursors for biosynthetic pathways. Understanding its regulation and integration with other metabolic pathways is crucial for biochemistry and physiology.

Regulation of PDHC and TCA

1. Regulation of the Pyruvate Dehydrogenase Complex (PDHC)

• PDHC is a key regulatory step between glycolysis and the TCA cycle.

• It catalyzes the reaction:

Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

• Irreversible step: once pyruvate is converted to acetyl-CoA, it cannot be used to make glucose.

• PDH (E₁) is regulated by covalent modification by enzymes that are allosteric

  • Product inhibition – NADH and Acetyl-CoA

  • Covalent modification – phosphorylation and dephosphorylation of E1 (PDH)

• Regulation of PDHC:

1. Allosteric Regulation

• Inhibitors (high energy state):

  • NADH and Acetyl-CoA (products of the reaction)

• Activators (low energy state):

  • Ca²⁺, ADP, Insulin (indicate a need for energy production)

2. Covalent Modification (Reversible Phosphorylation)

• Pyruvate Dehydrogenase Kinase (PDHK):

  • Phosphorylates PDH → Inactivates it

  • Activated by NADH and Acetyl-CoA (high energy state)

  • Inhibited by Pyruvate (low energy state)

  • Mechanism:

    • Serine residue of E1-PDH gets phosphorylated

      • Blocks decarboxylation of pyruvate and halts formation of acetyl-CoA

  • Is permanently associated with the PDHC

• Pyruvate Dehydrogenase Phosphatase (PDHP):

  • Dephosphorylates PDH → Activates it

  • Activated by Ca²⁺ and Insulin (low energy state, need for ATP production)

    • Also associated with low NADH/NAD⁺ and Acetyl-CoA/CoA ratios

  • Dissociates from the PDHC when there are high concentrations of NADH and Acetyl-CoA (inactivates phosphatase)

  • Mechanism:

    • The phosphorylated Serine residue of E1 gets hydrolyzed

  • Hormonal Control

    • Insulin binding activates PDHP, which activates PDH

  • Not part of the complex, will associate and dissociate when needed

    • Ca²⁺ stabilizes the interactions of phosphatase to the complex

• Phosphorylation inactivates; Dephosphorylation activates.

• Product Inhibition – NADH and Acetyl-CoA

• Covalent Modification – phosphorylation and dephosphorylation of E1 (PDH)

2. Regulation of the TCA Cycle

• Three irreversible steps are key regulation points:

1. Citrate Synthase

• Inhibited by ATP, NADH, and Succinyl-CoA (indicating high energy state)

  • ATP and NADH → allosteric

  • Succinyl-CoA → feedback inhibition (binds to active site) & citrate synthase recognizes the CoA from Acetyl-CoA

2. Isocitrate Dehydrogenase

• Inhibited by ATP (high energy state)

• Activated by ADP and NAD⁺ (low energy state, need for ATP production)

3. α-Ketoglutarate Dehydrogenase

• Inhibited by NADH and Succinyl-CoA

  • Both bind to the active site

• Activated by AMP

  • Allosteric

• Energy State and Regulation

  • High ATP/NADH → Slows down TCA cycle

  • High ADP/Ca²⁺ → Speeds up TCA cycle

  • Ca²⁺ is an important activator in muscle contraction (signaling a need for ATP).

  • Citrate leaks into the cytosol from the mitochondria when too much is made, which then inhibits the glycolysis enzyme PFK1

3. TCA Cycle Provides Intermediates for Biosynthesis

• Many TCA cycle intermediates serve as precursors for biosynthesis:

  • α-Ketoglutarate → Amino acids (Glutamate, Proline, Arginine), Purines

  • Succinyl-CoA → Heme synthesis (Porphyrins)

  • Fumarate & Oxaloacetate → Amino acids (Aspartate, Asparagine), Pyrimidines

  • Malate → Glucose

  • Citrate → Fatty acids, Cholesterol

• All 20 common amino acids can be made from metabolites derived from the TCA cycle.

4. Anaplerotic Reactions (Replenishing TCA Cycle Intermediates)

• Because TCA intermediates are used for biosynthesis, they need to be replenished.

  • The “filling up” reactions

  • Building up TCA intermediates

• Key anaplerotic reactions include:

  • Pyruvate carboxylase → Converts pyruvate (3C) to oxaloacetate (4C) (only in mitochondria).

    • CO₂ + ATP → ADP + P_i

    • Irreversible

    • Requires biotin

    • Activated by high levels acetyl-CoA because is signals the cell has plenty of “fuel”

  • PEP carboxylase → Converts PEP (3C) to oxaloacetate (4C).

    • CO₂ → P_i

  • Malic enzyme → Converts pyruvate (3C) to malate (4C).

    • CO₂ + NADPH → NADP⁺

5. Transport of Metabolites Across the Mitochondrial Membrane

• The inner mitochondrial membrane (IMM) is impermeable to most metabolites.

  • Cytosol → Mitochondria (and vice versa)

• Shuttles are required for transport:

1. Malate-Aspartate Shuttle (liver & heart)

• Transfers NADH equivalents between cytosol & mitochondria.

• Pyruvate → Oxaloacetate (NADH) → Malate (NAD+) → cross IMM→ Malate (NAD+) → Oxaloacetate (NADH) → Gluconeogenesis

• Aspartate Aminotransferase

  • Does NOT require NAD+/NADH

  • Liver has variation of this for a deamination reaction via urea cycle before conversion to OAA

• Malate Dehydrogenase

  • Requires NAD+/NADH, thus reducing equivalents can be transported across the IMM

2. Glycerophosphate Shuttle (muscle & brain)

• Transfers electrons from NADH to FADH₂ for oxidative phosphorylation via a reaction catalyzed by 3-phosphoglycerol dehydrogenase

  • Ketone converted to alcohol (reduction); NADH gets oxidized to NAD+

    • 3-phosphoglycerol dehydrogenase

  • Alcohol converted back to ketone as FAD is reduced to FADH₂

    • Flavoprotein dehydrogenase

  • FADH₂ transports 2 electrons to the ETC

    • Flavoprotein dehydrogenase

6. Glyoxylate Cycle (Alternative to TCA in Some Organisms)

• Plants, bacteria, and fungi use the glyoxylate cycle to convert acetyl-CoA into glucose.

• Bypasses CO₂-releasing steps of the TCA cycle.

  • 2 carbons in, 2 carbons out

• Key (short-circuiting) enzymes:

  • Isocitrate lyase

    • Isocitrate (6C) → Glyoxylate (2C) + Succinate (4C)

  • Malate synthase

    • Glyoxylate (2C) + Acetyl-CoA (2C) → Malate (4C) + CoA

• Humans & animals lack this pathway, meaning they cannot convert fat (acetyl-CoA) into glucose.

• The succinate molecule that acts as a byproduct (not in the cycle) leaves the glyoxylate cycle and enters the TCA cycle (succinate → fumarate → malate), leaves the mitochondria via the malate transporter and converts to oxaloacetate to enter the gluconeogenesis pathway to make glucose.

7. Physiological Relevance of Regulation (Athlete vs. Sedentary Person)

• In an athlete:

  • Epinephrine & exercise → low blood glucose → increased glycolysis & TCA activity.

• In a sedentary person:

  • Excess food & no exercise → high blood glucose → excess fat storage.

• High-fructose corn syrup metabolism favors fat synthesis over energy production.

Final Summary

• PDHC is regulated by phosphorylation (kinase/phosphatase) & allosteric effectors.

• TCA cycle is regulated at three key irreversible steps (Citrate Synthase, Isocitrate DH, α-KG DH).

• TCA cycle provides intermediates for biosynthesis, which must be replenished via anaplerotic reactions.

• Metabolite transport & shuttles are essential for cellular energy balance.

• Glyoxylate cycle allows plants/microbes to convert acetyl-CoA into glucose, which humans cannot do.

• Energy demand (exercise) vs. excess nutrients (sedentary lifestyle) affects metabolic pathways.

  Electron Transport Chain and Oxidative Phosphorylation

1. Overview of Electron Transport and ATP Synthesis

• Electron Transport Chain (ETC) and Oxidative Phosphorylation occur in the inner mitochondrial membrane.

• NADH and FADH₂ donate electrons to the ETC, creating a proton gradient that drives ATP synthesis (oxidative phosphorylation).

• The process follows Mitchell’s Chemiosmotic Hypothesis, where the proton gradient is used to generate ATP.

1. Electron path through the ETC is extremely favorable (-ΔG)

2. ETC generates the proton motive force (PMF), which pumps protons against the gradient

   • Protons go from Matrix (negative/low [H⁺]) → Intermembrane Space (positive/high [H⁺])

3. ETC releases H⁺ gradient (energy) to produce ATP (oxidative phosphorylation)

2. Electron Transport Chain (ETC) Components

• NADH + H⁺ + ½O₂ → NAD⁺ + H₂O 

• Reduction Potentials:

  • Positive Reduction Potential = “happy” to accept e^– (ex: O₂)

  • Negative Reduction Potential = will only accept e^– if you add large amounts of energy; prefers to donate electrons (ex: NADH)

• Coenzyme Q:

  • Q = ubiquinone → “none”; ready to accept e^–; oxidized form

  • QH∙ = ubisemiquinone → has 1e^–

  • QH₂ = ubiquinol → “all”; has 2e^–; reduced form

• The ETC consists of four major protein complexes:

  • Complex I: NADH-UQ Reductase (NADH Dehydrogenase)

    • Transfers electrons from NADH to Ubiquinone (Q).

      • Reduced by NADH

      • Oxidized by Coenzyme Q

    • Pumps 4 H⁺ into the intermembrane space per 2e^–.

    • Uses FMN and iron-sulfur clusters as electron carriers.

      • FMN accepts 2 electrons but donates 1 at a time to the FeS clusters

        • “Middle Man”

      • FeS can only accept 1 electron at a time

    • NADH (2e^–) → FMN (2e^–) → FeS clusters (1e^–) → CoQ (Q→QH₂) (2e^–)

  • Complex II: Succinate-UQ Reductase (Succinate Dehydrogenase)

    • Transfers electrons from FADH₂ to Ubiquinone (Q).

    • No proton pumping occurs at Complex II.

    • Contains FAD and iron-sulfur clusters.

    • Succinate DH is a component of e^– transport Complex II

      • Where FADH₂ gets its electrons from

    • Succinate (2e^–) → FADH₂ (2e^–) → FeS clusters (1e^–) → Heme (1e^–) → CoQ (2e^–)

  • Complex III: Ubiquinol-Cytochrome c Reductase

    • Transfers electrons from QH₂ (Ubiquinol) to Cytochrome c.

      • Reduced by QH₂ (donates e^–)

        • QH₂ = lipid-soluble electron carrier

      • Oxidized by Cyt C (accepts e^–)

        • Cytochrome C = water-soluble electron carrier

          • Only mitochondrial cytochrome that is water soluble

          • Shuttles electrons from Complex III to Complex IV by loosely associating with the inner mitochondrial membrane (IMM)

    • Pumps 4 H⁺ into the intermembrane space per 2e^–.

    • Uses cytochromes and Fe-S clusters as electron carriers.

    • CoQ (QH₂ → Q) (2e^–) → FeS cluster (1e^–) → Cyt c₁ (1e^–) → Cyt C (1e^–)

    • The Q-Cycle is the path electrons take through Complex III

    • Oxidation process occurs in two steps

      • First Half

        • UQH₂ → Rieske Fe-S → Cyt c

          • Releases 2H⁺, leaves UQ∙^–

        • 1 e^– transferred from UQ∙^– to b_L heme then to b_H heme

      • Second Half

        • Second UQH₂ is oxidized same way as first half

        • After electron is transferred to b_H heme, it is transferred to QH∙^– to reform UQH₂

          • Releases 2H⁺

    • CoQ (QH₂ → Q) (2e^–) → Cyt b_L (1e^–) → Cyt b_H (1e^–) → QH∙^– or QH₂ (Q pool)

  • Complex IV: Cytochrome c Oxidase

    • Transfers electrons from Cytochrome c to O₂, forming H₂O.

      • Reduced by Cyt C (donates e^–)

      • Oxidized by O₂ (accepts e^–)

    • Pumps 2 H⁺ into the intermembrane space per 2e^–.

      • But always works in batches of 4e^– to run full cycle

      • 4e^– are transferred sequentially

    • Uses cytochromes and copper centers as electron carriers.

    • Cyt C (1e^–) → Cu_A (1e^–) → Cyt a (1e^–) → Cyt a₃–Cu_B (1e^–) → (½O₂ + 2H⁺ → H₂O)

      • Some intermediates in O₂ → H₂O are extremely reactive free radicals

        • Buildup of these radicals can cause diseases

• Electron movement follows an increasing redox potential, meaning electrons flow from low to high electron affinity.

3. ATP Production and Proton Gradient (Chemiosmotic Hypothesis)

• Proton Gradient Formation:

  • NADH donates electrons → 10 H⁺ pumped across the membrane.

    • Complex I (4H⁺) → Complex III (4H⁺) → Complex IV (2H⁺)

  • FADH₂ donates electrons → 6 H⁺ pumped across the membrane.

    • Complex II  → Complex III (4H⁺) → Complex IV (2H⁺)

• Proton Motive Force (PMF):

  • Drives ATP synthesis via ATP synthase.

  • pH gradient (ΔpH ≈ 0.75 units) + Charge gradient (Δψ ≈ 0.17V).

    • Both of these drives the synthesis of ATP via the PMF

• ATP Yield per Molecule:

  • Usually 3H⁺ makes 1 ATP molecule, however, we should account for the transport costs of protons needed for other molecules

    • This transport costs 1 extra H⁺, so therefore the cost to make 1 ATP molecule is 4H⁺

  • NADH → ~2.5 ATP.

    • 10H⁺/4H⁺ = 2.5 ATP

  • FADH₂ → ~1.5 ATP.

    • 6H⁺/4H⁺ = 2.5 ATP

4. The Q-Cycle in Complex III

• Complex III passes electrons through a Q-cycle mechanism:

  • Step 1: QH₂ donates electrons → one electron to cytochrome c, another to a semiquinone intermediate.

  • Step 2: A second QH₂ donates electrons, completing the reduction of the semiquinone to QH₂.

  • Result: 4 H⁺ pumped across the membrane.

5. ATP Synthase Mechanism

• ATP Synthase (Complex V) consists of:

  • F₀ Subunit: Rotates as protons flow through.

    • This is the part of the protein that is embedded in the membrane

    • Includes:

      • a-subunit → where protons enter (inlet half) from the matrix

      • c-subunits → where the protons from the inlet half a-subunit transfer to; rotate to deliver proton to the outlet half of a-subunit; c-ring

      • b₂-subunit → stabilizes protein and connects the F₀ subunit (a-subunit) to the F₁ subunit (σ-subunit)

  • F₁ Subunit: Catalyzes ATP synthesis.

    • This is the part of the protein that actually does the ATP synthesis

    • Includes:

      • δ-subunit → keeps F₁ stationary while the rotor turns and anchors the ⍺₃β₃ hexamer in place (prevents F₁ region from spinning with the 𝛾-subunit)

      • 𝛾-subunit → central axle that rotates the ⍺₃β₃ hexamer; connected to c-ring, allowing it to rotate when protons move through F₀; 120° rotation induces the conformational changes in the three β-subunits (L, T, O)

      • ε-subunit → at bottom of 𝛾-subunit; more significant in bacteria; can inhibit excessive ATP hydrolysis when the proton gradient is low by locking the 𝛾-subunit, preventing unnecessary ATP breakdown

      • ⍺-subunits → play a structural and regulatory role; interacts with the 𝛾-subunit to regulate rotation

      • β-subunits → the catalytic sites where ATP synthesis occurs; three β-subunits cycles through three conformational states as 𝛾-subunit rotates:

• Rotation of the γ-subunit changes the conformation of αβ subunits:

  • Loose (L) → ADP + Pi bind.

  • Tight (T) → ATP is synthesized.

  • Open (O) → ATP is released.

• 1 full rotation (revolution) = 360° = (9 H⁺) → 3 ATP synthesized.

  • Every 120° rotation = 1 ATP made

6. ATP Transport and Energy Cost

• ATP-ADP Translocase:

  • Transports ATP out of mitochondria into cytosol and ADP into mitochondria

  • Each ATP exported costs 1 additional H⁺.

• Total Cost for ATP Synthesis and Export:

  • 1 ATP = 4 H⁺.

  • NADH → ~10 H⁺ → ~2.5 ATP.

  • FADH₂ → ~6 H⁺ → ~1.5 ATP.

7. Reactive Oxygen Species (ROS) and Protection Mechanisms

• ETC intermediates can generate reactive oxygen species (ROS):

  • Superoxide radical (O₂⁻∙).

    • O₂ + 1e^– → O₂⁻∙

  • Hydrogen peroxide (H₂O₂).

    • O₂⁻∙ + 1e^– + 2H⁺ → H₂O₂

  • Hydroxyl radical (∙OH).

    • H₂O₂ + 1e^– → OH^– + ∙OH

  • Water Product

    • H⁺ + ∙OH + 1e^– → H₂O

• Cells counteract ROS with antioxidants:

  • Superoxide dismutase (SOD) → Converts O₂⁻∙ to H₂O₂.

  • Catalase and Glutathione Peroxidase → Convert H₂O₂ to H₂O.

8. ETC Supercomplexes ("Respirasomes")

• Recent evidence suggests ETC complexes form supercomplexes for:

  • Greater efficiency in electron transfer.

  • Reduced electron leakage, decreasing ROS formation.

  • Improved stability of the respiratory chain.

Final Summary

• ETC creates a proton gradient that powers ATP synthase (chemiosmosis).

• NADH contributes ~2.5 ATP, while FADH₂ contributes ~1.5 ATP.

• ATP synthase uses a rotational mechanism to produce ATP.

• Reactive oxygen species (ROS) are byproducts, but antioxidant enzymes protect cells.

• Supercomplexes in the ETC improve efficiency and minimize ROS.

ETC & Oxidative Phosphorylation Inhibitors

1. Overview of Electron Transport Chain (ETC) & ATP Synthesis

• ETC & Oxidative Phosphorylation occur in the inner mitochondrial membrane.

• NADH & FADH₂ donate electrons to the ETC, generating a proton gradient.

• ATP synthesis is tightly regulated by:

  • [ATP] & [NADH] concentrations.

  • ADP availability (rate-limiting step).

  • Mitochondrial transport systems (e.g., ATP-ADP translocase).

2. Mitochondrial Transport Systems

• ATP-ADP Translocase:

  • Exports ATP from mitochondria & imports ADP.

  • Uses 1 H⁺ per ATP transported.

  • Total cost of making & exporting ATP = 4 H⁺.

• Electron Shuttle Systems for Cytosolic NADH:

  • Glycerophosphate Shuttle:

    • Converts cytosolic NADH to FADH₂, bypassing Complex I.

    • Yields ~1.5 ATP per NADH (loses energy compared to mitochondrial NADH).

    • Used in skeletal muscle & brain.

  • Malate-Aspartate Shuttle:

    • Transfers electrons from NADH to oxaloacetate → malate.

    • Yields ~2.5 ATP per NADH.

    • Used in liver, kidney, and heart.

3. Complex Inhibitors in the ETC

• Inhibitors reveal mechanisms of oxidative phosphorylation.

• Three main types of inhibitors:

  1. Complex inhibitors – Block electron transport.

  2. ATP Synthase inhibitors – Prevent ATP synthesis.

  3. Uncouplers – Disrupt the proton gradient.

Complex Inhibitors

4. Specific Inhibitors & Their Effects

Rotenone (Complex I Inhibitor)

• Found in plant roots, used by indigenous groups to paralyze fish.

• Prevents Fe-S clusters from reducing CoQ.

• Disrupts mitochondrial respiration → cell death.

Demerol (Pain Medication)

• Opioid (similar to morphine).

• Inhibits Complex I, reducing ATP synthesis.

• Highly addictive, linked to opioid crisis.

Amytal (Barbiturate Sedative)

• CNS depressant used for sedation & insomnia.

• Inhibits Complex I, reducing electron flow.

• Highly addictive, risk of coma & overdose.

Cyanide, Azide, & Carbon Monoxide (Complex IV Inhibitors)

• Bind to cytochrome a₃, preventing O₂ reduction.

• Cyanide poisoning symptoms:

  • Sudden collapse, cherry-red skin (due to oxygen retention in blood).

• Carbon monoxide poisoning:

  • Binds to Fe²⁺ in hemoglobin, reducing oxygen transport.

5. ATP Synthase Inhibitor

Oligomycin

• Binds to ATP synthase F₀ subunit, blocking proton flow.

• Prevents ATP synthesis → Cell death.

• Used as an antibiotic.

6. Uncouplers (Disrupt Proton Gradient)

• Uncouplers transport protons back into the matrix, bypassing ATP synthase.

• Energy is released as heat instead of ATP.

• Examples:

  • Dinitrophenol (DNP) – Weight-loss drug (banned in 1938 due to toxicity).

  • Thermogenin (UCP1 in brown fat) – Used by hibernating animals for heat production.

7. Biological & Medical Relevance

Hibernation & Thermogenesis

• Hibernating animals (e.g., bears, squirrels) use uncoupling proteins (UCP1) to generate heat.

• Plants (e.g., skunk cabbage) use proton uncoupling to warm floral spikes for pollination.

Obesity Research & Brown Adipose Tissue (BAT)

• BAT generates heat via uncoupling.

• Inversely related to BMI – higher BAT activity = lower obesity risk.

• Challenges in using BAT for weight loss:

  • Counter-regulatory mechanisms (increased appetite).

  • Excessive heat generation & sweating.

8. Reactive Oxygen Species (ROS) & Antioxidant Defense

• ROS are byproducts of ETC:

  • Superoxide (O₂⁻∙)

  • Hydrogen peroxide (H₂O₂)

  • Hydroxyl radical (∙OH)

• Fenton Reaction (Iron & H₂O₂ generate hydroxyl radicals): H2O2+Fe2+→∙OH+OH−+Fe3+H₂O₂ + Fe²⁺ → ∙OH + OH⁻ + Fe³⁺

• Antioxidant Defense Systems:

  • Superoxide Dismutase (SOD): Converts O₂⁻∙ → H₂O₂.

  • Glutathione Peroxidase & Catalase: Convert H₂O₂ → H₂O.

  • Glutathione (GSH): Neutralizes ROS.

• Oxidative stress is linked to aging & degenerative diseases.

Final Summary

• ETC inhibitors block electron flow, preventing ATP production.

• ATP Synthase inhibitors (Oligomycin) stop ATP generation.

• Uncouplers (DNP, UCP1) dissipate the proton gradient as heat.

• ROS damage cells, but antioxidant enzymes counteract oxidative stress.

• Thermogenesis & uncoupling proteins play roles in metabolism & weight regulation.

• Understanding these processes has medical applications in toxicity, obesity, & aging research.