Cellular Respiration: PDH, TCA Cycle, ETC & Oxidative Phosphorylation

What is the primary definition of cellular respiration?

The cellular consumption of O2 and release of CO2 coupled to the oxidation of organic fuels and ATP synthesis.

What are the three successive stages of cellular respiration?

Stage 1: Oxidation of fuels to Acetyl-CoA; Stage 2: Oxidation of acetyl groups in the TCA cycle; Stage 3: Electron transfer through the ETC coupled to oxidative phosphorylation.

Under aerobic conditions, what is the fate of pyruvate produced by glycolysis?

Pyruvate is transported into the mitochondrial matrix and converted into Acetyl-CoA by the Pyruvate Dehydrogenase (PDH) Complex.

Under anaerobic conditions, what is the fate of pyruvate in human cells?

Lactate dehydrogenase reduces pyruvate to lactic acid (lactate) in the cytosol.

What is the physiological purpose of converting pyruvate to lactate under anaerobic conditions?

To re-oxidize NADH back to NAD+ so that the GAPDH step of glycolysis can continue generating ATP.

What specific transporter brings pyruvate across the inner mitochondrial membrane into the matrix?

The Mitochondrial Pyruvate Carrier (MPC).

What is the net chemical reaction catalyzed by the Pyruvate Dehydrogenase (PDH) Complex?

Pyruvate + CoA-SH + NAD+ -> Acetyl-CoA + CO2 + NADH + H+.

Is the PDH reaction reversible in animal cells?

No, it is chemically irreversible in animals; Acetyl-CoA cannot be converted back into pyruvate.

What are the names of the three catalytic enzyme subunits of the PDH complex?

E1: Pyruvate dehydrogenase; E2: Dihydrolipoyl transacetylase; E3: Dihydrolipoyl dehydrogenase.

Which of the three PDH enzymes perform decarboxylation and generation of Acetyl-CoA?

E1 and E2 generate Acetyl-CoA; E3 performs a series of redox reactions to regenerate the complex and produce NADH.

List the 5 coenzymes required by the Pyruvate Dehydrogenase Complex.

1. Thiamine pyrophosphate (TPP); 2. Lipoic acid (lipoamide); 3. Coenzyme A (CoA-SH); 4. FAD; 5. NAD+.

Which subunit of the PDH complex uses Thiamine Pyrophosphate (TPP) as a cofactor?

E1 (Pyruvate dehydrogenase).

What vitamin is TPP derived from, and what is its functional reactive group?

Vitamin B1 (Thiamine); its functional reactive group is the thiazolium ring.

Which coenzymes are associated with the E2 subunit of the PDH complex?

Lipoic acid and Coenzyme A (CoA-SH).

How is lipoic acid attached to the E2 subunit?

It is covalently bonded via an amide linkage to a specific Lysine residue, forming a flexible lipoamide/lipoyllysine arm.

What are the three distinct chemical forms of lipoic acid in the PDH complex?

1. Oxidized form (contains a closed disulfide ring -S-S-); 2. Reduced form (contains two free dithiol groups -SH); 3. Acetylated form (carries an acetyl group via a thioester bond).

What three structural components make up Coenzyme A (CoA-SH)?

A nucleotide (adenosine 3',5'-bisphosphate), Pantothenic acid (Vitamin B5), and a mercaptoethylamine group.

What is the reactive functional group of Coenzyme A?

The terminal thiol/sulfhydryl group (-SH).

What type of chemical bond joins the acetate group to CoA in Acetyl-CoA?

A high-energy thioester bond with a large negative standard free energy of hydrolysis.

Which coenzymes are associated with the E3 subunit of the PDH complex?

FAD (Flavin adenine dinucleotide) and NAD+ (Nicotinamide adenine dinucleotide).

What vitamin precursors give rise to FAD and NAD+?

FAD is derived from Riboflavin (Vitamin B2); NAD+ is derived from Niacin / Nicotinic acid (Vitamin B3).

Compare the physical mobility of FAD versus NAD+ in the PDH complex.

FAD is an immobile prosthetic group tightly bound to E3; NAD+ is a soluble, mobile electron carrier that binds transiently and diffuses away as NADH.

How many electrons can FAD accept versus NAD+?

FAD accepts 2 electrons one at a time; NAD+ accepts 2 electrons all at once.

What happens in Step 1 of the PDH mechanism (catalyzed by E1)?

E1 decarboxylates pyruvate, releasing the first CO2 of respiration and transferring the remaining 2 carbons onto TPP to form hydroxyethyl-TPP.

Which step of the PDH complex is the rate-limiting step?

Step 1 (the decarboxylation of pyruvate catalyzed by E1).

What happens in Step 2 of the PDH mechanism (catalyzed by E1 and E2)?

The oxidized lipoamide arm of E2 swings to E1; E1 oxidizes the hydroxyethyl group to an acetyl group while reducing the lipoamide disulfide bond.

What happens in Step 3 of the PDH mechanism (catalyzed by E2)?

The transacetylase activity of E2 transfers the acetyl group from the lipoamide arm to the -SH group of CoA-SH, generating Acetyl-CoA.

What happens in Step 4 of the PDH mechanism (catalyzed by E3)?

The reduced dithiol lipoamide arm swings to E3, where electrons are transferred from dihydrolipoamide to enzyme-bound FAD, generating FADH2.

What happens in Step 5 of the PDH mechanism (catalyzed by E3)?

Enzyme-bound FADH2 passes its electrons to mobile NAD+, forming NADH + H+.

What is the physical role of the lipoyllysine "swinging arm" on E2?

It acts as a flexible molecular tether that channels substrate intermediates between the active sites of E1, E2, and E3.

What allosteric metabolites INHIBIT the PDH complex?

High energy/product signals: ATP, Acetyl-CoA, NADH, and fatty acids.

What allosteric metabolites ACTIVATE the PDH complex?

Low energy/substrate signals: AMP, CoA-SH, and NAD+ (plus Ca2+).

How is the E1 subunit of PDH regulated by covalent modification?

It is inactivated by phosphorylation of regulatory Serine residues by PDK, and activated by dephosphorylation by PDP.

What enzyme phosphorylates and inactivates the PDH complex?

Pyruvate Dehydrogenase Kinase (PDK).

What enzyme dephosphorylates and activates the PDH complex?

Pyruvate Dehydrogenase Phosphatase (PDP).

What conditions or effectors stimulate Pyruvate Dehydrogenase Kinase (PDK)?

High energy/product states (high ATP, Acetyl-CoA, and NADH).

What effectors stimulate Pyruvate Dehydrogenase Phosphatase (PDP)?

Ca2+ (especially in contracting skeletal muscle) and Insulin (in liver/metabolic tissues).

How do Phosphatase 1 and Phosphatase 2 of PDP differ in tissue distribution?

Phosphatase 1 is regulated by Ca2+ in muscle; Phosphatase 2 is dominant in liver and sensitive to hormones.

How does arsenic (arsenite) poison the PDH complex?

It binds with high affinity to the -SH groups of reduced lipoyllysine on E2, deactivating the complex.

What reaction is catalyzed by Citrate Synthase (TCA Step 1)?

Acetyl-CoA + Oxaloacetate + H2O -> Citrate + CoA-SH.

What is the chemical logic of the Citrate Synthase reaction?

Condenses the 2-carbon acetyl group with 4-carbon oxaloacetate into a 6-carbon citrate skeleton.

How does Citrate Synthase display an induced fit mechanism?

Oxaloacetate must bind first, inducing a conformational change that creates the binding site for Acetyl-CoA.

What happens to TCA cycle flux in the liver during low blood glucose?

The liver diverts oxaloacetate into gluconeogenesis; with oxaloacetate depleted, Acetyl-CoA will not enter the TCA cycle.

What allosteric factors regulate Citrate Synthase?

Inhibited by ATP, NADH, Succinyl-CoA, and Citrate; activated by ADP.

What reaction is catalyzed by Aconitase (TCA Step 2)?

Citrate < = > cis-Aconitate < = > Isocitrate.

What is the chemical logic of the Aconitase reaction?

Citrate possesses a tertiary alcohol that cannot be oxidized; aconitase dehydrates and rehydrates it into isocitrate, a secondary alcohol that can be oxidized.

Why is Citrate converted to Isocitrate?

To reposition the -OH hydroxyl group so that it is in a more favorable position (secondary alcohol) to be oxidized to alpha-ketoglutarate by Isocitrate Dehydrogenase.

What reaction is catalyzed by Isocitrate Dehydrogenase (TCA Step 3)?

Isocitrate + NAD+ -> alpha-Ketoglutarate + CO2 + NADH + H+.

What unstable intermediate forms during the Isocitrate Dehydrogenase reaction?

Oxalosuccinate (an unstable intermediate created by oxidizing the secondary alcohol to a ketone, which then undergoes decarboxylation).

Does the CO2 released in Step 3 of the TCA cycle come from Acetyl-CoA?

No; the CO2 released originates from oxaloacetate carbons (both Acetyl-CoA carbons remain in alpha-ketoglutarate and are released in later turns).

How is Isocitrate Dehydrogenase regulated?

Inhibited by ATP and NADH; activated by ADP and Ca2+ (in muscle).

What reaction is catalyzed by the alpha-Ketoglutarate Dehydrogenase Complex (TCA Step 4)?

alpha-Ketoglutarate + CoA-SH + NAD+ -> Succinyl-CoA + CO2 + NADH + H+.

Why is the alpha-Ketoglutarate Dehydrogenase Complex homologous to PDH?

It uses analogous E1, E2, E3 subunits and requires the same 5 coenzymes: TPP, lipoic acid, CoA-SH, FAD, and NAD+.

What regulates the alpha-Ketoglutarate Dehydrogenase Complex?

Inhibited by its direct products (Succinyl-CoA and NADH) as well as ATP; activated by Ca2+.

What reaction is catalyzed by Succinyl-CoA Synthetase (TCA Step 5)?

Succinyl-CoA + GDP (or ADP) + Pi < = > Succinate + GTP (or ATP) + CoA-SH.

What is the chemical logic of the Succinyl-CoA Synthetase reaction?

Conserves the free energy released from cleavage of the high-energy thioester bond to drive substrate-level phosphorylation without carbon loss or redox chemistry.

How does nucleoside triphosphate production in Step 5 vary between tissues?

Actively respiring cells form ATP from ADP; biosynthetic cells form GTP from GDP.

What reaction is catalyzed by Succinate Dehydrogenase (TCA Step 6)?

Succinate + FAD < = > Fumarate + FADH2.

What is unique about Succinate Dehydrogenase's location?

It is bound to the inner mitochondrial membrane and serves as Complex II of the ETC, linking the TCA cycle to oxidative phosphorylation.

What chemical change does Succinate Dehydrogenase introduce?

It oxidizes a central alkane into a trans double bond (alkene) in fumarate without releasing carbon.

What reaction is catalyzed by Fumarase (TCA Step 7)?

Fumarate + H2O < = > L-Malate.

What is the chemical logic of the Fumarase reaction?

Stereospecific hydration across the double bond adds H2O to form a secondary alcohol in L-malate positioned for oxidation in the final step.

What reaction is catalyzed by Malate Dehydrogenase (TCA Step 8)?

L-Malate + NAD+ < = > Oxaloacetate + NADH + H+.

Why can the Malate Dehydrogenase reaction proceed in both directions?

It oxidizes the secondary alcohol to regenerate oxaloacetate for another turn of the TCA cycle and is reversible to participate in gluconeogenesis.

What is the net yield of high-energy products from one turn of the TCA cycle?

3 NADH, 1 FADH2, 1 GTP (or ATP), and 2 CO2.

How many pairs of electrons are harvested per Acetyl-CoA in the TCA cycle?

4 pairs of electrons (total of 8 electrons: 6 via NADH and 2 via FADH2).

What does it mean that the Citric Acid Cycle is amphibolic?

It functions in both catabolism and anabolism.

Distinguish between cataplerotic and anaplerotic reactions.

Cataplerotic reactions drain intermediates; anaplerotic reactions replenish intermediates.

What biosynthetic pathway drains Citrate from the TCA cycle?

Fatty acid and cholesterol/sterol synthesis.

What biosynthetic pathway drains alpha-Ketoglutarate from the TCA cycle?

Nitrogen metabolism: it is aminated to Glutamate.

What biosynthetic pathway drains Succinyl-CoA from the TCA cycle?

Porphyrin and heme biosynthesis.

What biosynthetic pathways drain Oxaloacetate from the TCA cycle?

Gluconeogenesis and nitrogen metabolism.

What is the most important anaplerotic reaction in the liver and kidneys?

Pyruvate Carboxylase, which carboxylates pyruvate into oxaloacetate.

What cofactor is commonly employed by carboxylation enzymes?

Biotin, which activates CO2.

What is the ATP yield per NADH and per FADH2 oxidized in the ETC?

2.5 ATP for NADH; 1.5 ATP for FADH2.

Break down the total ATP yield from the complete oxidation of 1 glucose molecule.

32 ATP total (28 ATP from oxidation).

What are the permeability characteristics of the outer mitochondrial membrane?

Readily permeable to small molecules and ions.

What are the permeability characteristics of the inner mitochondrial membrane?

Impenetrable to small molecules and ions; houses all ETC complexes.

What is the purpose of mitochondrial cristae?

Increase surface area to enhance ATP production.

What is standard reduction potential (E'°)?

A measurable value that assesses the tendency of a chemical species to accept or donate electrons.

In what direction do electrons flow through the ETC?

From carriers with lower E'° to carriers with higher E'°.

What equation relates free energy change to the difference in reduction potential?

Delta G'° = -n * F * Delta E'°.

For the reaction of NADH donating electrons to O2, what are Delta E'° and Delta G'°?

Delta E'° = +1.14 V; Delta G'° = -220 kJ/mol.

What is Ubiquinone (Coenzyme Q) and where is it located?

A lipid-soluble benzoquinone that diffuses within the inner mitochondrial membrane.

What are the three oxidation states of Ubiquinone?

1. Ubiquinone (Q); 2. Semiquinone radical (QH radical); 3. Ubiquinol (QH2).

Why is Ubiquinone suited to couple electron flow to proton movement?

It can pick up 1 or 2 electrons and protons, carrying both across the inner membrane.

What are Cytochromes?

Electron-transferring proteins with strong absorption of visible light due to iron-containing heme groups.

What iron oxidation state transition occurs in cytochromes during electron transport?

The iron atom moves between Fe3+ (oxidized) and Fe2+ (reduced).

How many electrons can a cytochrome accept at a single time?

One electron.

What is unique about Cytochrome c compared to other respiratory cytochromes?

It is a soluble, mobile peripheral membrane protein located in the intermembrane space that accepts electrons from Complex III and moves to donate them to Complex IV.

What are Iron-Sulfur (Fe-S) centers, and how many electrons do they transfer?

Non-heme proteins containing iron coordinated with inorganic sulfur or cysteine sulfur residues; they participate in 1-electron transfers.

Is Coenzyme A (CoA-SH) a carrier in the respiratory electron transport chain?

No; Coenzyme A is not in the respiratory chain.

What is the full systematic name and cofactor composition of Complex I?

NADH:ubiquinone oxidoreductase (NADH dehydrogenase); a large L-shaped enzyme containing FMN (flavoprotein) and 8+ iron-sulfur (Fe-S) centers.

Trace the electron path through Complex I.

NADH is oxidized to NAD+ -> FMN is reduced to FMNH2 -> series of Fe-S centers -> Ubiquinone (Q) is reduced to Ubiquinol (QH2).

How many protons are pumped into the intermembrane space by Complex I per pair of electrons?

4 protons (4 H+) are pumped from the matrix into the intermembrane space.

What is the full systematic name and cofactor composition of Complex II?

Succinate dehydrogenase; a TCA cycle enzyme bound to the inner membrane containing FAD, Fe-S centers, and heme b.

Trace the electron path through Complex II.

Succinate oxidized to fumarate -> FAD reduced to FADH2 -> Fe-S centers -> Ubiquinone (Q) reduced to Ubiquinol (QH2).

How many protons are pumped by Complex II into the intermembrane space?

Zero protons (0 H+); the thermodynamics of FADH2 to Q are not sufficient to pump protons.

What is the full systematic name of Complex III and what are its key redox centers?

Cytochrome bc1 complex; contains cytochrome b, Rieske iron-sulfur proteins, and cytochrome c1.

What reaction does Complex III catalyze, and what does it reduce?

Oxidizes Ubiquinol (QH2) back to Ubiquinone (Q) and passes electrons to reduce soluble Cytochrome c.