Cellular Respiration, PDH, TCA Cycle & Intro to 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 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 as a single hydride ion.

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+ and regenerating oxidized FAD.

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+.

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), which is physically associated with E2.

What enzyme dephosphorylates and activates the PDH complex?

Pyruvate Dehydrogenase Phosphatase (PDP), which is physically associated with E2.

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

High energy ratios (ATP/ADP, Acetyl-CoA/CoA, NADH/NAD+).

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 vicinal -SH groups of reduced dihydrolipoamide on E2, blocking re-oxidation and halting 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 so that the otherwise inert methyl carbon can undergo oxidation in subsequent steps.

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 to prevent premature, wasteful hydrolysis of the thioester bond.

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

The liver diverts oxaloacetate into gluconeogenesis; with oxaloacetate depleted, incoming Acetyl-CoA cannot enter the TCA cycle and is shunted to ketone bodies.

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.

What prosthetic cofactor cluster is contained within Aconitase?

An iron-sulfur cluster ([4Fe-4S] center).

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 beta-keto acid intermediate that readily undergoes decarboxylation).

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

No; the CO2 released originates from oxaloacetate carbons (the incoming acetyl carbons remain in the cycle).

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 structurally and mechanistically homologous to PDH?

It uses analogous E1, E2, E3 subunits and requires the exact 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 tissues (like skeletal/heart muscle and brain) generate ATP; biosynthetic tissues (like liver) primarily generate GTP.

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

Succinate + FAD < = > Fumarate + FADH2.

What is unique about the cellular location of Succinate Dehydrogenase compared to other TCA enzymes?

It is an integral inner mitochondrial membrane protein (Complex II of the ETC); all other TCA enzymes are soluble in the matrix.

What stereoisomer of fumarate is generated by Succinate Dehydrogenase?

The trans double-bond isomer (fumarate).

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 trans double bond creates 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 does the Malate Dehydrogenase reaction proceed forward in cells despite a highly positive Delta G'°?

Rapid consumption of oxaloacetate by the highly exergonic Citrate Synthase reaction keeps steady-state [OAA] extremely low, pulling the equilibrium forward.

What is the net yield of high-energy products from one turn of the TCA cycle (per Acetyl-CoA)?

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 (oxidative breakdown for energy) and anabolism (supplying precursor intermediates for biosynthesis).

Distinguish between cataplerotic and anaplerotic reactions.

Cataplerotic reactions drain intermediates from the cycle for biosynthetic pathways; anaplerotic reactions replenish intermediates back into the cycle.

What biosynthetic pathway drains Citrate from the TCA cycle?

Fatty acid and cholesterol/sterol synthesis in the cytosol.

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

Amino acid synthesis (transamination to Glutamate) and purine nucleotide synthesis.

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

Porphyrin and heme biosynthesis.

What biosynthetic pathways drain Oxaloacetate from the TCA cycle?

Gluconeogenesis (conversion to PEP) and amino acid synthesis (transamination to Aspartate).

What is the most important anaplerotic reaction in mammalian liver and kidney?

Pyruvate Carboxylase: Pyruvate + HCO3- + ATP -> Oxaloacetate + ADP + Pi.

What cofactor and obligate allosteric activator are required by Pyruvate Carboxylase?

Biotin (Vitamin B7) carries the activated CO2; Acetyl-CoA is an absolute obligate allosteric activator.

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

1 NADH ~ 2.5 ATP; 1 FADH2 ~ 1.5 ATP.

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

Glycolysis: 2 ATP + 2 NADH (5 ATP); PDH: 2 NADH (5 ATP); TCA: 2 GTP (2 ATP) + 6 NADH (15 ATP) + 2 FADH2 (3 ATP) = 30 to 32 ATP total (28 ATP from oxidative phosphorylation).

What are the permeability characteristics of the outer mitochondrial membrane?

Readily permeable to small molecules and ions (< 5 kDa) due to porin channels.

What are the permeability characteristics of the inner mitochondrial membrane?

Strictly impermeable to most small molecules, polar compounds, and ions (including H+); requires dedicated membrane transport systems.

What is the structural and physiological purpose of mitochondrial cristae?

Extensive convolutions of the inner membrane that expand surface area to accommodate high numbers of ETC complexes and ATP synthase particles.

What is standard reduction potential (E'°)?

A measure (in Volts) of the affinity of a chemical species to accept electrons (tendency to be reduced).

In what direction do electrons flow spontaneously through the ETC in terms of reduction potential?

From carriers with a lower/more negative E'° (strong donors) to carriers with a higher/more positive E'° (strong acceptors).

What equation relates free energy change (Delta G'°) to the difference in reduction potential (Delta E'°)?

Delta G'° = -n * F * Delta E'°.

For the overall 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 / Q) and where is it located?

A small, lipid-soluble benzoquinone with a 10-isoprene hydrophobic hydrocarbon tail that freely diffuses within the inner mitochondrial membrane bilayer.

What are the three oxidation states of Ubiquinone?

1. Ubiquinone (Q, fully oxidized); 2. Semiquinone radical (QH radical, intermediate with 1 e- and 1 H+); 3. Ubiquinol (QH2, fully reduced with 2 e- and 2 H+).

Why is Ubiquinone uniquely suited to couple electron flow to proton pumping?

It carries both electrons and protons; reduction picks up matrix protons, and oxidation releases protons into the intermembrane space.

What are Cytochromes?

Electron-transferring proteins containing iron-protoporphyrin (heme) prosthetic groups that exhibit characteristic visible light absorption.

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

The central iron reversibly cycles between Fe3+ (ferric, oxidized) and Fe2+ (ferrous, reduced).

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

Exactly 1 electron at a time.

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

It is a water-soluble, peripheral membrane protein located in the intermembrane space that shuttles single electrons from Complex III to Complex IV.

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

Non-heme centers containing iron coordinated by cysteine sulfurs or inorganic sulfides; they transfer 1 electron at a time.

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

No; CoA-SH is an acyl-group carrier, not a redox carrier in the ETC.

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

NADH:ubiquinone oxidoreductase (NADH dehydrogenase); contains an FMN flavoprotein and at least 8 iron-sulfur (Fe-S) centers.

Trace the electron path through Complex I.

NADH -> FMN -> Fe-S centers -> Ubiquinone (Q) to form QH2.

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

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

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

Succinate:ubiquinone oxidoreductase (Succinate dehydrogenase); contains bound FAD, Fe-S centers, and heme b.

Trace the electron path through Complex II.

Succinate -> FAD -> Fe-S centers -> Ubiquinone (Q) to form QH2.

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

Zero protons (0 H+); the standard reduction potential difference between succinate and ubiquinone is too small to drive proton translocation.

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

Ubiquinol:cytochrome c oxidoreductase (Cytochrome bc1 complex); contains cytochrome b, Rieske Fe-S center, and cytochrome c1.

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

Oxidizes QH2 back to Q and transfers electrons to reduce soluble Cytochrome c.