Lecture 12 (Citric Acid to Oxidative Phosphorylation)

The bridging step: Oxidative Decarboxylation

PDH (pyruvate dehydrogenase) converts pyruvate to acetyl CoA.

One carbon released as CO₂.

NAD⁺ reduced to NADH to supply energy to oxidative phosphorylation.

5 coenzymes in PDH complex

Prosthetic group (tightly together): TPP (thiamine pyrophosphate), Lipoamide (lipoyllysine), FAD

Co-substrate: NAD⁺ AND CoA-SH

PDH (phosphate dehydrogenase) complex

Large multienzyme (E1, E2, E3) complex in mitochondrial matrix

E1: Decarboxylates pyruvate, release CO_{2; require TPP. (Decarboxylates pyruvates)}

_{E2: Dihydrolipoyl transacetylase, Transfers acetyl group to CoA forming acetyl-CoA; uses covalently attached lipoamide. (Transfers acetyl group)}

_{E3: Dihydrolipoyl dehydrogenase, Regenerates oxidized lipoamide and transfers electrons to NAD+; requires FAD. (regenerates lipoamide)}

Regulation of Pyruvate dehydrogenase

PDH kinase phosphorylates E1 and inactivates the complex

PDH phosphatase removes the phosphate and reactivates PDH

High energy (ATP, Acetyl CoA, NADH) inhibits PDH

Low energy (ADP, pyruvate) promotes PDH

Some tissues activate PDH through Ca²⁺, helping activate PDH when energy demand rises.

TCA cycle Beginning reaction

Citrate synthase catalyzes C-C bond formation

Citrate Synthase Regulation

• Substrate Availability: reaction rate dependent on oxaloacetate and Acetyl CoA concentration

• Product inhibition: Accumulated citrate inhibits citrate synthase

• Feedback Inhibition: Succinyl-CoA competes with acetyl CoA, reducing citrate synthase activity

• High energy signals: ATP and NADH lowers TCA cycle.

Citrate to Isocitrate

Aconitase catalyzes reversible isomerization of Citrate to Isocitrate

Happens in 2 steps:

• Dehydration: H₂O removed, form double bond cis - aconitate intermediate.

• rehydration: H₂O added back stereotypically, making isocitrate

Delta G positive but reaction continues due to rapid consumption of isocitrate by isocitrate dehydrogenase.

Why turn Citrate into isocitrate

Because citrate is a tertiary alcohol which cannot readily undergo oxidation while isocitrate is a secondary alcohol which can be oxidized

Isocitrate to Alpha ketoglutarate (Oxidative decarboxylation)

Isocitrate dehydrogenase catalyzes conversion of isocitrate to alpha ketoglutarate

Reaction involves 2 steps:

• Oxidation: isocitrate oxidized into ketone, generate NADH

• Decarboxylation: Carbon released as CO₂.

Delta G negative, Irreversible

What is the intermediate between citrate and isocitrate during oxidative decarboxylation

Cis-Aconitate

Different forms of Isocitrate dehydrogenase

• IDH1 and IDH2: NADP⁺ dependent isozyme (IDH1 in cytosol and IDH2 in mitochondria), generates NAPDH, supports biosynthesis and antioxidant defense.

• IDH3: TCA cycle uses mitochondrial NAD⁺ dependent isocitrate dehydrogenase, generate NADH for oxidative phosphorylation.

Regulation for IDH3

Activated by: ADP and Ca²⁺

Inhibited by: ATP and NADH

Alpha Ketoglutarate to Succinyl-CoA

α-Ketoglutarate dehydrogenase converts alpha ketoglutarate (5C) to succinyl-CoA (4C).

Same process as going from isocitrate to α-Ketoglutarate.

α-ketoglutarate has similar enzyme complex as PDH

delta G negative, irreversible

Regulation for α-Ketoglutarate dehydrogenase

• Inhibited by: NADH and succinyl CoA

• Promoted by: Ca²⁺, particularly in muscle mitochondria (muscle contraction)

• Substrate availability: α-Ketoglutarate, CoA-SH, NAD⁺

Generation of GTP through Thioester (Succinyl CoA to succinate)

Succinyl CoA synthetase converts Succinyl CoA to Succinate by breaking the thioester bond and releasing CoA-SH.

Only substrate level phosphorylation in TCA

GTP is energetically equivalent to ATP.

G of succinyl CoA = -36 KJ/mol

G of Succinate = -2.9 KJ/mol

Delta G of Succinyl CoA = -33.5 KJ/mol

Why is the overall ΔG°′ much less negative?

the energy released from breaking the thioester bond drives the unfavorable reaction forward.

How is GTP converted to ATP

using Nucleoside Diphosphate kinase and Mg²⁺.

Oxidation of an Alkane to alkene

Succinate dehydrogenase oxidizes Succinate to fumarate forms C=C.

FAD is reduced to FADH₂ (2 hydrogen removed during the process)

SDH is Complex II of ETC (electron transport chain) and the only TCA-cycle enzyme embedded in the inner mitochondrial membrane.

FAD remains bound to SDH

Hydration across Double bond of Fumarate to L-malate

Fumarase catalyzes fumarate to malate

H₂O is added across C=C, introduces -OH and -H.

reaction is stereospecific, only produces L-malate stereoisomer

Oxidation of Malate to OAA

Malate dehydrogenase catalyzes L-malate to oxaloacetate

reaction generates the third NADH of the TCA cycle and regenerates OAA

delta G positive, reaction continues due to citrate synthase rapidly using OAA.

What is the net ATP produced in TCA? How much ATP per NADH, GTP and FADH

net ATP for TCA cycle is 20 ATP

2GTP: 2 ATP

6NADH: 15 ATP

2FADH₂: 3 ATP

TCA cycle and its importance in Cancer Metabolism

• Altered TCA cycle activity: cancer cell increases TCA cycle flux to support synthesis of amino acids

• Metastatic adaptation: Cancer cells reprogram TCA-cycle metabolism to meet changing energy and biosynthetic demands

• Fumarase deficiency: Loss of fumarase causes fumarate accumulation, disrupting metabolic regulation and promote cancer-associated signaling

Complex I: NADH oxidation and proton Pumping

• Complex I (NADH: ubiquinone oxidoreductase): large L shaped inner mitochondrial membrane complex

• FMN accepts 2 electrons from NADH

• Electrons pass through a series of Fe - S centers to ubiquinone (Q), forming ubiquinol (QH₂).

• Energy released by ET pumps 4H from matrix (N side) to intermembrane space (P side)

FADH + 5H⁺_N + Q —— FAD⁺ + 4 H₂ + QH⁺_P

Complex II: Succinate Oxidation and Ubiquinone Reduction

SDH bounded to Complex II, does reaction 6,

Electrons transferred through Fe - S chain to ubiquinone (Q), produces ubiquinol (QH₂)

Complex II contains Heme B

Complex II does not pump H⁺ across the inner mitochondrial membrane.

Succinate + Q —— Fumarate + QH₂

Complex III: Ubiquinone to Cytochrome C

transfers electrons from ubiquinol (QH₂) to cytochrome C

QH₂ is a mobile electron carrier within the inner mitochondrial membrane obtained from Complex I and II.

Complex III contains cytochrome b, cytochrome c₁, and the Rieske Fe - S protein.

Electron transfer occurs through Q cycle, QH₂ follows 2 different pathways

1. Pass through Reiske Fe - S chain to cytochrome c₁ to cytochrome c

2. Pass through cytochrome b, contribute to Q reduction

Q cycle results in 4H⁺ released to intermembrane space

2H⁺ taken by the matrix

QH₂ + 2 cyt c(ox) + 2H^+_N —— Q + 2 cyt c(red) + 4H⁺_P

Complex IV: Cytochrome c to O₂

transfers electrons from cytochrome c to molecular oxygen (O₂), forming H₂O

Important redox centers:

• Heme a and heme a₃

• Copper centers CuA and CuB

Pathway: Cytochrome c — CuA — heme a — heme a₃ — CuB — O₂

O₂ is the final electron acceptor

4 electrons and 4 matrix H⁺ are required to reduce 1 O₂ to 2 H₂O

Chemiosmotic Coupling: Electron Transport Drives ATP synthesis

• Chemiosmotic coupling: Electron transport through Complexes I, III, and IV generates an electrochemical proton gradient across the inner mitochondrial membrane

• Proton-motive force: The H⁺ concentration gradient and membrane potential store energy released during electron transport

• ATP synthesis H⁺: H⁺ flows back into mitochondrial matrix through ATP synthase

ATP synthase: Structure and Rotary Mechanism

F₀: Membrane-embedded proton channel containing a rotating c-ring

F₁: Catalytic headpiece facing the mitochondrial matrix; contains the catalytic beta subunits where ATP is synthesized.

• Protons flow through F₀ from intermembrane space into matrix

• Proton flow drives rotation of the c-ring and central stalk (gamma) while catalytic α₃ β₃ head remains stationary

F₁ contains 3 catalytic β subunits, which cycle through 3 conformational states:

• β-ADP: Binds ADP and Pi

• β-ATP: Tightly binds newly synthesized ATP

• β-empty: Releases ATP