CM14 - Oxidative Phosphorylation

Biomedical Sciences I

What are the two phases of energy production from fuel oxidation?

• Production of reduced NADH and FADH2

• Use of generated energy to produce ATP = oxidative phosphorylation

How is energy produced in oxidative phosphorylation?

• Electrons flow from NADH and FADH2 through carriers to reach O2

• Energy from electron transfer is used to pump protons (H+) into the intermembrane space

• Energy released when protons reenter the mitochondrial matrix is used to synthesize ATP

• ETC coupled with ATP synthesis = oxidative phosphorylation

How are reducing agents transported into the mitochondria if the IMM lacks an NADH transporter?

Via the Glycerol 3-P shuttle (NADH → FADH2) and the Malate shuttle (NADH → NADH)

Describe the mechanism of the Glycerol 3-P shuttle (NADH → FADH2).

• Electrons from NADH are transferred to DHAP by cytosolic glycerol 3-P dehydrogenase

• Glycerol 3-P is then oxidized by mitochondrial isoenzyme, turning FAD → FADH2

Describe the mechanism of the Malate shuttle (NADH → NADH)

• Oxaloacetate reduced to malate using NADH

• Malate enters mitochondria and is oxidized back to oxaloacetate, reforming NADH

How are ATP and ADP transported across the IMM?

• Adenine nucleotide antiporter: imports 1 ADP into the matrix, exports 1 ATP to cytosol

• Phosphate transporter: carries phosphate from cytosol into the matrix

Where is the ETC located and what are its main components?

• Located in the inner mitochondrial membrane

• 4 large multiprotein complexes (I–IV)

• 2 small carriers: coenzyme Q (CoQ) and cytochrome c

• Prosthetic groups:

  • FAD, FMN: complexes I & II

  • Heme groups: complexes III & IV

  • Copper ion: complex IV

• Carriers transfer electrons between complexes → final combination with O2 + H+ → H2O

What occurs in Complex I (NADH:CoQ oxidoreductase)?

• Giant protein complex in IMM

• Electrons from NADH are passed to complex I, energy is lost → used to pump 4 H+ from matrix into IMS

What occurs in Complex II (Succinate dehydrogenase)?

• Oxidizes succinate → fumarate (TCA cycle), producing FADH2

• No protons pumped (no energy lost here)

• Provides a parallel entry for electrons into the ETC

• Electrons passed to CoQ one at a time

What is Coenzyme Q (ubiquinone)?

• Quinone derivative from cholesterol

• Only lipid-soluble, non-protein-bound ETC component

• Mobile carrier of electrons from Complex I/II → Complex III

• Carries 2 electrons at a time

What occurs in Complex III (Cytochrome bc1)?

• 2 electrons from ubiquinone transferred: ubiquinone → cytochrome b → cytochrome c1 → cytochrome c

• Cytochrome c is a mobile electron carrier (1 electron at a time) to Complex IV

• Large energy drop → 4 H+ pumped into IMS

What occurs in Complex IV (Cytochrome oxidase, cytochrome a+a3)?

• Conducts electrons through cytochromes a and a3, finally reducing O2

• With 4 electrons, 4 protons are reduced and O2 is split →  forms 2 H2O

• Pumps additional 2 H+ per H2O into IMS

What are Reactive Oxygen Species (ROS)?

• O2 can accept 4 electrons → reduced in 4 steps

• CoQ can accidentally interact with O2 → superoxide

• Partially reduced oxygen is unstable and electron-hungry = ROS

What is oxidative stress?

• Imbalance between ROS production and removal mechanisms

• Results in free-radical mediated damage:

  • Lipid peroxidation

  • Protein oxidation/degradation/aggregation

  • DNA damage (base oxidation, double-strand breaks)

What defenses exist against oxygen toxicity?

• Enzymes: glutathione peroxidase, catalase, superoxide dismutase

• Antioxidants: vitamins A, C, E

What are the inhibitors of the ETC?

• Block electron flow to oxygen → inhibit ATP synthesis

• Complex I: rotenone, barbiturates

• Complex III: antimycin A

• Complex IV: cyanide (CN⁻), carbon monoxide (CO)

What is the chemiosmotic theory?

• Energy for ADP → ATP phosphorylation is produced by proton flow down an electrochemical gradient

• Proton gradient established by H+ pumped into IMS during electron transport (Complexes I, III, IV)

• Electron flow is coupled to proton flow, and proton flow is coupled to ADP phosphorylation

What is the structure of ATP synthase (Complex V)?

Multisubunit enzyme with two domains:

• Fo (membrane domain): rotor + H+ channel

• F1 (matrix domain): protruding sphere with 3 αβ-subunits, each β with catalytic site

What is the mechanism of ATP synthase (Complex V)?

• H+ reenters matrix through Fo H-channel → drives rotation of c ring

• Rotation causes conformational changes in F1 catalytic sites → ADP + Pi → ATP

• One complete c ring rotation = 3 ATP molecules

What are the requirements for oxidative phosphorylation?

• Electron donors: NADH, FADH2

• Electron acceptor: O2

• Intact mitochondrial membrane

• Functional ETC components

• ATP synthase

What happens if ATP synthase is inhibited or ADP is inadequate?

• ATP synthesis inhibited

• O2 not consumed

• ETC components accumulate in reduced states

What is the function of oligomycin (poison)?

Binds Fo domain, closes H+ channels → blocks H+ reentry → inhibits ATP synthesis and oxidative phosphorylation

What are oxidative phosphorylation uncouplers?

Proteins/channels that allow H+ reentry into matrix without ATP synthesis

What are the effects of oxidative phosphorylation uncouplers?

• ATP production ↓

• O2 consumption and ETC rate ↑

• Energy released as heat (non-shivering thermogenesis)

What are some examples of oxidative phosphorylation uncouplers?

• UPC1/thermogenin: brown adipose tissue heat production

• Dinitrophenol: lipophilic H+ carrier disrupting proton gradient

How many proteins involved in oxidative phosphorylation are encoded by mitochondrial DNA (mtDNA)?

13 proteins are encoded by mtDNA and synthesized in the mitochondrial matrix.

Why does mtDNA have a higher mutation rate than nuclear DNA?

• It lacks protective histones

• Has limited repair mechanisms

• Is directly exposed to ROS generated by the ETC

What are the main consequences of mtDNA mutations affecting oxidative phosphorylation?

• Defective oxidative phosphorylation → ATP deficiency

• Affects tissues with high ATP demand (muscle and nerve)

• Leads to:

  • Lactic acidosis (cells shift to anaerobic glycolysis → lactate buildup)

  • Myopathy (muscle weakness, fatigue, exercise intolerance)

  • Neuropathy/neurodegeneration (since neurons need continuous ATP)

What is Leber’s Hereditary Optic Neuropathy (LHON)?

• Caused by mutations in Complex I of the ETC

• Leads to bilateral degeneration of retinal ganglion cells and optic nerve atrophy

• Clinical presentation: sudden, painless central vision loss, usually in young adults

• Optic nerve especially vulnerable because of its high ATP demand

What is Leigh Syndrome?

• Often linked to mutations in the Fo portion of ATP synthase (Complex V), though other ETC mutations can cause it

• Clinical features:

  • Optic nerve atrophy

  • Hypotonia (“floppy baby”)

  • Ataxia (unsteady movements, poor coordination)

  • Respiratory abnormalities (due to ATP-dependent brainstem centers)

• It is a progressive, neurodegenerative disorder of infancy/early childhood