🧬 Mitochondrial Function & Bioenergetics: Inhibitors, Assays, and Complex
Assembly – A Comprehensive Study Guide
🔹 I. Mitochondrial Function and Role of the Electron Transport Chain (ETC)
Mitochondria are critical organelles often referred to as the "powerhouses" of the cell due
to their central role in aerobic ATP production through oxidative phosphorylation
(OXPHOS). This process relies on a series of protein complexes (I–V) embedded in the
inner mitochondrial membrane, collectively known as the Electron Transport Chain
(ETC).
Electrons from metabolic substrates (via NADH and FADH ₂) are transferred through these
complexes, ultimately reducing molecular oxygen to water at Complex IV. The energy
released is used to pump protons (H⁺) from the matrix to the intermembrane space,
establishing the Proton Motive Force (PMF) that drives ATP synthesis via ATP synthase
(Complex V).
🔹 II. Membrane Disruption and Protein Complex Isolation Using Digitonin
Digitonin is a mild non-ionic detergent used in biochemical research to permeabilize
cholesterol-rich membranes while preserving the integrity of membrane-associated
protein complexes. It is particularly useful for:
Solubilizing mitochondrial inner membranes without denaturing ETC
complexes.
, Preserving supramolecular assemblies, such as respirasomes and ATP
synthesome.
Facilitating Blue Native PAGE (BN-PAGE) to analyze the composition and
molecular weight of intact protein complexes.
✅ Color Identification in Native Gels:
Complexes I and II: Appear violet.
Complexes III and IV: Contain heme iron, giving them a red coloration due to the
presence of cytochrome c.
🔹 III. Key Inhibitors of the Electron Transport Chain (ETC)
Understanding how specific inhibitors block the ETC is crucial for both diagnostic tools and
experimental controls.
Target
Inhibitor Mechanism of Action Biological Impact
Complex
Prevents electron transfer Disrupts NADH oxidation;
Rotenone Complex I from NADH to ubiquinone used in Parkinson’s disease
(CoQ) models
Inhibits electron transfer Halts downstream ETC
Antimycin A Complex III
from cytochrome b to c₁ activity, increasing ROS
Cyanide (CN⁻) & Complex IV Prevents O₂ reduction,
Bind irreversibly to
Carbon Monoxide halts respiration; toxic to
Assembly – A Comprehensive Study Guide
🔹 I. Mitochondrial Function and Role of the Electron Transport Chain (ETC)
Mitochondria are critical organelles often referred to as the "powerhouses" of the cell due
to their central role in aerobic ATP production through oxidative phosphorylation
(OXPHOS). This process relies on a series of protein complexes (I–V) embedded in the
inner mitochondrial membrane, collectively known as the Electron Transport Chain
(ETC).
Electrons from metabolic substrates (via NADH and FADH ₂) are transferred through these
complexes, ultimately reducing molecular oxygen to water at Complex IV. The energy
released is used to pump protons (H⁺) from the matrix to the intermembrane space,
establishing the Proton Motive Force (PMF) that drives ATP synthesis via ATP synthase
(Complex V).
🔹 II. Membrane Disruption and Protein Complex Isolation Using Digitonin
Digitonin is a mild non-ionic detergent used in biochemical research to permeabilize
cholesterol-rich membranes while preserving the integrity of membrane-associated
protein complexes. It is particularly useful for:
Solubilizing mitochondrial inner membranes without denaturing ETC
complexes.
, Preserving supramolecular assemblies, such as respirasomes and ATP
synthesome.
Facilitating Blue Native PAGE (BN-PAGE) to analyze the composition and
molecular weight of intact protein complexes.
✅ Color Identification in Native Gels:
Complexes I and II: Appear violet.
Complexes III and IV: Contain heme iron, giving them a red coloration due to the
presence of cytochrome c.
🔹 III. Key Inhibitors of the Electron Transport Chain (ETC)
Understanding how specific inhibitors block the ETC is crucial for both diagnostic tools and
experimental controls.
Target
Inhibitor Mechanism of Action Biological Impact
Complex
Prevents electron transfer Disrupts NADH oxidation;
Rotenone Complex I from NADH to ubiquinone used in Parkinson’s disease
(CoQ) models
Inhibits electron transfer Halts downstream ETC
Antimycin A Complex III
from cytochrome b to c₁ activity, increasing ROS
Cyanide (CN⁻) & Complex IV Prevents O₂ reduction,
Bind irreversibly to
Carbon Monoxide halts respiration; toxic to
