Electron Transport and Oxidative Phosphorylation Study Guide

Shuttles for Cytosolic NADH Reoxidation

• Glycerol 3-Phosphate Shuttle
      * Cytosolic glycerol 3-phosphate enters the mitochondrion.
      * It is converted back to dihydroxyacetone phosphate by mitochondrial glycerol 3-phosphate dehydrogenase, which is an FAD-linked enzyme.
      * The dihydroxyacetone phosphate then diffuses back into the cytosol.
      * The enzyme-linked $FADH_2$ is reoxidized by transferring its electrons to ubiquinone in the electron transport chain.
      * Because these electrons enter the chain via $FADH_2$, only approximately two Molecules of ATP are synthesized per molecule of cytosolic NADH.

• Malate-Aspartate Shuttle
      * Used in the heart and liver to reoxidize cytosolic NADH.
      * Oxaloacetate in the cytosol is reduced to malate by NADH.
      * Malate enters the mitochondrion via a malate-$\alpha$-ketoglutarate carrier.
      * Inside the mitochondrial matrix, malate is reoxidized to oxaloacetate by $NAD^+$, which is converted back to NADH.
      * This results in a net transfer of electrons from the cytosol to the matrix.
      * The oxaloacetate is converted to aspartate via transamination, leaves the mitochondrion, and is reconverted to oxaloacetate in the cytosol through another transamination reaction.

Overview of Electron Transport and Oxidative Phosphorylation

• Location and Purpose
      * In eukaryotes, these processes occur in the inner membrane of the mitochondria.
      * These processes re-oxidize NADH and $FADH_2$ produced by:
          * The Citric Acid Cycle (mitochondrial matrix).
          * Glycolysis (cytoplasm).
          * Fatty acid oxidation (mitochondrial matrix).
      * Energy released during reoxidation is trapped as ATP.
      * Oxidative phosphorylation is the cell's major source of ATP.
      * In prokaryotes, these components are located in the plasma membrane.

Redox Potential and Energetics

• Principles of Oxidation-Reduction
      * Oxidation is the loss of electrons; reduction is the gain of electrons.
      * In a reaction, if one molecule is oxidized, another must be reduced (redox reaction).
      * Example reaction: $NADH + H^+ + \frac{1}{2}O_2 \rightarrow NAD^+ + H_2O$.
          * NADH is oxidized to $NAD^+$ (loses electrons).
          * Molecular oxygen is reduced to water (gains electrons).

• Oxidation-Reduction Potential ($E$)
      * A measure of a substance's affinity for electrons, measured relative to hydrogen.
      * Positive Redox Potential: Indicates a higher affinity for electrons than hydrogen ($H_2$); the substance will accept electrons from hydrogen.
      * Negative Redox Potential: Indicates a lower affinity for electrons than hydrogen; the substance will donate electrons to $H^+$ to form hydrogen.
      * NADH is a strong reducing agent with a negative redox potential (tends to donate electrons).
      * Oxygen is a strong oxidizing agent with a positive redox potential (tends to accept electrons).

• Standard Redox Potential ($E_o^{'}$)
      * Measured under standard conditions at pH 7, expressed in volts ($V$).
      * The total voltage change ($\Delta E_o^{'}$) is the sum of the individual redox steps.

• Free Energy Correlation
      * The standard free energy change ($\Delta G^{o'}$) can be calculated using the formula:
          * $\Delta G^{o'} = -nF \Delta E_o^{'}$
          * $n$ is the number of electrons transferred.
          * $\Delta E$ is in volts ($V$).
          * $\Delta G$ is in kilocalories per mole ($kcal\,mol^{-1}$).
          * $F$ is the Faraday constant ($23.06\,kcal\,V^{-1}\,mol^{-1}$).
      * A reaction with a positive $\Delta E_o^{'}$ has a negative $\Delta G^{o'}$ (exergonic).
      * For the oxidation of NADH:
          * $\Delta E_o^{'} = +1.14\,V$
          * $\Delta G^{o'} = -52.6\,kcal\,mol^{-1}$
      * ATP synthesis ($ADP + P_i + H^+ \rightarrow ATP + H_2O$) requires $\Delta G^{o'} = +7.3\,kcal\,mol^{-1}$.
      * NADH oxidation releases enough energy to drive the synthesis of several ATP molecules.

The Electron Transport Chain (Respiratory Chain)

• Chain Structure
      * Electrons are not transferred directly to oxygen; they move through a series of carriers.
      * Consists of three large protein complexes in the inner mitochondrial membrane:
          1. NADH Dehydrogenase (Complex I)
          2. Cytochrome $bc_1$ Complex (Complex III)
          3. Cytochrome Oxidase (Complex IV)
      * Two mobile carries link these complexes:
          * Ubiquinone (Coenzyme Q or CoQ): Small lipid-soluble molecule.
          * Cytochrome c: A small peripheral membrane protein.

Detailed Pathway of Electron Flow

• NADH to NADH Dehydrogenase (Complex I)
      * Complex I consists of at least 30 polypeptides.
      * Binds NADH and re-oxidizes it to $NAD^+$.
      * Two electrons are passed to a prosthetic group called FMN (flavin mononucleotide) to produce $FMNH_2$.
      * Electrons and hydrogen ions ($H^+$) are accepted together.
      * Electrons are then transferred within the complex to iron-sulfur clusters (FeS) in iron-sulfur proteins (nonheme iron proteins).
      * Iron atoms in FeS clusters alternate between ferric ($Fe^{3+}$) and ferrous ($Fe^{2+}$) states as they accept and pass electrons.

• NADH Dehydrogenase to Ubiquinone (CoQ)
      * Electrons from FeS clusters are passed to Ubiquinone (CoQ).
      * CoQ can accept up to two electrons and two $H^+$ ions, converting to ubiquinol ($CoQH_2$).

• Ubiquinol to Cytochrome $bc_1$ Complex (Complex III)
      * The complex is also called cytochrome reductase.
      * It contains cytochrome $b$, cytochrome $c_1$, and an FeS protein.
      * Cytochromes contain a heme group with an iron atom ($Fe^{3+} \rightleftharpoons Fe^{2+}$).
      * Transfer is complicated because ubiquinol is a two-electron carrier, while cytochromes are one-electron carriers.
      * Pathway: $CoQH_2 \rightarrow$ ubisemiquinone ($CoQH^{\cdot}$) $\rightarrow CoQ$, releasing protons.

• Complex III to Cytochrome c to Cytochrome Oxidase (Complex IV)
      * Cytochrome c is loosely bound to the outer surface of the inner membrane.
      * It accepts electrons from the $bc_1$ complex and donates them to Cytochrome oxidase.

• Cytochrome Oxidase to Oxygen
      * Contains two cytochromes ($a$ and $a_3$) and two copper atoms ($Cu_A$ and $Cu_B$).
      * Iron cycles between $Fe^{3+}/Fe^{2+}$ and copper cycles between $Cu^{2+}/Cu^+$.
      * Final reaction: Four electrons from four cytochrome c molecules and four $H^+$ ions are transferred to molecular oxygen to form two water molecules.
      * Equation: $4\,cyt.\,c(Fe^{2+}) + 4H^+ + O_2 \rightarrow 4\,cyt.\,c(Fe^{3+}) + 2H_2O$

Proton Gradient and Energy Coupling

• Directional Flow
      * Carriers interact according to redox potentials.
      * The accepting carrier always has a higher affinity for electrons than the donating carrier.
      * Potential becomes more positive along the chain, ensuring unidirectional flow from NADH to oxygen.

• Proton Pumping
      * The potential falls in three large steps at the three main protein complexes (I, III, and IV).
      * The free energy change at these steps is used to pump $H^+$ ions from the matrix, across the inner membrane, into the intermembrane space.
      * This creates an electrochemical proton gradient consisting of a chemical gradient (pH) and an electrical charge potential (membrane potential).

• Electron Transport from $FADH_2$ (Complex II)
      * Succinate dehydrogenase (Citric Acid Cycle) contains bound FAD reduced to $FADH_2$.
      * Reoxidation occurs via succinate-coenzyme Q reductase (Complex II), an integral membrane protein.
      * Electrons pass from $FADH_2$ to FeS clusters, then to CoQ.
      * Complex II is NOT a proton pump because the free energy change is too small.
      * Other flavoproteins (glycerol phosphate dehydrogenase, fatty acyl CoA dehydrogenase) also feed electrons into CoQ.

Oxidative Phosphorylation and ATP Synthase

• Chemiosmotic Hypothesis
      * Proposed by Peter Mitchell in 1961.
      * Energy from electron transport creates a proton gradient which drives ATP synthesis.
      * Proton-motive force (sum of pH gradient and membrane potential) drives the process.

• ATP Stoichiometry
      * Past Belief: $3\,ATP$ per NADH; $2\,ATP$ per $FADH_2$.
      * Recent Measurements: $2.5\,ATP$ per NADH; $1.5\,ATP$ per $FADH_2$.

• ATP Synthase structure ($F_oF_1\,ATPase$)
      * $F_1$ Unit: Spherical projection into the matrix; consists of five polypeptides in the ratio $(\alpha\beta)_3 \gamma \delta \epsilon$. It has ATPase activity (hydrolyzes ATP) when isolated.
      * $F_o$ Unit: A proton channel spanning the inner mitochondrial membrane.
      * Rotatory Mechanism: The $F_1$ portion acts as the world's smallest rotatory engine; the $\gamma$ subunit rotates relative to the $(\alpha\beta)_3$ unit during ATP hydrolysis/synthesis.

Respiratory Control and Inhibitors

• Inhibitors
      * Rotenone and Amytal: Inhibit Complex I (NADH dehydrogenase); prevent NADH oxidation but allow $FADH_2$ oxidation (since $FADH_2$ enters at CoQ).
      * Antimycin A: Inhibits Complex III (cytochrome $bc_1$ complex).
      * Cyanide ($CN^-$), Azide ($N_3^-$), and Carbon Monoxide ($CO$): Inhibit Complex IV (cytochrome oxidase).

• Respiratory Control
      * Electron transport is usually tightly coupled to ATP synthesis.
      * The rate is regulated by the availability of ADP.
      * Low ADP leads to high ATP, which inhibits electron transport, causing NADH, $FADH_2$, and citrate to accumulate, subsequently inhibiting glycolysis and the citric acid cycle.

Uncoupling and Thermogenesis

• Uncoupling Agents
      * Chemicals like 2,4-dinitrophenol (DNP) stop ATP synthesis while allowing electron transport to continue.
      * DNP is a lipid-soluble $H^+$ ionophore that carries protons back across the membrane, bypassing ATP synthase and dissipating the gradient.
      * Energy is released as heat instead of being captured as ATP.

• Nonshivering Thermogenesis
      * Natural uncoupling in brown adipose tissue.
      * Contains the protein thermogenin (uncoupling protein) in the inner membrane.
      * Thermogenin allows $H^+$ ions to flow back into the matrix without making ATP, generating heat for the organism.