Biochemistry II Lecture Notes: Oxidative Phosphorylation

19.1 The Mitochondrial Respiratory Chain

The Chemiosmotic Theory

  • Chemiosmotic Theory: The concept that transmembrane differences in proton concentration serve as a reservoir for the energy derived from biological oxidation reactions.

Structure of Mitochondria

  • Mitochondria have two membranes:

    • Outer Membrane:

    • Characteristics:

      • Permeable to small molecules and ions.

      • Transport occurs through porins.

    • Inner Membrane:

    • Characteristics:

      • Impermeable to most small molecules and ions.

      • Requires specific transporters for molecule passage.

The Mitochondrial Matrix

  • Contents of the Mitochondrial Matrix:

    • Pyruvate Dehydrogenase Complex

    • Enzymes of the Citric Acid Cycle

    • Enzymes for Fatty Acid β-Oxidation

    • Enzymes for Amino Acid Oxidation

  • Function: The inner mitochondrial membrane segregates intermediates and enzymes of cytosolic and matrix metabolic pathways.

The Effect of Stress on Mitochondria

  • Mitochondrial Dynamics: During cell growth and division, mitochondria undergo fission.

  • Stress Response:

    • Can trigger:

    • Mitochondrial fission.

    • Mitophagy: Breakdown of mitochondria for recycling of amino acids, nucleotides, and lipids.

    • Under relieved stress: Small mitochondria can fuse to form elongated tubular structures.

Electron Transfer in Oxidative Phosphorylation

  • Types of Electron Transfers:

    • Direct transfer of electrons.

    • Transfer as a hydrogen atom (H++e−H^+ + e^-).

    • Transfer as a hydride ion (:H−:H^-).

  • Reducing Equivalent: Defined as a single electron equivalent transferred in an oxidation-reduction reaction.

Electron-Carrying Molecules in the Respiratory Chain

  • Five Types of Electron-Carrying Molecules:

    • NAD (Nicotinamide adenine dinucleotide)

    • Flavoproteins (e.g., FAD)

    • Ubiquinone (coenzyme Q or Q)

    • Cytochromes

    • Iron-sulfur proteins

Ubiquinone

  • Definition: A lipid-soluble benzoquinone with a long isoprenoid side chain.

  • Functions:

    • Can accept one or two electrons.

    • Freely diffuses within the inner mitochondrial membrane.

    • Plays a central role in coupling electron flow to proton movement.

Cytochromes

  • Definition: Proteins characterized by strong absorption of visible light due to their iron-containing heme prosthetic groups.

  • Carriers: Function as one-electron carriers, classified into three classes in mitochondria: a, b, and c.

  • Binding:

    • Heme groups of a and b are not covalently bound to proteins.

    • Heme c is covalently attached through Cys residues.

Iron-Sulfur Proteins

  • Definition: Proteins containing iron in association with inorganic sulfur atoms and/or sulfur from cysteine residues.

  • Function: Involved in one-electron transfers.

  • Rieske Iron-Sulfur Proteins: Feature one iron atom coordinated to two histidine residues.

Methods for Determining the Sequence of Electron Carriers

  • Approaches:

    • Measure the standard reduction potentials (E′°E'°) of individual electron carriers.

    • Reduce the chain by omitting O2O_2, then measure the oxidation rate when O2O_2 is reintroduced.

    • Assess effects of inhibitors on oxidation state of electron carriers.

Standard Reduction Potentials of Individual Electron Carriers

  • Overview: Electrons tend to flow from carriers with a lower standard reduction potential to those with a higher potential

The Sequence of Electron Carriers

  • Order of Carriers: Sequential transport of electrons occurs from:

    • NADH —> Q —> cytochrome b —> cytochrome c1 —> cytochrome c —> cytochrome a —> cytochrome a3 —> O2

Multienzyme Complexes in the Respiratory Chain

  • Complexes: Four unique electron-carrier complexes catalyze electron transfer:

    • Complex I: Electron transfer from NADH to ubiquinone.

    • Complex II: Electron transfer from succinate to ubiquinone.

    • Complex III: Electron transfer from ubiquinone to cytochrome c.

    • Complex IV: Electron transfer from cytochrome c to molecular oxygen.

Protein Components of the Mitochondrial Respiratory Chain

  • Table of Protein Components:

    • Complex I:

      • Enzyme: NADH dehydrogenase

      • Mass: 850 kDa

      • Subunits: 45 (14 prosthetic groups: FMN, Fe-S)

    • Complex II:

      • Enzyme: Succinate dehydrogenase

      • Mass: 140 kDa

      • Subunits: 4 (prosthetic group: FAD, Fe-S)

    • Complex III:

      • Enzyme: Ubiquinone: cytochrome c oxidoreductase

      • Mass: 250 kDa

      • Subunits: 11 (prosthetic groups: Hemes, Fe-S)

    • Cytochrome c:

      • Mass: 13 kDa

      • Subunits: 1 (prosthetic group: Heme)

    • Complex IV:

      • Enzyme: Cytochrome oxidase

      • Mass: 204 kDa

      • Subunits: 13 (3-4 prosthetic groups: Hemes; CuA, CuB)

Separation of Functional Complexes of the Respiratory Chain

  • Methods:

    • Treatment with digitonin for osmotic rupture.

    • Solubilization using detergent followed by ion-exchange chromatography for separation of inner and outer membrane fragments.

Complex I: NADH to Ubiquinone

  • Description:

    • Large L-shaped enzyme with over 40 polypeptide chains.

    • FMN-containing flavoprotein that accepts 2 electrons from NADH.

    • 8+ Fe-S centers transfer electrons to ubiquinone.

  • Reactions:

    • Exergonic transfer of a hydride ion from NADH and a proton from the matrix to ubiquinone:

    • extNADH+H++Q<br>ightarrowextNAD++QH2ext{NADH} + H^+ + Q <br>ightarrow ext{NAD}^+ + QH_2

    • Endergonic transfer of 4 protons from the matrix to the intermembrane space.

  • Functionality:

    • Complex I is a proton pump driven by the energy derived from electron transfer, facilitating the movement of protons across the membrane.

Complex II: Succinate to Ubiquinone

  • Mechanism:

    • Couples the oxidation of succinate to the reduction of ubiquinone.

    • Electrons move from FAD to Fe-S centers to ubiquinone without proton pumping.

  • Role: Also functions in converting succinate to fumarate in the citric acid cycle.

Heme b of Complex II

  • Function: Reduces the frequency of electron leakage, which would otherwise lead to the formation of reactive oxygen species (ROS) such as H2O2H_2O_2 and superoxide radical.

Complex III: Ubiquinone to Cytochrome c

  • Description:

    • Complex III (cytochrome bc1 complex): couples transfer of 2 electrons from ubiquinol to cytochrome c while transporting four protons into the intermembrane space.

    • Contains cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein.

Path of Electrons through Complex III

  • Details:

  • Ubiquinone diffuses through the membrane and transfers electrons to cytochrome b.

  • Electrons are relayed through Fe-S centers to cytochrome c, one at a time.

The Q Cycle

  • Stages:

    • Stage 1: Electron transfer with pumping protons into the intermembrane space.

    • Stage 2: Regeneration of ubiquinone from ubiquinol.

  • Net Equation of Q Cycle:

    • QH2+2cytcext(oxidized)+2H+<br>ightarrowQ+2cytcext(reduced)+4HP+QH_2 + 2 cyt c_{ ext{(oxidized)}} + 2H^+ <br>ightarrow Q + 2 cyt c_{ ext{(reduced)}} + 4H^+_{P}.

Complex IV: Cytochrome c to O2

  • Function:

  • Carries electrons from cytochrome c to molecular oxygen, reducing it to H2OH_2O.

  • Characteristics:

    • Large dimeric enzyme.

    • 3 conserved subunits through evolution.

    • Contains cytochromes a and a3, along with 2 copper ions.

Path of Electrons Through Complex IV

  • Mechanism:

  • Transfers electrons from cytochrome c, reducing oxygen to water while pumping protons into the intermembrane space.

Overall Reaction of Complex IV

  • Equation:

    • 4cytcext(reduced)+8H+<em>N+O2ightarrow4cytc</em>ext(oxidized)+4HP++2H2O4 cyt c_{ ext{(reduced)}} + 8 H^+<em>{N} + O_2 ightarrow 4 cyt c</em>{ ext{(oxidized)}} + 4 H^+_{P} + 2 H_2O.

Mitochondrial Complexes and Respirasomes

  • Definition: Respirasome is a supercomplex containing Complexes I, III, and IV that may enhance electron transfer efficiency and minimize ROS production.

  • Diffusion: Cytochrome c and ubiquinone diffuse between supercomplexes effortlessly.

Other Electron-Drawing Pathways

  • Pathways:

    • Acyl-CoA Dehydrogenase: Catalyzes β-oxidation and passes electrons to ETF which reduces ubiquinone.

    • Glycerol 3-Phosphate Dehydrogenase: Moves electrons to Q, yielding QH2 for the electron transport chain.

    • Dihydroorotate Dehydrogenase: Donates electrons to Q at the pump site.

Energy of Electron Transfer

  • Net Equation:

    • 2NADH+2H++O2<br>ightarrow2NAD++2H2O2 NADH + 2H^+ + O_2 <br>ightarrow 2 NAD^+ + 2H_2O.

Change in Standard Reduction Potential

  • Calculation:

    • riangleE′°=E′°(extelectronacceptor)−E′°(extelectrondonor)riangle E'° = E'°( ext{electron acceptor}) - E'°( ext{electron donor})

    • Result:

    • riangleE′°=0.816extV−(−0.320extV)=1.14extVriangle E'° = 0.816 ext{ V} - (−0.320 ext{ V}) = 1.14 ext{ V}

    • where E′°E'° for NAD+/NADHNAD^+/NADH is −0.320extV-0.320 ext{ V} and for O2/H2OO_2/H_2O is 0.816extV0.816 ext{ V}.

The Net Reaction is Highly Exergonic

  • Standard Free Energy Change:

    • riangleG′°=−nFriangleE′°riangle G'° = -nF riangle E'°

    • Result:

    • riangleG′°=−2(96.5extkJ/V∙extmol)(1.14extV)=−220extkJ/mol(ofNADH)riangle G'° = -2(96.5 ext{ kJ/V} \bullet ext{mol})(1.14 ext{ V}) = -220 ext{ kJ/mol (of NADH)}

Summary of Electron and Proton Flow Through the Respiratory Chain

  • Conclusion: Much of the free energy generated in the chain is conserved and stored as an electrochemical proton gradient across the inner mitochondrial membrane.