BMM339 Electron Transport and Oxidative Phosphorylation PDF

Lecture 23: Electron Transport and Oxidative Phosphorylation Electron (e-) Transport Chain (ETC) - 4 Complexes of ETC present in the inner membrane of mitochondria Complex 1: catalyzes the e- transfer from NADH coenzymes Q (UQ) Complex 2: catalyzes the e- transfer from FADH2 to coenzyme Q...

Lecture 23: Electron Transport and Oxidative Phosphorylation Electron (e-) Transport Chain (ETC) - 4 Complexes of ETC present in the inner membrane of mitochondria Complex 1: catalyzes the e- transfer from NADH coenzymes Q (UQ) Complex 2: catalyzes the e- transfer from FADH2 to coenzyme Q (UQ) Complex 3: transfers the e- from reduced UQ (UQH2) to cytochrome c (Cyt. c) Complex 4: catalyzes 4 e- to O2 to form H2O Electron Transfer by Complex-1 -Complex-1 (NADH Dehydrogenase Complex): catalyzes the e- transfer from NADH to enzyme Q (UQ) - During the e- transfer from NADH to UQ by complex-1, 4 H+ are pumped out from the matrix into the intermembrane space UQ: lipid-soluble e- carrier shuttles the e- between ETC complexes along the inner mitochondrial membrane Electron Movement by Complex-2 - Complex-2 (Succinate Dehydrogenase Complex): catalyzes the transfer e- from FADH2 to UQ - During the e- transfer from FADH2 to UQ, H+ is not pumped out of matric into the intermembrane space UQ can also get e- from acyl-CoA and glycerol-3-phosphate dehydrogenases Electron Movement by Complex-3 - Complex 3 (Cytochrome bc 1 Complex): transfers e- from reduced UQ (UQH2) to cytochrome c (Cyt c) Takes one e- at a time from UQH2 and transfers to Cyt c. - During e- transfer from UQH2 to Cyt c by complex-3, 4 H+ are pumped out from the matrix and in the intermembrane space Cyt c carries only 1 e- Cyt c is a water-soluble mobile e- carrier of the outer face of the intermembrane Electron Movement in Complex-4 - Complex-4 (Cytochrome Reductase): transfers 4 e- from 4 Cyt c to O2 to form H2O - During e- transfer from Cyt c to O2 by complex-4, 4 H+ are pumped out from matrix into intermembrane ATP can act as an allosteric inhibitor of cytochrome oxidase by binding to complex-4 and Cyt c Energy Relationships in ETC - NADH oxidation results in a substantial energy release This energy is used to pump H+ from matrix into the intermembrane space, which establishes H+ gradient to generate ATP 2.5 molecules of ATP are synthesized per NADH 1.5 molecules of ATP are synthesized per FADH2 Oxidative Phosphorylation and Chemiosmotic Theory - Oxidative Phosphorylation: process that conserves the energy of the ETC by phosphorylation of ADP → ATP As the e- pass through the ETC, H+ are pumped out of the matrix into the intermembrane space, generating H+ motive force - Chemiosmotic Coupling Theory: H+ moves back from intermembrane space into matrix across the membrane through ATP synthase driving ATP formation Explains how oxidative phosphorylation links the ETC and ATP synthesis Evidence for the Chemiosmotic Theory - pH drops in a weakly buffered mitochondria suspension when actively respiring - Disruption of inner membrane stops respiration - Uncouplers: (Dinitrophenol) picks up protons from one side and release on the other side to collapse the H+ gradient - Ionophores: (Gramicidin A) form a channel, allow for passage of proton to disrupt the proton gradient ATP Synthase Structure - ATP synthase consists of two rotors linked by a strong flexible stator - Two major components: F1 unit (ATP synthase) and F0 unit (transmembrane channel) F1 unit has 5 subunits: 3𝛼, 3𝛽, 𝛶, δ, ε F0 unit has 3 subunits: a, 2b, and 12c - F0 unit converts the proton motive force into rotational force of the central shaft (𝛶 and ε subunits) that, in turn, drives ATP synthase - 1 molecule of ATP synthesis requires translocation of 3 protons through the ATP synthase Cross section of ATP Synthase and Shaft Locations - ATP synthase is composed of 3𝛼 and 3𝛽 subunits All 3 𝛼 subunits (𝛼1, 𝛼 2, 𝛼 3) are the same size and shape All 3 𝛽 subunits (𝛽1, 𝛽2, 𝛽3) are the same size and shape. ^ 𝛽-subunits consist of: ADP binding site, Pi binding site Conformational changes of 𝛽-subunit - 𝛽 subunits of the ATP synthase goes through conformational changes: loose (L), tight (T) and open (O) When 𝛶-shaft touches 𝛽1: “L” conformation- ADP and Pi bind to their respective site When neither 𝛶 or ε has touches 𝛽1: 𝛽1 has “T” confirmation - ADP and Pi join to form ATP When 𝛽1 is in contact with ε: 𝛽1 has “O” confirmation - ATP released to matrix ATP, ADP and Pi Transport - ATP synthase synthesizes ATP delivered into matrix ATP exits the matrix though ADP-ATP translocator (ADP-ATP exchange) ADP gets into matrix through ADP-ATP translocator (ADP-ATP exchange) -The required Pi is transported as H2PO4 though phosphate translocase H2PO4 co-transport with H+ Regulation of Oxidative Regulation - High ADP (respiratory control) and Pi level in matrix activate oxidative phosphorylation - High ATP level in matrix inhibits the oxidative phosphorylation  The matrix ATP and ADP level is controlled by the ADP-ATP translocator The matrix H2PO4- level is controlled by phosphate translocase (H2PO4- /H+ symporter) Mechanisms to move cytoplasmic NADH into Matrix (Glycerol-Phosphate Shuttle) - NADH reduces Dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate Glycerol-3-Phosphate diffuses through the mitochondrial outer membrane and reduces FAD to FADH2. ^ Complex-2 of ETC transfer electron from FADH2 to UQH2 Mechanisms to move cytoplasmic NADH into Matrix (Malate-Aspartate Shuttle) - Cytoplasmic NADH reduces oxaloacetate → malate Malate is transported to the matrix - Malate is re-oxidizes to produce NADH Lecture 24: Reactive Oxygen Species and Antioxidants Reactive Oxygen Species - All living processes take place with a RedOx environment - Oxygen usage comes with the danger of forming: Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS) - ROS and RNS: oxygen containing molecules with uneven # of electrons (e- ← “Free Radicals”) Normal ROS level: help fight pathogens Normal RNS level: damages fat, protein, and nucleic acids Antioxidants and Oxidative Stress - Antioxidants: can donate e- to neutralize ROS without compromising their stability and mitigate oxidative damage - Oxidative Stress: an imbalance between ROS and antioxidants Under certain conditions, antioxidant mechanisms are overwhelmed → Oxidative Stress Oxidative damage has been linked to hundreds of human diseases Cell Function and Oxidative Stress - Many proteins of metabolic and signaling pathways are involved in redox rxns - Activation and inactivation of proteins (e.g. enzymes) are altered during redox rxns - Oxidation of sulfhydryl (SH) group in protein: Sulfenic (SOH) acid Sulfinic (R-SO2H) acid Sulfonic (R-SO3H) acid ^change in functional properties of these molecules - Redox change occurs during cellular processes: Cytoplasm of cells that enter cell division become more reduced Cytoplasm of differentiates cells are more oxidized - Intracellular Compartments are different redox conditions: Nucleus/mitochondria are more reduced (GSH/GSSH is High) ER is more oxidized (GSH/GSSH is low) Oxidative Phosphorylation and ROS Formation - Reactive Oxygen Species (ROS)/Free Radicals: O2- : Superoxide Radical H2O2 : Hydrogen Peroxide OH : Hydroxyl Radical - Electron (e-) leaks out of ETC due to aging/over exertion - E- could possibly exit ETC while the e- are transferred by complex-1 and complex-3 E- reacts with O2 and creates superoxide radical (O2-) Additional e- reacts with O2- → Hydrogen Peroxide (H2O2) H2O2 reacts with ferrous iron (Fe2+) → OH Reactive Nitrogen Species (RNS)/Free Radicals - Reactive Nitrogen Species: NO- : Nitric Oxide (good) NO2 : Nitrogen dioxide ONOO- : Peroxynitritrite - Superoxide radical (O2-) reacts with NO- to form peroxynitrite  O2- + NO- → ONOO- - “ONOO-” has nitrating and oxidizing functions. It damages protein and nucleic acid. - “NO-” is an important signaling molecule. It regulates blood pressure, inhibits blood clotting, and destruction of foreign cells by macrophages Radical Chain Reaction 1.) Lipid peroxidation rxns begins after the extraction of hydrogen atom from an unsaturated fatty acid (LH-L) 2.) Lipid radical (L ) rxts with O2 to form a peroxyl radical (L+ O2 → L-O-O) 3.) The radical chain rxn begins when the peroxyl radical extracts a hydrogen atom from another fatty acid molecule (L-O-O+L’H → L-O-OH+L’) 4.) In the presence of transition metal, such as Fe2+ initiates further radical formation (L-O-O-H+Fe2+ → LO+OH- + Fe3+) 5.) One of the most serious consequences of lipid peroxidation is the formation of 𝛼,𝛽-unsaturated aldehyde, which involves a radical cleavage rxn. 6.) The chain continues as the free radical product, then rxts with nearby molecule 7.) Reactive carbonyl products are also products of this process Respiratory Burst - ROS also generated during Respiratory Burst: Macrophages and neutrophils actively make large quantities of ROS to destroy pathogens (microorganisms) - Macrophages/Neutrophils phagocytose the pathogen and form phagosomes - The NADPH-oxidase present on the phagolysosome membranes convert O2 to O2- - O2- rxts with several other molecules to generate OH, -OCL, ONOO-, and NO2 radicals The free radicals destroy the bacteria ROS/RNS kills pathogens - Phagocytosis: bacterium → receptor → phagosome + lysosome → phagolysosomes →Exocytosis -debri Antioxidants - To protect against oxidative stress, living organisms have developed several antioxidants (defense mechanisms) Enzyme Systems: -Superoxide Dismutase -Catalase - Glutathione-centered system  - Thioredoxin-centered system Molecule Systems: - 𝛼-Tocopherol (Vitamin-E) - 𝛽-carotene (Vitamin A) - Ascorbic Acid (Vitamin C) Enzyme Systems Antioxidants Hydrogen peroxide (H2O2) generation and degradation - Superoxide Dismutase (SOD): catalyzes formation of H2O2 and O2 from superoxide radical 2O-2 + 2H+ →(SOD)→ H2O2 + O2 - Catalase degrades the H2O2 into H2O and O2. Catalase present in peroxisome 2 H2O2 → Catalase → 2 H2O + O2 Glutathione-centered System - Glutathione (GSH)-centered system consists of 2 enzymes: glutathione peroxidase and glutathione reductase: Glutathione Peroxidase: Uses GSH to reduce H2O2 to form water and transforms organic peroxides to alcohols. During this process GSH is oxidized to GSSG. 2 GSH + R-O-OH → (GPx) → G-S-S-G + R-OH + H2O Glutathione reductase: uses NADPH to reduce GSSG to GSH G-S-S-G + NADPH + H+ → (Glutathione Reductase) → 2 GSH + NAD+ Thioredoxin-Centered System - Thioredoxin-centered system: Consists of 2 enzymes: Perodiredoxin (PRX) and Thioredoxin Reductase TRX: proteins that act as a antioxidant -The oxidized TRXs (TRS-S2) are reduced by Thioredexin reductase enzyme that utilizes NADPH PRX: uses thiol-containing peptides like TRX to detoxify organic peroxides Chemicals/Molecules Antioxidants 𝛼-Tocopherol, 𝛽-carotene, Ascorbic Acid - 𝛼-Tocopherol (Vitamin-E): potent, lipid-soluble radical scavenger - protects membranes from lipid peroxyl radicals - 𝛽-carotene (Vitamin A): a carotenoid, is a precursor of vitamin A (retinol): a potent, lipid-soluble radical scavenger in membranes - Ascorbic Acid (Vitamin C): efficient antioxidant, present as ascorbate, scavenger variety of water soluble ROS Ascorbic Acid - Scavenges ROS and aqueous compartment of cells and extracellular fluids - Reacts with peroxyl radicals and protects membranes by preventing lipid peroxidation - enhances the antioxidant activity of vitamin E by regenerating Alpha-tocopherol - Protects membrane through two mechanisms: Scavenging a variety of ROS and aqueous environments enhancing the activity of alpha-tocopherol Lecture 25: Lipid Structure and Classification 1 Lipids and Lipid Classification - Lipids: Substances from living things that can be dissolved in nonpolar solvents Can be used for energy storage, membrane structure, chemical signals, vitamins, or pigments - Lipid Classification: 1.) Fatty Acids 2.) Triglycerides 3.) Wax Esters 4.) Phospholipids 5.) Sphingolipids 6.) Isoprenoids 1.) Fatty Acids - Fatty acids are: monocarboxylic acids, contain hydrocarbon chains of variable lengths (12-26 or more carbons) Most of the naturally occurring fatty acids have an even number of carbons in an unbranched chain - Saturated Fatty Acids: Fatty acids that contain only single carbon-carbon bonds - Unsaturated Fatty Acids: Fatty acids that contain one or more double bonds Numbering Systems of Fatty Acids - Numbering of fatty acids starts from Carboxyl C - The 𝛼-carbon is adjacent to the carboxyl group - The terminal methyl carbon is denoted as the omega (𝜔) carbon Fatty acids are important for triglycerides and phospholipids