Oxidative Phosphorylation PDF

Topic 7 ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION November 25th, 2021 Topic 7 Electron transport Consumption of electron generated from TCA Mitochondria Q, QH, QH2. Sequence of electron flow Transport means: 4 complexes and the Q cycle in compl...

Topic 7 ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION November 25th, 2021 Topic 7 Electron transport Consumption of electron generated from TCA Mitochondria Q, QH, QH2. Sequence of electron flow Transport means: 4 complexes and the Q cycle in complex III ATP synthesis ATP synthase C-ring rotation Potential E and free energy G of ATP synthesis Van Nguyen 1  Summary (Cellular) Respiration: ATP- generating from TCA and oxidative phosphorylation process. Oxidative phosphorylation captures the energy of high-energy electrons to synthesize ATP. COUPLING The flow of electrons from NADH and electron acceptor FADH2 to O2 occurs in the electron- Transport carriers Electron transport vs transport chain or respiratory chain. ATP synthesis energetics Sequence of electron flow Transport enzymes ATP synthase This exergonic set of oxidation– (4 complexes; Q cycle) C-ring rotation reduction reactions generates a proton gradient, and then the proton gradient is used to power the synthesis of ATP. Van Nguyen 2 Coupling of Electron Carrier Oxidation and ADP Phosphorylation The flow of electrons from reduced carriers such as NADH is highly exergonic. This is sufficient to power the rephosphorylation of multiple copies of ADP. The coupling of electron carrier oxidation with ADP rephosphorylation occurs with the participation of the inner mitochondrial membrane. Van Nguyen 3  Location: Mitochondria permeable to most small ions and molecules TCA, fatty acid oxidation E transport, ATP synthesis impermeable to most molecules Van Nguyen 4  Electron transport chain Complex I and II Complex III Complex IV Sequence of electron flow: Oxidized form Reduced form. pH 7, 25oC NADH + H+ à NAD+ + 2e- + 2H+ 2 steps FMNH2 à FMN + 2e- + 2H+ 2 steps FADH2 à FAD + 2e- + 2H+ Van Nguyen 5 Electron flow Van Nguyen 6  Electron transport chain Complex I and II Complex III Complex IV Van Nguyen 7 Ubiquinone (Q) Van Nguyen 8 Cytochromes with hemes Van Nguyen 9 Iron-sulfur centers Van Nguyen 10 Transport e to Q from NADH NADH + H+m + Q à NAD+ + QH2 4H+m à 4H+i Van Nguyen 11  Coupled Electron–proton Transfer Reactions through NADH-Q Oxidoreductase Electrons flow in Complex I from NADH through FMN and a series of iron–sulfur clusters to ubiquinone (Q), forming Q2−. The charges on Q2− are electrostatically transmitted to hydrophilic amino acid residues [shown as red (glutamate) and blue (lysine or histidine) balls] that power the movement of HL and βH components. This movement changes the conformation of the transmembrane helices and results in the transport of four protons out of the mitochondrial matrix. Van Nguyen 12 Transport e to Q from FADH2 Complex II is not a proton pump. Succinate dehydrogenase of the citric acid cycle is a part of the succinate-Q reductase complex (Complex II). FADH2 à Q Van Nguyen 13 Transport e to Q FADH2 à Q Van Nguyen 14 Complex III: Q-Cytochrome c oxidoreductase Van Nguyen 15 Complex III – The Q cycle QH2 carries two electrons, whereas cytochrome c carries only one electron electron transfer from QH2 to cytochrome c In the first half of the Q cycle, one electron from QH2 reduces cytochrome c and one reacts with Q to form QŸ−. In the second half of the cycle, another QH2 reduces cytochrome c and QŸ−. In one cycle, four protons are pumped out of the mitochondrial matrix and two are removed from the matrix. Van Nguyen 16 Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen to Water Cytochrome c oxidase accepts four electrons from four molecules of cytochrome c in order to catalyze the reduction of O2 to two molecules of H2O In the cytochrome c oxidase reaction, eight protons are removed from the matrix. Four protons, called chemical protons, are used to reduce oxygen. In addition, four protons are pumped into the intermembrane space Van Nguyen 17  Structure of Cytochrome c Oxidase Consists of 13 polypeptide chains; most are embedded in the membrane Two major prosthetic groups: heme a and heme a3–CuB Heme a3–CuB is the site of the reduction of oxygen to water. CuA/CuA: accept electrons from CO(bb) is a carbonyl group cytochrome c. of the peptide backbone. When the Fe of heme a3 and CuB are reduced (accept e), they bind oxygen as a peroxide bridge between them 2 electrons flow from cytochrome c à CuA/CuA à heme a à heme a3 à CuB. Van Nguyen 18 Cytochrome c Oxidase Mechanism 2 electrons flow from cytochrome c à CuA/CuA à heme a à heme a3 à CuB. Van Nguyen 19 Proton Transport by Cytochrome c Oxidase Van Nguyen 20 Summary of electron transport Total of 10 Protons pumped Van Nguyen 21 NADH & proton gradient coupling (*) Energy cost to pump 1 mol H+ is 20 kJ/mol (calculation at the end) 10 Van Nguyen 22  A Proton Gradient Powers the Synthesis of ATP The proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motive force. The proton-motive force powers the synthesis of ATP. The proton-motive force consists of a chemical gradient and a charge gradient. Heterologous experimental systems confirmed that proton gradients can power ATP synthesis. Van Nguyen 23  ATP synthase One of nature’s greatest enzymes: MW > 500 kDa Contains 2 motors: One driven by proton motive force (F0 subunit) One driven by ATP hydrolysis (F1 subunit) The rotation of 1 motor runs the other in reverse Stator to connect parts together (not shown) Van Nguyen 24  ATP synthase structure F0 subunit - proton motive force (embedded in inner membrane): Contains c ring: 10 – 15 c chains (hydrophobic polypeptides, depends on species) Each c chain composed of 2 alpha – helixes To one side of the c ring, there is a single chain called the a chain a chain has proton channels and isolate some c chains Van Nguyen 25  ATP synthase structure F1 subunit – ATP driven motor (in matrix): contains 5 types of chains γ, ε chains: stalk, asymmetric, α, β chains: form a ring of 6 chains (3 – 3 alternative); both chain types can bind ATP Stalk is intact with c ring and rotate through the 6-chain ring As the stalk turns, each β chain alternates through 1 of 3 conformations that change their ATP binding state Van Nguyen 26  ATP synthase structure Stator: partly determined hold the α, β ring still resist the rotation of F0. and the stalk Van Nguyen 27  ATP synthase structure summary F1: α, β, γ chains β catalyzes; γ connects F1 with F0. F0: a, b, c chains; transport protons c chains assembled (10 – 15 depending on species) to form c ring Van Nguyen 28  Free energy change of ADP à ATP of (E-S) of (E-P) P. S. Transitional states with both S & P Yellow (S à P): Unfavorable, require energy à easily convert ES to EP à need to release P from the EP à c ring rotation Van Nguyen 29 ATP synthase function: C-ring rotation Binding change model Van Nguyen 30  2 C-ring rotation 1 3 O state L state T state (open) (loose) (tight) Accept Trap Bound ADP + Pi, ADP + Pi tightly 4 Release ADP + Pi, 5 ATP ATP formed Stalk rotates 1200 per time For every full turn: 3 ATP released (1 ATP per β chain) Van Nguyen 31  Proton pump mechanism 2 of the 10 c chains are isolated by the a chain. a chain: 2 hydrophilic half channels, P side opens to the (i) and N-side opens to the (m) (i-intermembrane space, m-matrix) (+) charged Arginine residue Aspartate residues of the 2 c chains lie in contact with P & N-sides Van Nguyen 32  Proton pump mechanism Asp facing P-side is originally deprotonated; (-) and has electrostatic interaction with Arg. Meanwhile the Asp facing N-side is protonated and hence neutral. When [H+]i is high, H+ enter P-side half channel, propel Arg shifted to N-side, and Asp (P-side) is protonated. Arg in its new position triggers the deprotonation of Asp (N-side) and releases H+ into N-side half channel. Arg is now free to rotate by 1 c chain, move the deprotonated Asp to P-side. C ring rotation is Brownian motion (random) but its direction is determined by the proton gradient See video Van Nguyen 33 References https://courses.edx.org “HarvardX: MCB63X” Van Nguyen 38  Energetics of proton gradient Substrate Product CM: concentration in Matrix CI: concentration in Innermembrane Van Nguyen 39 Energetics of proton gradient Van Nguyen 40 Energetics of proton gradient The difference in pH between Matrix and Innermembrane Van Nguyen 41