Oxidative Phosphorylation Chapter 19 Page 690 718 1

Oxidative Phosphorylation Chapter 19 (Page 690 -718) 1

1. Energy from Reduced Fuels is used to Synthesize ATP in Animals • Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell. • Electrons from reduced fuels are transferred to reduced cofactors NADH or FADH 2. • In oxidative phosphorylation, energy from NADH and FADH 2 are used to make ATP.

2. Energy Flow in Cellular Respiration Stage 1: Oxidation of metabolic fuels yielding acetyl-Co. A. Stage 2: Oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. Stage 3: Electrons carried by NADH and FADH 2 are funneled into a chain of mitochondrial electron carriers-the respiratory chain-ultimately reducing O 2 to H 2 O. • e- flow drives the production of ATP

3. Oxidative Phosphorylation • Electrons from the reduced cofactors NADH and FADH 2 are passed to proteins in the respiratory chain. • In eukaryotes, oxygen is the ultimate electron acceptor for these electrons. • Energy of oxidation is used to phosphorylate ADP generating ATP. • This process was discovered in 1948 by Eugene Kennedy and Albert Lehninger.

3. Oxidative Phosphorylation

3 A. Chemiosmotic Theory ADP + Pi ATP Highly Thermodynamically Unfavorable How do we make it possible? • Phosphorylation of ADP is not a result of a direct reaction between ADP and some high-energy phosphate carrier. Rather the process involves: – Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient – The energy released by electron transport is used to transport protons against the electrochemical gradient

3 A. Chemiosmotic Theory Four e- carrying complexes. ATP Synthase Complex

3 B. Chemiosmotic Energy Coupling Requires Membranes • The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions. – Inner membrane in mitochondria • Membrane must contain proteins that couple the energetically “downhill” flow of electrons in the electrontransfer chain with the energetically “uphill” flow of protons across the membrane. • Membrane must contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP.

4. Structure of a Mitochondrion Double membrane leads to four distinct compartments: 1. Outer Membrane: – Relatively porous membrane allows passage of metabolites 2. Intermembrane Space (IMS): – similar environment to cytosol – higher proton concentration (lower p. H) 3. Inner Membrane – Relatively impermeable, with proton gradient across it – Location of electron transport chain complexes – Convolutions called Cristae serve to increase the surface area 4. Matrix – Location of the citric acid cycle and parts of lipid and amino acid metabolism – Lower proton concentration (higher p. H)

4. Structure of a Mitochondrion

5. Electron-transport Chain Complexes contain a Series of Electron Carriers • Each of the 4 complexes contains multiple redox centers consisting of: – Flavin Mononucleotide (FMN) or Flavin Adenine Dinucleotide (FAD) • • Initial electron acceptors for Complex I and Complex II Can carry two electrons by transferring one at a time Tightly bound, sometimes covalently, to flavoproteins Standard reduction potential is dependent on protein environment – Cytochromes a, b or c – Iron-sulfur cluster

5 A. Cytochromes • • One electron carriers (Fe 2+ ↔ Fe 3+) Iron coordinating porphoryin ring derivatives a, b or c are integral proteins but differ by ring additions c also has peripheral protein form

5 B. Iron-Sulfur Clusters • One electron carriers • Coordinate Fe 2+ by cysteines in the protein and inorganic S atoms – Centers defined by inorganic S coordination • Contain equal number of iron and sulfur atoms • Standard reduction potential of the iron varies depending on the center and interaction with protein. 1 Fe 2 Fe-2 S 4 Fe-4 S

6. Ubiquinone (Coenzyme Q) • Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons • Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol • Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side • Coenzyme Q is a mobile electron carrier transporting electrons from Complexes I and II to Complex III

6. Ubiquinone (Coenzyme Q)

6. Ubiquinone (Coenzyme Q)

7. Free Energy of Electron Transport Reduction Potential (E) ∆Eo′ = Eo′(e- acceptor) – Eo′(e- donor) ∆Go′ = –n. F∆Eo′ For negative G need positive E E(acceptor) > E(donor) § Electrons are transferred from lower (more negative) to higher (more positive) reduction potential. § Free Energy released is used to pump protons, storing this energy as the electrochemical gradient.

7. Free Energy of Electron Transport Order of e- can be deduced from E’°: NADH → Q → Cyt b → Cyt c 1 → Cyt c → Cyt a 3 → O 2

8. Composition of Multienzyme Complexes

8 A. Flow of Electrons from Biological Fuels into the Electron-Transport Chain

8 B. NADH: ubiquinone oxidoreductase, a. k. a. Complex I • L-shaped complex and starting point for NADH for electron transfer. • One of the largest macro-molecular assemblies in the mammalian cell. – Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes.

8 B. NADH: ubiquinone oxidoreductase, a. k. a. Complex I • NADH binding site located in the matrix side. – Noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH – Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site

8 BI. Complex I- NADH to Ubiquinone NADH NAD+ + H+

8 BII. NADH: Ubiquinone oxidoreducase is a proton pump • Transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of protons from the matrix (N for loss of protons) to the intermembrane space (P for gain of protons) • Experiments suggest that about four protons are transported per one NADH + Q + 5 H+N → NAD+ + QH 2 + 4 H+P – Reduced coenzyme Q picks up two protons

8 C. Succinate Dehydrogenase, a. k. a. Complex II • Complex II is the starting point for FADH 2 for electron transfer. • FAD accepts two electrons from succinate (recall the citric acid cycle).

8 C. Succinate Dehydrogenase, a. k. a. Complex II • Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone, which becomes reduced QH 2. • Does not transport protons.

8 CI. Complex II- Succinate to Ubiquinone § The complex consists of 4 subunits, two of which (C and D) are transmembrane. § Heme b is not on the main path of e- transfer but may protect against reactive oxygen species.

8 D. Ubiquinone: Cytochrome c Oxidoreductase, a. k. a. Complex III • Uses two electrons from QH 2 to reduce two molecules of cytochrome c. – Involves iron-sulfur clusters, cytochrome bs, and cytochrome cs. – Engages in a Q cycle, which results in four additional protons being transported to the intermembrane space.

8 DI. Complex III- Ubiquinone to Cyt C § The complex is a dimer of identical monomers, each with 11 different subunits. § The complex has two distinct binding sites for ubiquinone, QN and Qp. § Rieske iron-sulfur proteins have Fe-S clusters with His residue attachments in place of Cys.

8 DII. The Q Cycle