1.5.3. Oxidative Phosphorylation

Unit 1 9 of the ten metabolites of glycolysis are phosphorylated. Phosphorylated intermediates serve 3 functions. 1. The phosphoryl groups are ionized at physiological pH giving them a net negative electrostatic charge. Biological membranes are impermeable to charged molecules. Intermediates are held within the cell. 2. The transfer of phosphoryl groups conserves metabolic energy. The energy released in breaking the phosphoanhydride bonds of ATP is partially conserved in the formation of phosphate esters. 3. High-energy phosphate compounds formed in glycolysis donate phosphoryl groups to ADP to form ATP. 4. The enzymes of glycolysis use the binding energy of phosphate groups to lower the activation energy and increase the specificity of the enzyme reactions.

1.5.3. Oxidative Phosphorylation

Snapshot

1. Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including oxidative phosphorylation and photophosphorylation. The mechanism of energy coupling is similar in both cases: the energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton-motive force. 2. In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenases donate electrons to the respiratory (electron-transfer) chain, which transfers the electrons to molecular O2, reducing it to H2O. 3. Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH. Reducing equivalents from all NAD-linked dehydrogenations are transferred to mitochondrial NADH dehydrogenase (Complex I). 4. Reducing equivalents are then passed through a series of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the first carrier in Complex III. In this complex, electrons take two separate paths through two b-type cytochromes and cytochrome c1 to an Fe-S center. The Fe-S center passes electrons, one at a time, through cytochrome c and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a3, accumulates electrons, then passes them to O2, reducing it to H2O. 5. Some electrons enter this chain of carriers through alternative paths. Succinate is oxidized by succinate dehydrogenase (Complex II), which contains a flavoprotein that passes electrons through several Fe-S centers to ubiquinone. Electrons derived from the oxidation of fatty acids pass to ubiquinone via the electron-transferring flavoprotein. 6. Plants also have an alternative, cyanide-resistant NADH oxidation pathway. The flow of electrons through Complexes I, III, and IV results in pumping of protons across the inner mitochondrial membrane, making thematrix alkaline relative to the intermembrane space. This proton gradient provides the energy (in the form of the proton-motive force) for ATP synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane. ATP synthase carries out “rotational catalysis,” in which the flow of protons through Fo causeseach of three nucleotide-binding sites in F1 to cycle from (ADP _ Pi)–bound to ATP-bound to empty conformations. 7. ATP formation on the enzyme requires little energy; the role of the proton-motive force isto push ATP from its binding site on the synthase.  The complete oxidation of glucose by molecular oxygen is:  C6 H12O6 + 6O2 → 6CO2 + 6H2O ΔG°′ = -2823 kJ/mol

This equation is broken down into two half-reactions.  The glucose carbon atoms are oxidized by water molecules: o C6 H12O6 + 6H62O → 6CO2 + 24H+ + 24e- {Glycolysis and citric acid cycle}

The molecular oxygen is reduced by the protons and electrons produced by glucose oxidation:  6O2 + 24H+ + 24e-  12H2 O {Electron transport and oxidation}

Unit 1  The oxidation of glucose carbon atoms is carried out in glycolysis and the citric acid cycle,and the produced protons and electrons are stored in NADH and FADH2 molecules.  The NADH and FADH2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules because each contains a pair of electrons having a high transfer potential.  When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP.  Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.  This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.  For example, oxidative phosphorylation generates 26 of the 30 molecules of ATP that are formed when glucose is completely oxidized to CO2 and H2O.

The Mitochondrion

 Is located inside of cells.  Size: Ellipsoid of ~0.5 μm diameter and ~1 μm length. o Proteins that mediate electron transport and oxidative phosphorylation are bound to the inner membrane.  Inner compartment called matrix contains:  Soluble enzymes for oxidative metabolism.  substrates  nucleotide  cofactors  inorganic salts  DNA, RNA, ribosomes Outer membrane:

 Has porins which have non-specific pores that permit free diffusion up to 10 kD molecules.  Thus the concentration of ions in the intermembrane space and cytosol are nearly the same.  Inner membrane:

Figure 1.5.9. Catabolism of proteins, f ats, and carbohydrates in the three stages of cellular respiration. Stage 1: oxidation of f atty acids, glucose, and some amino acids yields acetyl-CoA. 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 FADH2 are f unneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain— ultimately reducing O2 to H2O. This electron f low drives the production of ATP.

- is ~75% protein.  Is impermeable to most hydrophilic substances, freely permeable only to O2, CO2 and H2O.  Contains respiratory chain proteins and transport proteins that control [ATP], [ADP],[pyruvate], [Ca2+], [Pi], and etc. - Generates ionic gradients between cytosol and mitochondria.

Oxidative Phosphorylation

 In the electron transport chain, 10NADH+10H++5O2→10NAD++10H2O, and 2FADH2+O2→2H2O.  Both reactions are highly exergonic (NADH oxidation has a ΔG° of -52.6kcal/mol, and FADH2 oxidation has a ΔG° of -46kcal/mol).  Oxidative phosphorylation combines the oxidation of NADH or FADH2 with the phosphorylation of ADP to produce ATP.  Energy captured from the exergonic oxidation reactions is stored in the terminal phosphate group of ATP.  Substrate-level phosphorylation captures energy directly from highly-energetic substrates and stores it in ATP.  Oxidative phosphorylation takes place in the mitochondria, and specifically in the inner (semipermeable) membrane, or matrix membrane.

Electron Transport Chain

 The electron transport chain (ETC) begins on the matrix side of the inner mitochondrial membrane. Figure 1.5.10. Biochemical anatomy of a mitochondrion. Cytosolic NADH can only enter into oxidative phosphorylation as FADH2 produced by the malateaspartate shuttle.  Complex IV of the electron transport chain receives electrons from cytochrome C (from Complex III ← CoQ ← Complex I ← NADH), and utilizes oxygen as the final electron acceptor, reducing it to water.  The -52.6kcal/mol of energy liberated by NADH oxidation is released slowly by the electron transport chain, rather than at any single point, and immediately stored by the production of a hydrogen gradient by pumping protons from the matrix to the intermembrane space (and hence, to the cytoplasm).  Complex I pumps four protons, Complex III pumps two protons, and Complex IV pumps four protons, resulting in ten protons pumped per electron pair (NADH molecule) entering the chain.  Because of this proton gradient, matrix pH is much higher than cytosolic pH (8.4 versus 7.4, or a tenfold difference in proton concentration).  A membrane potential across the inner mitochondrial membrane is set up due to the mass moving of positive charges out of the matrix, negatively charging the matrix space, resulting in an electrochemical proton gradient (or "proton-motive force").  Oxidative phosphorylation is the culmination of a series of energy transformations that are called cellular respiration or simply respiration in their entirety.

Unit 1  First, carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential. Then, this electron-motive force is converted into a proton-motive force and, finally, the proton-motive force is converted into phosphoryl transfer potential.  The conversion of electron-motive force into proton-motive force is carried out by three electron-driven proton pumps—NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase.  These large transmembrane complexes contain multiple oxidation-reduction centers, including quinones, flavins, iron-sulfur clusters, hemes, and copper ions.  The final phase of oxidative phosphorylation is carried out by ATP synthase, an ATPsynthesizing assembly that is driven by the flow of protons back into the mitochondrial matrix.  Components of this remarkable enzyme rotate as part of its catalytic mechanism. Oxidative phosphorylation vividly shows that proton gradients are an interconvertible currency of free energy in biological systems.

Succinate and FADH2

 Succinate as an electron donor enters the ETC through Complex II (succinate dehydrogenase), passing its electrons via Complex II to CoQ (coenzyme Q).  FADH2 as an electron donor transfers its electrons either to CoQ or to Complex III.  Both succinate and FADH2 pump only six protons up the gradient, since they bypass Complex I, which pumps four protons.

ATP Synthesis

 To capture the energy resident in the proton gradient, adenine nucleotide translocase (ANT) exchanges cytosolic ADP for matrix ATP, making ATP available to the cell and ADP available to Complex V (ATP Synthase), which phosphorylates ADP using energy captured from proton influx through its central channel.  ANT dissipates the proton gradient by one proton per nucleotide pair exchanged.  While Complex V only requires the transport of three protons for synthesis of each ATP, ANT requires the transport of one additional proton to make that ATP available to the cell, resulting in a net use of four protons from the gradient for each ATP produced. Thus oxidation of NADH results in the production of 2.5 ATP, and oxidation of FADH2 results in the production of 1.5 ATP.

Chemiosmotic Theory

 Forty years ago, the inner mitochondrial membrane was suggested to be semipermeable. In particular, it was thought to be impermeable to protons.  The ETC pumps protons out of the matrix, setting up a proton gradient across the matrix membrane.  Energy from the oxidation of electron carriers is converted to the energy of the chemiosmotic gradient.  The chemiosmostic theory received the Nobel prize when it was proven correct.

Complex V Blockers

 Complex V blockers dramatically increase the magnitude of the proton-motive force, since they block ATP-synthase-driven dissipation of the gradient.  This stops ATP synthesis and shuts down the ETC, since it does not have enough energy to pump protons up such a steep gradient.  NADH oxidation will be shut down since NADH will accumulate, oxygen will not be used, and will then accumulate to toxic levels.

Figure 1.5.11. Essence of Oxidative Phosphorylation Oxidation and ATP synthesis are coupled by transmembrane proton f luxes.

Unit 1

Proton Gradient Uncouplers

 Proton gradient uncouplers shut down ATP synthesis by providing alternate paths for gradient dissipation which do not produce ATP.  Complex V is "coupled" to the electron transport chain, since the ETC requires Complex V to dissipate the proton gradient it produces.  Proton channels and proton shuttles uncouple ATP synthase from the ETC.  Electron-carrier oxidation and ETC activity continues, since the ETC doesn't care if ATP is synthesized or not. In fact, NADH oxidation is stimulated, since the proton gradient is completely broken down, and there is very little resistance to proton transport.  Oxygen utilization is increased when NADH oxidation increases.

 Dinitrophenol is an uncoupler, since it is a membrane-soluble proton acceptor, so it can act as a proton shuttle, facilitating the movement of protons down the gradient.  Uncouplers increase temperature, since the energy released is no longer stored as chemical energy, but is simply released as heat.  Thermogenin, a human protein involved in thermoregulation, is a carefully-controlled uncoupler which redirects ETC-produced energy to heat production from ATP production.

Malate-aspartate shuttle

- is the mammalian system. - is more energy efficient than glycerophosphate shuttle.

Figure 1.5.12. Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons f rom NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron f low is accompanied by proton transf er across the membrane, producing both a chemical gradient (pH) and an electrical gradient (). The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through protonspecif ic channels (Fo). The proton-motive f orce that drives protons back into the matrix provides the energy f or ATP synthesis, catalyzed by the F1 complex associated with Fo.