Chapter 6 Biological Oxidation Oxidation removal of electrons Reduction gain of electrons NADH and FADH2 formed in glycolysis, fatty acid oxidation, and citric acid cycle can be used for reductive biosynthesis

Biological Oxidation The deducing potential of mitochondrial NADH is most often used to supply the energy for ATP synthesis via oxidative phosphorylation. Oxidation of NADH with phosphorylation of ADP to form ATP are processes supported by the mitochondrial electron transport assembly and ATP synthase witch are integral protein complexes of the inner mitochondrial membrane.

Principles of Reduction/Oxidation (Redox) Reactions Redox reactions involve the transfer of electrons from one chemical species to another.

Principles of Reduction/Oxidation (Redox) Reactions Oxidation of NADH by the electron transport chain NADH + (1/2)O2 +H+  NAD+ + H2O The reduction potential is –52.6 kcal/mol

Principles of Reduction/Oxidation (Redox) Reactions ADP + Pi  ATP is kcal/mole Direct chemical analysis has shown that for every 2 electrons transferred from NADH to oxygen, 2.5 equivalents of ATP are synthesized and 1.5 for FADH2

Principals of Reduction/Oxidation (Redox) Reactions Redox reactions involve the transfer of electrons from one chemical species to another. The oxidized plus the reduced form of each chemical species is referred to as an electrochemical half cell. Two half cells having at least one common intermediate comprise a complete, coupled, redox reaction. Coupled electrochemical half cells have the thermodynamic properties of other coupled chemical reactions. If one half cell is far from electrochemical equilibrium, its tendency to achieve equilibrium (i.e., to gain or lose electrons) can be used to alter the equilibrium position of a coupled half cell. An example of a coupled redox reaction is the oxidation of NADH by the electron transport chain: NADH + (1/2)O 2 + H > NAD + + H 2 O

The thermodynamic potential of a chemical reaction is calculated from equilibrium constants and concentrations of reactants and products. Because it is not practical to measure electron concentrations directly, the electron energy potential of a redox system is determined from the electrical potential or voltage of the individual half cells, relative to a standard half cell. When the reactants and products of a half cell are in their standard state and the voltage is determined relative to a standard hydrogen half cell (whose voltage, by convention, is zero), the potential observed is defined as the standard electrode potential, E 0. If the pH of a standard cell is in the biological range, pH 7, its potential is defined as the standard biological electrode potential and designated E 0 '. By convention, standard electrode potentials are written as potentials for reduction reactions of half cells. The free energy of a typical reaction is calculated directly from its E 0 ' by the Nernst equation as shown below, where n is the number of electrons involved in the reaction and F is the Faraday constant (23.06 kcal/volt/mol or 94.4 kJ/volt/mol):  G 0' = -nF  E 0 '

For the oxidation of NADH, the standard biological reduction potential is kcal/mole. With a free energy change of kcal/mole, it is clear that NADH oxidation has the potential for driving the synthesis of a number of ATPs since the standard free energy for the reaction below is +7.3kcal/mole: ADP + P i > ATP Classically, the description of ATP synthesis through oxidation of reduced electron carriers indicated 3 moles of ATP could be generated for every mole of NADH and 2 moles for every mole of FADH 2. However, direct chemical analysis has shown that for every 2 electrons transferred from NADH to oxygen, 2.5 equivalents of ATP are synthesized and 1.5 for FADH 2.

Electron transport and Oxidative phosphorylation The final piece of the puzzle Take a deep breath and push on

Major Energy Pathways Glycolysis Oxidative phosphorylation pyruvate 3 NADH Glucose Galactose Fructose Mannose Fatty Acids 1 FADH 2 Lactate Amino Acids O2O2 H2OH2O Anaerobic Aerobic Krebs Cycle Acetyl-CoA

Electron Transport and Oxidative Phosphorylation 1. The absolute heart of aerobic metabolism 2. Three Functional Phases Electron transfer from NADH, FADH 2 to O 2 Energy preserved as a proton gradient Proton gradient energy makes ATP We are making ATP from ADP and P i by tapping the oxidative energy generated in the transfer of electrons to O 2

Anatomy of Mitochondria Mitochondria are composed of a dual membrane system: Outer: Porous to all molecules < 10 kDa Inner: Transporter-dependent transport

Diagrammatic representation of the flow of electrons from either NADH or succinate to oxygen (O 2 ) in the electron transport chain of oxidative phosphorylation. Complex I contains FMN and iron-sulfur (Fe-S) proteins in 5-7 clusters. Complex II contains FAD and 7-8 Fe-S proteins in 3 clusters and cytochrome b 560. Complex III contains cytochrome b, cytochrome c 1 and one Fe-S protein. Associated with complex III by electrostatic interaction is cytochrome c, the ultimate electron acceptor in complex III. Complex IV contains cytochrome a, cytochrome a 3 and 2 copper ions. As the two electrons pass through the proteins of complex I, four protons (H + ) are pumped into the intramembrane space of the mitochondrion. Similarly, four protons are pumped into the intramembrane space as each electron pair flows through complexes III and as four electrons are used to reduce O 2 to H 2 O in complex IV. The free energy released as electrons flow through complex II is insufficient to be coupled to proton pumping. These protons are returned to the matrix of the mitochondrion, down their concentration gradient, by passing through ATP synthase coupling electron flow and proton pumping to ATP synthesis.

Complex I - NADH-Q reductase The first step in the electron transport chain is the oxidation of NADH to NAD+. The electrons are transferred to flavin mononucleotide (FMN), producing the reduced form of this compound (FMNH2):

The reduced FMNH2 is oxidized back to FMN by transferring the electrons to an iron-sulfur cluster. These clusters are contained in iron-sulfur proteins (or non-heme iron proteins): they contains either one, two or four iron molecules coordinated to the sulfhydryl groups of four cysteine residues, with two or four inorganic sulfide groups in the case of the two and four iron clusters, respectively. The iron in these clusters cycles between the +2 and +3 states.

The electrons in these clusters are then transferred to a tightly-bound coenzyme Q (or ubiquinone (Q)) molecule, reducing it to form ubiquinol. Ubiquinone has a long isoprenoid tail (50 carbons in mammals) which anchors it to the mitochondrial membrane in the case of the mobile form:

The electrons from this bound ubiquinol are transferred through two iron-sulfur clusters to mobile ubiquinone located in the inner mitochondrial matrix. These molecules can then shuttle around in the membrane to pass the electrons to another protein complex. The net result of this transfer is four protons being pumped out of the matrix and into the intermembrane space for each molecule of NADH which is oxidized:

Complex II - Succinate - coenzyme Q reductase The second complex in the electron transport chain is an enzyme of the TCA cycle which uses a tightly bound FAD to oxidize succinate to fumarate. The electrons from this reaction are passed through an Fe-S center before being transferred to mobile ubiquinone in the mitochondrial membrane. Similarly, electrons from the FAD-mediated oxidation of fatty acids and glycerol 3- phosphate are passed to mobile, membrane ubiquinone. No protons are pumped out during these reactions because the free-energy change is too small.TCA cycle

The ubiquinol formed by complexes I and II can migrate to complex III and transfer their electrons to cytochrome c in the next step of this process.

Complex III - Cytochrome reductase Complex III (cytochrome reductase, ubiquinol- cytochrome c reductase) is used to transfer the electrons from ubiquinol, oxidizing it back to ubiquinone, and passes these electrons to cytochrome c in a two-step process:

The first half of this reaction is the migration of ubiquinol to the Qp site of cytochrome c reductase. Two electrons and two protons are released, resulting in an oxidation to a semiquinone intermediate and finally to ubiquinone, which can leave the site and enter the membrane pool. One electron is passed to an iron-sulfur protein, through cytochrome c1 and finally to mobile cytochrome c in the intermembrane space. The other electron is passed through cythochromes bL and bH, reducing ubiquinone to a semiquinone intermediate in the Qn site of the enzyme.

In the second step of this reaction, another molecule of ubiquinol enters the Qp site and is oxidezed to ubiquinone in the same manner as in step one. This time, however, the second electron is used to reduce the semiquinone intermediate to ubiquinol, pulling two protons out of the matrix and returning ubiquinol to the membrane pool. The net result for these reactions is four protons being pumped out of the matrix for each molecule of ubiquinol which is oxidized. The reason for the complexity of this process is to transfer the two electrons from ubiquinol to two molecules of the one-electron carrier, cytochrome c. Cytochrome c contains a heme group attached to the protein by thioether linkages:

Complex IV - cytochrome c oxidase Cytochrome c is reduced in complex III, and is oxidized by complex IV, cytochrome c oxidase, in a process which results in two more protons being pumped out of the mitochondrial matrix :

Two molecules of the reduced form of cytochrome c pass their electrons to a copper-heme a complex and then to a copper-heme a3 group. This last group is responsible for the reduction of oxygen to produce water in a multi-step reaction which uses four electrons and four protons for each molecule of oxygen which is reduced :

The heme of cytochrome a is slightly different than that of cytochrome c, having a long, hydrophobic side chain:

The electron transport chain is used to oxidize NADH and reduce molecular oxygen, resulting in the production of water and regenerating NAD+. The net reaction is: This energy is used to create phosphoryl potential in ATP by ATP synthase.

ATP synthase

How is ATP made? ADP + P i ATP + H 2 O F o F 1 ATPase Complex (ATP Synthase) 1. An ATP making machine 2. Driven by a proton gradient 3. Attached to the inner mitochondria membrane F 1 = stalk and lollypop F o = base

How is the energy of Oxidation Preserved for the synthesis of ATP? ANS: Electron transfer to oxygen is accompanied by the formation of a high energy proton gradient. The Gradient arises by having protons pumped from the matrix side of the mitochondria to the inner membrane spaces Back flow of the protons to the matrix leads to the synthesis of ATP.

F1F1 FOFO F O F 1 ATPase (ATP Synthase) Matrix Intermembrane space H+H+ Binding-Change Model 3 non-equivalent sites

ADP + Pi ATP 3-Site Model of ATP Synthesis Loose Site (ADP and P i bind) Tight Site (ATP is formed and held) Open Site (ATP is released)  The flow of protons through F 1 makes the sites alternate much like a spinning propeller. F1F1

P/O Ratios P is phosphate taken up (incorporated into ATP) O is the oxygen taken up (measured as atomic oxygen) What is it? What is the significance? Compares substrate efficacy to form ATP Examples: P/O NADH~3 FADH 2 ~2 Succinate ~2 Assumed to be whole intergers based on the “coupling site” model of ATP synthesis (Equated to a pair of electrons traveling to O 2 )

Chemiosmotic Adjustment to P/O 10 protons are pumped for each electron pair from NADH 6 protons are pumped for each electron pair from FADH 2 4 protons are required to make one ATP 1 of the 4 is used in transport of ADP, Pi and ATP across mitochondrial membrane Therefore, 10/4 or 2.5 is the P/O ratio for NADH Therefore, 6/4 or 1.5 is the P/O ratio for FADH 2

Inhibitors and Uncouplers Any compound that stops electron transport will stop respiration…this means you stop breathing Electron transport can be stopped by inhibiting ATP synthesis An uncoupler breaks the connection between ATP synthesis and electron transport

What is an Uncoupler? Uncouplers break the connection between electron transport and phosphorylation Electron transport is a motor Phosphorylation is the transmission Uncouplers let you put the car in NEUTRAL

O NO 2 2,4-dinitrophenol – a proton ionophore H+H+ O NO 2 H O HO H+H+ Inner Membrane Matrix

Brown Adipose Tissue Uncoupling a proton gradient from F O F 1 ATPase Produces Heat! Thermogenin

Staying Alive Energy Wise We need 2000 Cal/day or 8,360 kJ of energy per day Each ATP gives 30.5 kJ/mole of energy on hydrolysis We need 246 moles of ATP Body has less than 0.1 moles of ATP at any one time We need to make moles of ATP Each mole of glucose yields 38 ATPs or 1160 kJ We need 7.2 moles of glucose (1.3 kg or 2.86 pounds) Each mole of stearic acid yields 147 ATPs or 4,484 kJ We need 1.86 moles of stearic acid (0.48 kg or 1.0 pound of fat)

Control of Oxidative phosphorylation What makes us breathe faster? How does ATP synthesis in the mitochondria adjust to the needs of the cell?

Regulation of Oxidative Phosphorylation Since electron transport is directly coupled to proton translocation, the flow of electrons through the electron transport system is regulated by the magnitude of the PMF. The higher the PMF, the lower the rate of electron transport, and vice versa. Under resting conditions, with a high cell energy charge, the demand for new synthesis of ATP is limited and, although the PMF is high, flow of protons back into the mitochondria through ATP synthase is minimal. When energy demands are increased, such as during vigorous muscle activity, cytosolic ADP rises and is exchanged with intramitochondrial ATP via the transmembrane adenine nucleotide carrier ADP/ATP translocase. Increased intramitochondrial concentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge of the cell. In turn the energy charge, or more precisely ADP concentration, normally determines the rate of electron transport by mass action principles. The rate of electron transport is usually measured by assaying the rate of oxygen consumption and is referred to as the cellular respiratory rate. The respiratory rate is known as the state 4 rate when the energy charge is high, the concentration of ADP is low, and electron transport is limited by ADP. When ADP levels rise and inorganic phosphate is available, the flow of protons through ATP synthase is elevated and higher rates of electron transport are observed; the resultant respiratory rate is known as the state 3 rate. Thus, under physiological conditions mitochondrial respiratory activity cycles between state 3 and state 4 rates.

[ATP] [ADP][P i ] = ATP mass action ratio Low: Energy debt, Signifies high ADP or low ATP High: Energy sufficient, Signifies high ATP HIGH Mass Action Ratio: WHAT IS THE ATP MASS ACTION RATIO? Oxidized cytochrome C [C 3+ ] is favored Cytochrome oxidase is low because of low C 2+ O 2 uptake low LOW Mass Action Ratio: Reduced cytochrome C [C 2+ ] is favored Cytochrome oxidase stimulated because of high C 2+ Oxygen uptake high

ATP [ADP][P i ] [NAD + ] [NADH] ½ Keq = [c 2+ ] [c 3+ ] ½NADH + Cyt c (Fe 3+ ) + ADP + Pi ½ NAD + + Cyt c (Fe 2+ ) + ATP  G o’ = 0 Control of Oxidative Phosphorylation Equilibrium [ATP] can control its own production Cytochrome c oxidase step is irreversible and is controlled by reduced cytochrome c (c 2+ ) Because of equilibrium, concentration of c 2+ depends on [NADH]/[NAD + ] and [ATP]/[ADP][P i ]

[c 2+ ] [c 3+ ] [NADH] [NAD + ] ½ [ADP][P i ] [ATP] = Keq NADH ATP mass action ratio Mass Action ration NADH [c 2+ ]/[c 3+ ] equilibrium ADP[c 2+ ]/[c 3+ ] ATP[c 2+ ]/[c 3+ ] equilibrium Cytochrome oxidase controls the rate of O 2 uptake which means this enzyme determines how rapidly we breathe. Cytochrome oxidase controls the rate of O 2 uptake which means this enzyme determines how rapidly we breathe. Control of Cytochrome Oxidase (Cox) Stimulates Cox Suppresses Cox

Energy from Cytosolic NADH In contrast to oxidation of mitochondrial NADH, cytosolic NADH when oxidized via the electron transport system gives rise to 2 equivalents of ATP if it is oxidized by the glycerol phosphate shuttle and 3 ATPs if it proceeds via the malate aspartate shuttle. The glycerol phosphate shuttle is coupled to an inner mitochondrial membrane, FAD-linked dehydrogenase, of low energy potential like that found in Complex II. Thus, cytosolic NADH oxidized by this pathway can generate only 2 equivalents of ATP. The shuttle involves two different glycerol-3-phosphate dehydrogenases: one is cytosolic, acting to produce glycerol-3-phosphate, and one is an integral protein of the inner mitochondrial membrane that acts to oxidize the glycerol-3-phosphate produced by the cytosolic enzyme. The net result of the process is that reducing equivalents from cytosolic NADH are transferred to the mitochondrial electron transport system. The catalytic site of the mitochondrial glycerol phosphate dehydrogenase is on the outer surface of the inner membrane, allowing ready access to the product of the second, or cytosolic, glycerol-3-phosphate dehydrogenase.glycerol phosphate shuttlemalate aspartate shuttle In some tissues, such as that of heart and muscle, mitochondrial glycerol-3-phosphate dehydrogenase is present in very low amounts, and the malate aspartate shuttle is the dominant pathway for aerobic oxidation of cytosolic NADH. In contrast to the glycerol phosphate shuttle, the malate aspartate shuttle generates 3 equivalents of ATP for every cytosolic NADH oxidized. In action, NADH efficiently reduces oxaloacetate (OAA) to malate via cytosolic malate dehydrogenase (MDH). Malate is transported to the interior of the mitochondrion via the  - ketoglutarate/malate antiporter. Inside the mitochondrion, malate is oxidized by the MDH of the TCA cycle, producing OAA and NADH. In this step the cytosolic, NADH-derived reducing equivalents become available to the NADH dehydrogenase of the inner mitochondrial membrane and are oxidized, giving rise to 3 ATPs as described earlier. The mitochondrial transaminase uses glutamate to convert membrane-impermeable OAA to aspartate and  -ketoglutarate. This provides a pool of  - ketoglutarate for the aforementioned antiporter. The aspartate which is also produced is translocated out of the mitochondrion.

O2O2 O2O2 O :: O Octet Rule..... O :: O... Molecular Oxygen = O 2 - Unpaired electron Oxygen Radicals Superoxide Anion Partially reduced oxygen species

What is a Free Radical ? Highly Reactive Powerful Oxidant Any chemical species with one of more unpaired electrons……. Short half life (nanoseconds) Can exist freely in the environment

EXAMPLES OF FREE RADICALS H. Hydrogen atom O2O2. Superoxide (oxygen centered). OH Hydroxyl radical (most reactive). NO Nitric Oxide

PRO-OXIDANTS (Generates Free Radicals) Fe 2+ + H 2 O 2 Ascorbic acid + Fe 2+ Paraquat Agent Orange Ozone Generates hydroxyl radical Generates superoxide radical

WHAT ARE ANTIOXIDANTS? ENZYMES Superoxide dismutase Catalase Peroxidases O2-O2- H2O2H2O2 R-OOH