1 Electron Transport System

2 There are 2 Ways to Make ATP 1. Substrate phosphorylation 2. Electron transfer-dependent oxidative phosphorylation

3 2 Glycolytic Reactions Make ATP by Substrate-level Phosphorylation --1,3-BPG is an energy –rich molecule with a greater phosphoryl-transfer potential than that of ATP. Thus, it can be used to power the ATP synthesis from ADP. --This is called substrate-level phosphorylation because the phosphate donor is a Substrate with high phosphoryl-transfer potential.

4 2 Glycolytic Reactions Make ATP by Substrate-level Phosphorylation PEP has high phosphoryl-transfer potential, pyruvate (ketone) is much more stable than enol form.

5 There are 2 Ways to Make ATP 1. Substrate phosphorylation 2. Electron transfer-dependent oxidative phosphorylation

6 How do we obtain lots of ATP? Glucose Reduced coenzymes (NADH + H +, FADH 2 ) O2O2 H2OH2O  Glycolysis  TCA  ETC ATP Food (carbohydrates) ATP Little Lots (~4 ATP) (~28-30 ATP) After TCA cycle, energy is extracted In the form of reduced Coenzymes, FADH2 and NADH Electron transport and Oxidative phosphorylation: Involved many steps, Sequestered in special environment. Glycolysis

7 Minimal TCA CycleGlucose Pyruvate NADH + H + CO 2 NADH + H + CO 2 GDP GTP FADH 2 NADH + H + 6C4C 4C (2C) CoASH CH 3 C-SCoA O 1 GTP 1 GTP 3 NADH 3 NADH +1 FADH 2 10 ATP/cycle And releases two CO 2 NOTE: 1 NADH  2.5 ATP; 1 FADH2  1.5 ATP; 1 GTP  1 ATP so get = 10 ATP/cycle NADH + H +

8 Where in the cell does electron transport and oxidative phosphorylation occur?

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10 Mitochondria Permeable Outer Mtch Membrane Intermembrane Space Inner Mtch Membrane Matrix TCA enzymes  -oxidation ATP synthase e - transport chain M DNA

11 Mitochondria --A mitochondrion is bounded by a double membrane, with an intermembrane space. --Outer M: permeable to most ions and small molecules --The inner membrane: highly impermeable, Highly folded “cristae”. most molecules require transporters (exceptions: O2, CO2). provide large surface area for the transport proteins, several FAD-dependent dehydrogenases and all enzymes and proteins of oxidative phosphorylation --The matrix is the fluid-filled interior of the mitochondrion. oxidative enzymes like pyruvate dehydrogenase (acetyl Co A formation) glutamate dehydrogenase, TCA cycle enzymes, fatty acid oxidation enzymes --Note that glycolysis occurs outside the mitochondrion in the cytosol, whereas the citric acid cycle occurs in the matrix. --The electron transport system is located on the cristae, both TCA cycle and oxidative phosphorylation occur within the mitochondrion.

12 Electron Transport System (ETS)  The electron transport system is located in the cristae of mitochondria  It is a series of protein/prosthetic group carriers that pass electrons from one to the other.  Electrons are donated to the ETS by NADH and FADH 2  As a pair of electrons is passed from carrier to carrier, energy is released and is used to form ATP  At the end of the electron transport chain, oxygen receives the energy-spent electrons, resulting in the production of water. ½ O e- + 2 H + → H 2 O (Oxygen is the final electron acceptor)

Redox Reactions e- ++ A A B B oxidation reduction O oxidation I is L loss of electrons R reduction I is G gain of electrons Reductant (A): is oxidized, electron donor Oxidant (B): is reduced, electron acceptor

14 How are redox potentials determined?

15 Half cell reactions measure electromovtive force SampleReference Neg value = oxidized form has a lower affinity for electrons than does H 2 (e.g., NADH a strong reducing agent has a negative reduction potential) Pos value = oxidized form has a higher affinity for electrons than does H 2 (e.g., Oxygen a strong oxidizing agent has a positive reduction potential) Standard: 1M H+ 1atm H 2 gas E 0 ’ of H + /H 2 is 0 volts Reductant Oxidant Ethanol gives up e to H + to form H2 H2 gives up e to Fe 3+ to form H +

16 A strong reducing agent, NADH is poised to donate electrons, has a negative reduction potential, whereas a strong oxidizing agent O2 is ready to accept electrons and has a positive reduction potential. --Biochemists use E 0 ’, the value at pH 7. --Chemists use E 0, the value in 1M H +. --The prime denotes that pH 7 is the standard state. --Thus, these values are different in chem textbooks.

17 Partial reactions By convention, reduction potentials (as in Table 18.1) refer to partial reactions are written as:Table 18.1 oxidant + e - reductant OVERALL REACTION

18 Redox reactions  Redox pairs act as e - carriers  Reductant + oxidant  oxidized reductant + reduced oxidant  Free energy is released in the transfer of e - e- ++ A A B B oxidation reduction (OIL) (RIG)

19 Standard free-energy changes of an oxidation- reduction reaction can be determined  G 0 ’ = -nF  E’ 0   G 0 ’ = standard free-energy change  F= faraday constant = kcal/mol/V (required to remember!)  n = number of electrons   E’ 0 = Change in reduction potential   G 0’ : standard free energy change –for a redox reaction is related to the difference in E 0 between the e - acceptor and donor

20 Determining:  G 0’ : standard free energy change  E’ 0 = E’ 0 (acceptor) - E’ 0 (doner)  G 0 ’ = -nF  E’ 0  F= faraday constant = kcal/mol/V  n = number of electrons G0’G0’ = -2 x kcal/mol/V x [-0.19 – (-0.32) V ] -6.0 kcal/mol Pyruvate NADH = -2 x kcal/mol/V x 0.13V =

Volt potential favors formation of proton gradient Note:  G 0 ’ = -7.3 kcal/mol for the hydrolysis of ATP Acceptor donor  G 0 ’ = -nF  E’ 0 = -nF (E’ 0 acceptor – E’ 0 donor ) = -2 x kcal/mol/V x [0.82V- (-0.32V)] = -2 X kcal/mol/V x 1.14V = kcal/mol The driving force of oxi phos is the elec-trans potential of NADH or FADH2 rel. to that of O2. The released energy is used to generate a proton gradient, then for ATP synthesis

22 Driving e - Transport  Electron carriers at the beginning of the chain are more - E 0 ’ than those at the end –so e - flow spontaneously from NADH (E’ 0 = –0.32 v) or FADH 2 (E’ 0 = –0.22V) to O 2 (E’ 0 = volts)  Neg reduction potential = oxidized form has a lower affinity for electrons and so transfers them most easily to an acceptor  Pos reduction potential = will be the strongest oxidizing substance and have a higher affinity for electrons

23  The electron transport system consists of four protein complexes and two mobile carriers.  NADH-Q Oxidoreductase  Succinate-Q reductase  Q-cytochrome c Oxidoreductase  Cytochrome c Oxidase  Coenzyme Q  Cytochome c  The mobile carriers transport electrons between the complexes, which also contain electron carriers.  The carriers use the energy released by electrons as they move down the carriers to pump H+ from the matrix into the intermembrane space of the mitochondrion. complexes carrier

24 NAD+/NADH Fumarate/ Succinate Cytochrome C (+3) / (+2)

25  A very strong electrochemical gradient is established with few H+ in the matrix and many in the intermembrane space.  The cristae also contain an ATP synthase complex through which hydrogen ions flow down their gradient from the intermembrane space into the matrix.  The flow of three H+ through an ATP synthase complex causes a conformational change, which causes the ATP synthase to synthesize ATP from ADP + P.

26  Mitochondria produce ATP by chemiosmosis, so called because ATP production is tied to an electrochemical gradient, namely an H+ gradient.  Once formed, ATP molecules are transported out of the mitochondrial matrix.

27 Mitchell’s Postulates for Oxidative Phosphorylation 1.The respiratory and photosynthetic electron transfer chains should be able to establish a proton gradient 2.The ATP synthases should use the proton-motive force to drive the phosphorylation of ADP 3.Energy-transducing membranes should be “impermeable” to protons. If proton conductance is established (uncouplers), a proton-motive force should not form and ATP synthesis should not occur. 4.Energy-transducing membranes should possess specific exchange carriers to permit metabolites to permeate in the presence of high membrane potential ADP ATP H+H+ H+H+ H+H+ e-e- H+H+ ADP + P i ATP Mitochondrial matrix ATP-ADP Antiporter Intermembrane