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5.19.06 Electron Transport and Oxidative Phosphorylation Reading Assignment: Start reading Chapter 14: Energy Generation in Mitochondria and Cholorplasts See animation 14.3 on your text CD ATPsynthase animation and lots of other stuff http://vcell.ndsu.nodak.edu/animations/home.htm

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What does the citric acid accomplish? • Carbons in pyruvate are fully oxidized to CO2 • Some GTPs (which may be later converted to ATPs)

are generated by substrate level phosphorylation • 8 NADH and 2 FADH2 are stockpiled NADH and FADH2 are high energy molecules and they can be used as reducing agents by the cell. But much of the stored energy is not directly “accessible” to the cell in this form What happens in the mitochondria to convert the potential energy in NADH into the form of ATP?

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NADH and FADH2 are high energy molecules:

As electrons drop from the top to the bottom of the scale, energy is released

• Substances vary in their tendancy to become oxidized or reduced.

• This tendancy is expressed as the reduction potential. As electrons drop from the top to the bottom of the scale, energy is released.

• Glucose has a reduction potential of (-0.43 V) – so the exergonic oxidation of glucose is coupled to the endergonic reduction of NAD+

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How to power ATP synthesis • build a dam • pile up protons on one side • poke a hole -- use the rush of protons through the hole to turn a turbine which then makes ATP

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The inner mitochondrial membrane is 70% protein and 30% phospholipid by weight

An electron micrograph of the inside surface of the inner mitochondrial membrane in a plant cell. Densely packed particles are visible -- due to protruding portions of ATP synthase and the respiratory enzyme complexes

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A Closer look at the inner mitochondrial membrane

ATP synthase ATP Transporter The inner mt membrane is 70% protein and 30% phospholipid by weight • many of the proteins belong to the electron transport chain • also includes ATP synthase: converts the stored potential

energy in the electrochemical proton gradient into chemical energy

matrix space between inner membranes

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THE ELECTRON TRANSPORT CHAINS CONSISTS OF MEMBRANE ASSOCIATED ELECTRON CARRIERS These systems have two basic functions:

1. to accept electrons from an electron donor and to transfer them to an electron acceptor

2. to conserve some of the energy released during electron tranasfer for the synthesis of ATP

The electron transport chain reoxidizes the coenzymes NADH and FADH and channels the free energy into the synthesis of ATP • NADH and FADH gained electrons when

oxidizing other compounds • they transfer these electrons to the electron-

transport chain: electron transport chain reoxidizes the coenzymes NADH and FADH and channels the free energy into the synthesis of ATP

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NADH is a high energy molecule:

• Substances vary in their tendancy to become oxidized or reduced. • This tendancy is expressed as the reduction potential. As

electrons drop from the top to the bottom of the scale, energy is released.

• NOTICE the molecule at the bottom!

Brock 5.19 Reduction potential of the components of the electron transport chain of the mitochondrion of eukaryotic cells and the plasma membrane of some bacterial cells. By breaking up the complete oxidaton into a series of discrete steps, energy “recapture” is possible

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How to power ATP synthesis • build a dam • pile up protons on one side • poke a hole -- use the rush of protons through

the hole to turn a turbine which then makes ATP

Oxidative Phosphorylation: the production of ATP using energy derived from the redox reactions of an electron transport chain Chemiosmosis: the production of ATP from ADP using the energy of hydrogen ion gradients

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H-bonding is always with us proton wires: net translocation of protons can occur over a long distance through a protein by hopping between pairs of hydrogen bonded donor and acceptor residues; a string of such residues connected by hydrogen bonds can be thought of as a proton wire..... Matrix (inside the inner membrane of the mt) is above the membrane (gray bar). The intermembrane space is below the membrane. animation of electron transport http://www.sp.uconn.edu/~terry/images/anim/ETS_slow.html

How protons can be pumped across membranes: As an electron passes along an electron-transport chain embedded in a lipid-bilayer, it can bind and release a proton at each step. In this diagram electron carrier B picks up a proton (H+) from one side of the membrane when it accepts an electron from carrier A. It releases the proton to the other side of the membrane when it donates its electron to carrier C

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ALBERTS animation 14.2

• The energy released is used to transport H+ ions across the

inner mitochondrial membrane to the space between the two membranes

• In this way, a gradient of H+ ions is maintained across the

inner membrane • This gradient serves as a source of energy (like a battery) that

is tapped to drive a variety of energy-requiring reactions • The most prominent of these reactions is the generation of

ATP ADP + Pi -----> ATP •

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How to power ATP synthesis • build a dam • pile up protons on one side • poke a hole -- use the rush of protons

through the hole to turn a turbine which then makes ATP

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Potential energy in gradient converted to mechanical energy which via conformtional changes in the cytoplasmic portion of the ATPase is converted to chemical energy in ATP

• ATP synthase is imbedded in the inner mitochondrial membrane

• below & left in this figure is the matrix of the mitochondria (compartment contained within the inner mitochondrial membrane)

• water soluble catalytic domain is in matrix

• spinning ion transport channel (embedded in lipid bilayer)

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ATP synthase: enzyme that uses energy from the proton gradient to produce ATP from ADP + Pi • inner mitochondrial membrane of all

eukaryotic cells • the thylakoid membrane of chloroplasts of

plant cells • the plasma membrane of prokaryotic cells

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Now for some serious quaternary structure! ATP synthase — energy converter. The enzyme consists of two rotary motors, F0 and F1 which are coupled via their drive shafts. The transmembrane F0 motor has one a, two b and nine to twelve c subunits. The soluble F1 motor has three α and three β subunits, and one each of the other subunits. During ATP synthesis, F0 channels protons across the membrane to drive rotation.

Nature 410: 878 4/19/01

• The rotating subunits are the c polypeptide in Fo and the γ polypeptide in F1

• The rotation of Fo (caused by movement of protons) drives the rotation of γ

• This rotation drives the conformational transitions of the catalytic subunits which, in turn, alters the nucleotide binding site affinities.

• As a consequence, conformational energy flows from the catalytic subunit into the bound ADP and Pi to promote their dehydration into ATP.

ALBERTS animation 14.3 HTTP://WWW.TCD.IE/BIOCHEMISTRY/IUBMB-NICHOLSON/SWF/GLYCOLYSIS.SWF Step 32 in this animation substrate level ATP synthesis in glycolysis

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MORE optional Stuff on ATP synthase for those who are amused by this protein: Look at First two links ATP synthase do cross section alpha, beta gamma http://www.cnr.berkeley.edu/~hongwang/Project/ATP_synthase/ ATP synthase http://rsb.info.nih.gov/NeuroChem/biomach/ATPsyn.html This cartoon is adapted from fig. 2 of Cross. The 3 shades of red represent the 3 different conformational states of the catalytic subunits. The central asymmetric black object represents the gamma subunit which is caused to rotate by themitochondrial proton efflux. This rotation drives the conformational transitions of the catalytic subunits which, in turn,alters the nucleotide binding site affinities. As a consequence, conformational energy flows from the catalytic subunit into the bound ADP and Pi to promote their dehydration into ATP. http://teddy.berkeley.edu:1024/ATP_synthase/ ATP synthase: the rotory engine in the cell: http://www.res.titech.ac.jp/~seibutu/main.html?right/~seibutu/projects/f1_e.html in vitro rotation of an actin filament attached to ATP synthase

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What fraction of the potential energy can a respiring cell extract from a glucose molecule? A few billion years of evolution have ensured that the aerobic system is 40 - 54% efficient

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The complete oxidation of 1 mole of glucose generates about 38 moles of ATP (synthesized from ADP) ATP yield from complete oxidation of glucose Process

Direct product

Final ATP

Glycolysis 2 NADH (cytosolic) 2 ATP

3 or 5* 2

Pyruvate oxidation (2 per glucose)

2 NADH (mitochondrial matrix)

5**

Acetyl CoA oxidation (Citric Acid cycle) two per glucose

6 NADH (mitochondrial matrix) 2 FADH2 2ATP or 2 GTP

15 3 2

Total ATP yield per glucose molecule

30 -32***

* depends on which "shuttle system" transfers reducing equivalents into the mitochondria ** 3 protons per ATP *** This number varies from reference to reference..

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Why is ATP “high energy” ATP has stored potential energy: ATP ADP + Pi + energy ΔGo = - 7.3 kcal/mole exergonic reaction (corresponds to an Keq of >105 Under cellular conditions, the hydrolysis of ATP creates two molecules of much lower energy and releases a great deal of usable energy • The phosphates in ATP can be considered to exist

in an activated state: the presence of four negative charges in close proximity destabilizes the molecule -- electrostatic repulsion between negative charges favors hydrolysis

• Increased hydration ADP and P -- energetically favored

• Release of a phosphate increases entropy because the PO4 molecule released is capable of resonance forms (delocalized proton and oxygen binding) not possible when phosphate is bound to another molecule

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What do cells do with ATP?

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• Drives anabolic (endergonic) chemical

reactions • Used to do work (move stuff around –

molecular motors) • Used for active transport to make/maintain

ion and solute gradients • Lots of other roles

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Cells drive active transport in three main ways

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Remember lysosomes? • Lysosomes contain hydrolytic enzymes that are

active under acidic conditions. • The interior of this organelle is maintained at an

acidic pH (high proton concentration) by a H+ ATPase in the membrane that pumps protons against the concentration gradient

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WHAT HAPPENS DURING GLUCOSE OXIDATION IF NO OXYGEN IS PRESENT? AEROBIC AND ANAEROBIC ORGANISMS One basic metabolic distinction among organisms is whether or not they can use O2 as an electron acceptor in energy producing pathways AEROBES: CAN USE O2 Obligate Aerobe: O2 is obligatory for life ANAEROBES: CAN SUBSIST WITHOUT O2 Facultative Anaerobe: can adapt to anaerobic conditions by substituting other electron acceptors for O2 (yeast, bacteria) Obligate Anaerobe: cannot use O2 and are poisoned by it

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BEAKER CONTAINS YEAST AND SUGAR What is the gas being produced in the beaker? WHAT HAPPENS DURING GLUCOSE OXIDATION IF NO OXYGEN IS PRESENT? Cell has a limited amount of NADH which must be recycled if glycolysis is to continue under anaerobic conditions FERMENTATION: an anaerobic biological reaction process

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FERMENTATION: THE ANAEROBIC FATE OF PYRUVATE Commercially Valuable fermentation reactions:

Alcoholic fermentation by yeast used in brewing and winemaking Bacteria also can carry out alcoholic fermentation under anaerobic conditions

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This type of fermentation occurs in some fungi and bacteria (used to make yogurt and cheese) and in human muscle cells when oxygen is limiting

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The three common metabolic fates of pyruvate generated by glycolysis: • Under aerobic conditions, the pyruvate is completely

oxidized via the citric acid cycle to CO2 and H2O [NADH acts as a high energy compound]

• Under anaerobic conditions, pyruvate must be converted to a reduced end product in order to reoxidize the NADH produced by the GAPDH reaction

• alcoholic fermentation: in yeast, pyruvate is converted to ethanol + CO2 [free energy of NADH oxidation is dissipated as heat]

• in muscle cells, under anaerobic conditions, pyruvate is reduced to lactate [free energy of NADH oxidation is dissipated as heat]

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FERMENTATION: an anaerobic biological reaction process in which a reduced organic compound (like glucose) acts as an electron donor and another organic compound acts as an electron acceptor An even more formal definition of fermentation fermentation: catabolic reactions producing ATP in which organic compounds serve as both the primary electron donor and ultimate electron acceptor and ATP is produced by substrate level phosophorylation Note: fermentation is extremely inefficient compared to aerobic respiration. From first principles: Why?

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The potential energy drop between glucose and an electron acceptor like pyruvate is a fraction of the potential energy drop that occurs during cellular respiration where O2 is the electron acceptor