Lecture 12 1. Mitochondrion Structure 2. Electron Transport Chain 3.

ATP Synthase and ATP

Exam II Oct 6th Need Scantron Card

Evolution of Modern Animal and Plant Cells a. The origin of life is not known (many theories). b. 3.8 BYA life was anaerobic and in an atmosphere of reduced molecules: H2, NH3, CH4, CO2, H2O c. Energy metabolism was w/o oxygen (glycolysis, and fermentation). d. 2.4-2.7 BYA-cyanobacteria appeared and used photosynthesis for energy metabolism releasing molecular O2 into the air. e. Significant amounts of O2 in atmosphere is toxic to anaerobic life. (O2 radicals react and destroy biological molecules and cells). f. O2 in atmosphere selected for life forms that used O2 in energy metabolism and that detoxified O2 radicals. g. Anaerobes could only extract limited amounts of energy from food and secreted lactic acid or ethanol as waste, although still rich in energy. h. Aerobes, however, used O2 in energy metabolism to completely oxidized foods to CO2 and H2O and extracted much more energy in the process. This occurred in mitochondria, a special organelle. i. All oxygen-dependent prokaryotes & eukaryotes are descendants of these ancient aerobes. Modern Cell! Development! Evolutionary Time (2.7 BYA)!

Mitochondrion Structure

Outer membrane - contains many channel forming proteins (porins) for passage of large molecules (ATP, NAD+, Ca2+, etc) into the mitochondria. *Membrane resembles bacterial outer cell wall. Inner membrane has high membrane potential (220 mV inside) to show it is highly impermeable to ions and small molecules. Transmembrane transport proteins selectively regulate passage of materials into or out of matrix. Resembles bacterial plasma membrane because rich in cardiolipin. *Cristae form by invaginations and is where the Electron Transport Chain and ATP Synthases are found for Oxidative Phosphorylation. Matrix - Large internal compartment, gel-like due to its high viscosity from soluble proteins. That is, it contains hundreds of enzymes for the oxidation of pyruvate, fatty acids, proteins and for the Krebs Cycle intermediates. *Mitochondrial genome encodes: ribosomes, tRNAs, and other enzymes. Intermembrane Space a narrow space where H+ accumulate for transport back into the matrix for ATP synthesis. It is of variable size due to H+ concentration.

Outer and Inner Mitochondrial Membranes

Outer Mitochondial Membrane: Beta-barrel proteins make porin channel 1. Bacterial-like membrane with ~50% lipid and protein mix and has many unusual enzymes like those oxidizing epinephrine, degrading tryptophan, etc. 2. Porins - these are large integral membrane proteins that have large aqueous channels and show regulated opening/closing. Passes molecules <5,000 daltons. 3. Freely passes when open ATP, NAD+, coenzyme A. etc.

Inner Mitochondrial Membrane: 1. ~100 different polypeptides and a high protein/lipid ratio (>3:1 by weight) 2. Enriched in cardiolipin (a lipid found in bacteria). 3. Highly impermeable to ions and small molecules (specific transporters used). eg. Ca2+ ATPase.

The Mitochondrion: ! Structure:!

100nm!

1. The mitochondria is present in virtually all eukaryotic cells (animal, plant, algae). 2. Functions in the synthesis of ATP by Electron Transport Chain and Oxidative Phosphorylation. *Functions also to temporarily store Ca2+ from the cytosol.

Overview Energy Generation by ATP Synthesis

1. 2. 3. ATP is the main energy currency used by cells. In plants, light energy is converted into chemical bond energy by Photosynthesis in the chloroplasts. In animals, energy-rich food is broken down by oxidation in Aerobic Respiration in the cytosol and mitochondria. *Photosynthesis & Aerobic Respiration mechanisms first appeared almost 3.5 billion years ago in bacteria. Bacteria acquired a fundamental ability to make energy long ago in evolutionary history through a membrane-based mechanism described by 2 linked stages. a. Electron Transport Chain These consist of electron carriers embedded in inner membrane of mitochondria that carry e which transfer H+ (protons) across the inner membrane using the released energy from the various electron carriers with each e transfer. *Result: H+ electrochemical gradient is made that can be used to do work. b. ATP Synthase An enzyme protein complex that catalyzes the synthesis of ATP using the energy stored in the H+ electrochemical gradient. *Note: No ATP hydrolysis is used in formation of ATP by this enzyme complex. 5. The above mechanism is Chemiosmotic Coupling and refers to the link between membrane e transport processes, H+ electrochemical gradient and ATP synthesis.

4.

Glucose Metabolism in Eukaryotic Cells

Energy Released Overview of Pathways and NAD+ Reduced from Glucose Bonds Glycolysis > 2 high energy e pairs carried by NADH TCA Cycle > 5 high energy e pairs carried by NADH > > 2 ATP (net synthesis) >30 ATP

Energy Metabolism in Cells

In Cytoplasm (Glycolysis):
a. Glucose metabolized to Pyruvate and yields very little ATP in the process. (i.e. 2 net ATP molecules/Glucose). b. Less than 10% of available energy from glucose is extracted by glycolysis.

In Mitochondria (Oxidative Phosphorylation):

a. Glucose is completely oxidized to give >30 net ATP molecules/glucose.

General Overview of Mitochondria Biochemical Process:

a. Pyruvate (from glucose) > fuel after transport into Matrix > converted to acetyl CoA. b. Fatty Acids (from fats) > fuel after transport into Matrix > converted to acetyl CoA. c. Acetyl CoA > acetyl groups are oxidized in Krebs Cycle to CO2 (by-product) and metabolism generates high-energy es carried by NADH & FADH2 (reduced). d. NADH and FADH2 > delivers high-energy es to prosthetic group e carriers in the Electron Transport Chain and NAD+ and FAD (oxidized) are regenerated again. e. High-energy es > passed down series of electron complex carriers (complex I-IV) forming the electron transport chain > e energy released at each e transfer is conserved by pumping H+ across inner membrane (proton gradient made) f. Molecular O2 (strong oxidizing agent) > final electron acceptor to bind weak-energy es that then makes H2O (by-product). g. Proton gradient > H+ gradient is used to drive ATP synthesis by ADP + Pi addition (called Oxidative Phosphorylation).

Enzyme e carrier complexes have progressively higher affinity as electron acceptors > transport is energetically favored down chain

outer mitochondrial membrane

High-energy e carrier

*Produces H+ EC gradient. *Inner membrane separates ions *Machinery exists to use stored gradient energy (ATP made).

!O2 + 2H+ > H2O (O2 strong oxidizing agent; high e affinity) !

O2 a high affinity e acceptor to make H2O

An Overview Of Oxidative Phosphorylation !

NAD+ reduced to NADH (NADH strong reducing agent; low e affinity)!

a. The oxidation of food matter generates CO2 and high energy es in NADH and FADH2. b. The high energy es transfer to e carriers in the Electron Transport Chain and convert their energy to transport of H+ across the inner membrane (potential energy for work).

Three Respiratory Enzyme Complexes - drive electron transport

1. Over 40 proteins are involved in e transport along with prosthetic groups and metal ions. 2. Three enzyme complexes shown are sites of H+ pumping as high-energy es move through the chain guided by a series of oxidation-reduction reactions in the complexes.

14_10_resp_enzy_comp.jpg!
mobile e carrier mobile e carrier

NAD+ = oxidized NADH = reduced *Reduction-oxidation reactions in e carriers a. NADH has a low affinity for e pair and easily passes them to NADH dehydrogenase complex that has a higher e affinity (a redox reaction). b. Other e transporters in the electron transport chain have progressively higher e affinities to guide e pair through the chain. Molecular O2 has a very high affinity for es and acts as the final electron acceptor for the energy depleted es (see next slide).

Stepwise flow of electrons through the electron transport chain from NADH, succinate, and FADH2 to O2 (blue arrows)
high energy electrons!

Complex I ! Complex II ! Redox potential (mV)! Free energy (Kcal/ml)!

energy depleted electrons!

Complex III !

Complex IV !

Proton Pumping Generates a H+ Electrochemical Gradient (Proton Motive Force)!

*Shows that proton pumping creates a charge difference across the ! Inner Membrane. A membrane potential difference is created that ! is as high as 220mV. !

*Shows that proton pumping creates a 10-fold proton! concentration gradient across membrane (!pH values).!

Consequently, the electrochemical gradient for H+ is energetically steep to power the ! synthesis of ATP and other transport processes in the mitochondria.!

The proton electrochemical gradient across the Inner Membrane drives the synthesis of ATP by the exergonic movement of H+ back to the matrix after passing through a large enzyme complex called the ATP Synthase.

14_13_electroch_gradie.jpg!

The movement of e pairs through the Electron Transport Chain and the pumping of H+ into the Intermembrane Space ultimately to be used for the synthesis of ATP is called Chemiosmotic Coupling

Experimental Evidence that Proton Gradients Power ATP Production! 1.!

Light causes ! H+ pumping! into vesicle!

2.!
Light causes No! H+ pumping! into vesicle and! No ATP synthesis!

Fuse! 3.!
Light causes ! H+ pumping! into vesicle to! establish a ! H+ electro! chemical! gradient and ! power ATP! synthesis!

4.!

Light causes causes! H+ pumping across! membrane, but DNP! eliminates the H+! gradient & prevents! ATP synthesis. The! absence of the H+! gradient removes the! energy imput needed ! to power ATP ! formation. !

*1978 - Peter Mitchell received Nobel Prize for showing Chemiosmotic Coupling in ATP generation.!

ATP Synthesis is by the Binding Change Mechanism *Paul Boyer (1979) first proposed this hypothesis.
1. The energy released by the movement of protons is not used directly to drive ADP Phosphorylation for ATP formation. *Instead, the binding affinity in the catalytic site for ATP synthesis is changed by the energy from proton movement. 2. Each active site involved in ATP formation progresses successively through three distinct conformations that have different affinities for substrates (ADP + Pi) & products (ATP). 3. ATP synthesis is by rotational catalysis where one part of the ATP synthase rotates relative to the other parts. *The energy for rotational catalysis is provided by protons as they move down their steep electrochemical gradient.

Energetics of ATP Synthesis in Mitochondrion Inner Membrane

1. ATP synthesis in aqueous environment: soluble ADP + soluble Pi > soluble ATP + H2O

*Requires large energy input of +7.3 kcal/mol energy. 2. ATP synthesis in a non-aqueous environment (i.e. the ATP Synthase): enzyme-bound ADP + enzyme-bound Pi > enzyme-bound ATP + H2O *"G = 0, ATP formation occurs spontaneously (energetically favorable reaction) *That is, a condensation reaction between ADP + Pi occurs inside the ATP synthase (in the ATP catalytic site) and does not spontaneously occur in the cytosol aqueous environment. *Note: This does not mean the synthesis of ATP does not require energy. In this case, the energy is used to release bound ATP from its Active Site.

The ATP Synthase

14_14_ATPsynthase1.jpg! central stalk *The & subunit forms a

F0!

1. F1 is the ATP synthesizing enzyme. *Contains 5 different polypeptides (#3$3%&'). *The #3 $3 subunits alternate in arrangement. *$3 subunits are catalytic sites for ATP synthesis. that runs through the middle of the F0 base and the F1 head and links these domains together. *In the F0 portion, the c-subunits rotate due to proton flow through it to cause the & subunit to rotate and affect the F1 head for ATP formation. 2. F0 is embedded in the Inner Membrane. *Contains 3 differrent polypeptides (ab2c12). *The a and c subunits and create a channel for the movement of protons from Inner Membrane space to the Matrix. *The c subunits organized in a ring with the a and b subunits on outside of the ring. *The b2 subunits are structural proteins forms a stalk to link together the F0 & F1 groups. *The % subunit participates with the b subunits to hold the F1 head in place.

F1!

ATP Synthase
a. F0 portion of the enzyme is embedded in the inner membrane.

Intermembrane Space (high H+ conc.)!

Ring of! c subunits!
H+ H+ H+ H+

Subunit a !

Asp61!

H+

Matrix!

The ATP Synthase

14_14_ATPsynthase1.jpg!

Cross-section of $ subunits (next slide)

Binding Change Mechanism of ATP synthase ($ Subunits only)

Three conformations in F1 Sphere (!-subunits): O (open) - low affinity for ATP so ATP comes off L (loose) - ADP + Pi loosely bound to subunit binding sites T (tight) - tight ADP + Pi binding to force ATP synthesis. *All states exist and are sequentially changed by the rotation of the gamma subunit (Rotational Catalysis).

ATP Synthase - F1 Domain with "-subunit and !-subunits in action

1. Every shift and touch of &-subunit to a $-subunit changes the $-subunit from O, L and T confirmations. 2. The &-subunit rotation is powered by H+ transport down its EC gradient.

ATP Synthase & Proton Motive Force in ATP Synthesis *Chemiosmotic Coupling

ATP Synthase & Proton Motive Force in ATP Synthesis *Chemiosmotic Coupling

The Electron Transport Chain and ATP Synthesis *Transport into matrix of basic materials for ATP formation

14_16_coupled_transpor.jpg!

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