biochemiipart3ppspring 2011final

Fig. 19-1, p. 564

AEROBIC RESPIRATION OF GLUCOSE

Comparison of Anaerobic and Aerobic Catabolism of Glucose Anaerobic Glycolysis (Exergonic Component):

1 Glucose → 2 Lactate ΔGo’ = - 197 KJ/Mole

Aerobic Respiration (Exergonic Component):

1 Glucose + 6 O2 → 6 CO2 + 6 H2O ΔGo’ = - 2870 KJ/Mole

Phases of Aerobic Respiration

Phase I – Anaerobic Respiratory Phase (in cytoplasm)

1 Glucose → 2 Pyruvate (Aerobic Glycolysis, Embden-Meyerhof Pathway)

Phase II – Aerobic Respiratory Phase (in mitochondrion)

A. Pyruvate Activation B. Krebs Cycle C. Oxidative Phosphorylation

Fig. 20-1, p. 593

Complete Aerobic Respiration of Glucose

A. Aerobic Glycolysis – already completed.

B. Pyruvate Activation Step transport 2 pyruvate (cytoplasm) ------> 2 pyruvate (mitochondrial matrix)

CH3 O | PDH || 2 C = O + 2 NAD+ + 2 CoA-SH → 2 CH3 — C — S-CoA + 2 CO2 + | Complex COOH 2 NADH + 2 H+

ΔGo’ = -33.5 KJ/MolePDH – Pyruvate Dehydrogenase

Pyruvate Acetyl CoA

PYRUVATE DEHYDROGENASE(Reaction links aerobic glycolysis to Krebs Cycle)

1. Reaction represents the only way that pyruvate can be activated for entry into Krebs Cycle. 2. Extremely important for tissues in which glucose is the only energy source. Brain and nerous system. Brain itself requires a lot of energy, and glucose is the only source that is burned for energy.3. Irreversible reaction. Absolutely has to be oxidized aerobicly. 38 ATP vs 2ATP.4. Pyruvate Dehydrogenase is a cluster of three enzymes (E1-E2-E3) – a multienzyme complex. The three enzymes are: a. E1 – Pyruvate Dehydrogenase b. E2 − Dihydrolipoyl Transacetylase c. E3 − Dihydrolipoyl Dehydrogenase5. Major Product – Acetyl CoA a. As thioester, is unstable, therefore, has a high group transfer potential. More likely to go through hydrolysis. b. Can enter Krebs Cycle (if energy charge of cell is low).

Fig. 17-5, p. 517

PYRUVATE DEHYDROGENASE – COENZYME REQUIREMENTS

1. NAD+ - derivative of Niacin or Vitamin B3

2. CoA-SH – derivative of pantothenic acid (Vitamin B5) 3. TPP – derivative of thiamine (Vitamin B1)

4. FAD – derivative of riboflavin (Vitamin B2)

5. Lipoic Acid – functions as a coenzyme, technically not a true vitamin.

p. 570

Vitamin B5

CoA - SH

LIPOIC ACID

of E2

p. 570

E1 - Pyruvate DehydrogenaseE2 - Transacetylase (Dihydrolipoyl)E3 - Dehydrogenase (Dihydrolipoyl)

Fig. 19-4, p. 567

REGULATION OF PYRUVATE DEHYDROGENASE COMPLEX

Allosteric:Inhibited by: Acetyl CoA, NADH, ATP, GTPActivated by: AMP or ADP, NAD+, CoA-SH

Covalent: (Serine)

Pyruvate Dehydrogenase – P Catalytically Inactive In the liver – Kinase – activated by glucagon Phosphatase – activated by insulin

THIAMINE DEFICIENCY

1. Enzymes Affected a. Cannot activate Pyruvate Dehydrogenase Complex b. Transketolase (HMP Shunt Pathway) -will not be able to do that. c. α – Ketoglutarate Dehydrogenase (Krebs Cycle enzymes)

2. Deficiency Disorders a. Wernicke-Korsakoff Syndrome (more common in alcoholics) – loss of muscle coordination, visual problems, memory loss, altered perception of reality. b. Beriberi (I can’t, I can’t) – pain in limbs, muscle weakness, skin sensations, often with enlarged heart and edema.

INHIBITION OF PYRUVATE DEHYDROGENASE COMPLEX BY SELECTED ENVIRONMENTAL SUBSTANCES

1. Bind to Lipoic Acid “Arms”, inhibiting E2 – both effect nervous system!!! a. Arsenite (AsO3H2

-) bind to sulfurs and block reaction there… b. Mercury (Hg) – Mad Hatter Syndrome (mercury nitrate was used to soften felt used in hats)

http://www.youtube.com/watch?v=sBUni7Flbts

2. Effects of Inhibition(especially if energy charge is low)?

SOURCES AND FATES OF ACETYL CoA

Glucose

Certain Amino Acids Fatty Acids

synthesis β-oxidation

ACETYL CoA

Aerobic Respiration Ketone Bodies Cholesterol (Krebs Cycle)

Fig. 19-2, p. 565

C6

C5

C4

* *

* *

* *

* *

* *

C4

C2

A Tricarboxylic Acid and Tertiary Alcohol

Secondary Alcohol

Multienzyme complex

Substrate Level Phosphorylation

Fig. 19-6, p. 571

CITRATE SYNTHASE REACTION

A Regulatory Step in the Pathway

Reaction is highly exergonic and essentially irreversible.

Fig. 19-8a, p. 573

ACONITASE REACTION

Citrate (a tertiary alcohol) is converted to Isocitrate (a secondary alcohol). The oxygen of the secondary alcohol will be more readily oxidized (next step).

Fig. 19-10a, p. 574

-An oxidative decarboxylation- C6 → C5

- Quite exergonic - A regulatory step

p. 575

- Oxidative decarboxylation - C5 → C4

- Multienzyme Complex, similar to the Pyruvate Dehydrogenase Complex- Important Regulatory Step- Succinyl CoA – a high energy thioester, high group transfer potential- Highly exergonic

p. 575

-Cleavage of high energy thioester drives synthesis of GTP- Note the symmetry of Succinate.

p. 576

-Only Krebs Cycle Enzyme which is membrane bound (inner mitochondrial membrane)-Enzyme Marker for Mitochondria- Plant Acid (malonate) is a competitive inhibitor.

p. 577

Water is added across the double bond.

p. 578

-Final reaction in Krebs Cycle- Free reversible in vivo

Table 19-1, p. 572

KREBS CYCLE FUNCTIONS IN:

- CATABOLISM ( 2 decarboxylation steps, 4 oxidation steps)

- ANABOLISM

Fig. 19-16, p. 581

Fig. 19-17a, p. 583

THE MOST IMPORTANT “ANAPLEROTIC” REACTION

Fig. 19-18, p. 585

REGULATION OF KREBS CYCLE (AND PYRUVATE ACTIVATION STEP)

1. All are either inhibited by ATP and/or activated by AMP or ADP.

2. All are inhibited by NADH.

FATES OF PRODUCTS OF PYRUVATE ACTIVATION STEP AND KREBS CYCLE

1. Acetyl CoA - enters Krebs Cycle

2. CO2 – exhaled (most)

3. GTP - converted into ATP: Nucleoside Diphosphate Kinase

GTP + ADP ATP + GDP⇄. 4. NADH + H+ - must be reoxidized to NAD+. The ultimate electron acceptor in this process is O2.

5. FADH2 - must be reoxidized to FAD. Again, the ultimate electron acceptor is O2.

Fig. 19-1, p. 564

AEROBIC RESPIRATION OF GLUCOSE

Electrons will be removed from NADH and FADH2.

OXIDATIVE PHOSPHORYLATION

Oxidation: The removal of electrons from NADH AND FADH2

2 e- ‘s carrier #1 carrier #2 carrier #3 etc. 2 e- ‘s + ½ O2 + 2H+ H2O energy energy energy

Energy Released is used for Phosphorylation:

ADP + Pi → ATP + H2O

OXIDATIVE PHOSPHORYLATION:

THE PROCESS IN WHICH ATP IS FORMED AS ELECTRONS ARE TRANSFERRED FROM THE REUDCED COMPOUNDS, NADH AND FADH2 TO OXYGEN THROUGH A SERIES OF ELECTRON CARRIERS

Inner mitochondrial membrane with its large surface area (many cristae or folds) contains multiple functional electron transport chains.

DRIVING FORCE FOR ELECTRON TRANSPORT

What makes electron transport and therefore, oxidative phosphorylation, happen?

The electron transfer ability or tendency to give up electrons of FADH2 and NADH can be measured in “volts” as the “standard reduction (or redox) potential” – Eo

’.

Eo’ is the tendency of a reducing agent, such as NADH or FADH2, to give up electrons

or the tendency of an oxidizing agent, such as oxygen, to accept electrons.

A substance with a negative Eo’ will tend to give up electrons and a substance with a

positive Eo’ will tend to accept electrons. E o

’ is measured at a pH of 7.0 and at a temperature of 20-30o C.

The net redox potential of a complete system or reaction can be calculated from the individual redox potentials of the reactants/products. Once the net redox potential is determined it can be used to calculate the Standard Apparent Free Energy change, G o’ , for the reaction.

Table 20-1, p. 595

→

→

→

Calculation of the Net Redox Potential for the Reduction of Oxygen by NADH

Overall Reaction: ½ O2 + NADH + H+ H2O + NAD+ ΔEo’ = +1.14 Volts

ΔEo’ (net redox potential = Eo

’ (couple undergoing - Eo’ (couple undergoing

of complete reaction) reduction) oxidation)

Couple undergoing reduction: ½ O2 + 2H+ +2e- H2O Eo

’ = 0.82 V

Couple undergoing oxidation: NAD+ + H+ + 2e- NADH Eo

’ = -0.32V

Eo’ = 0.82 - ( - 0.32) = +1.14 V

G o’ = -nF Eo’

n = # of electrons transferred; F = Faraday = 96.485 KJ/V . Mole G o’ = -2( 96.485)( 1.14) = -220 KJ/Mole

ELECTRON CARRIERS(MOST are proteins)

1. NADH Dehydrogenase (E-FMN)

a. Flavoprotein which contains FMN as its coenzyme prosthetic group( E-FMN). b. Accepts 2 H+’s and 2 electrons.

NADH + H+ +E-FMN ------> E-FMNH2 + NAD+

This enzyme (flavoprotein) in combination with Fe-S Proteins (see below) spans the inner mitochondrial membrane, but the binding site for the substrate, NADH, is on the inner face of the membrane.

ELECTRON CARRIERS (Cont.)

2. Coenzyme Q - Ubiquinone.

a. Small, lipid soluble molecule, which diffuses easily through the phospholipid bilayer of the inner mitochondrial membrane.

b. Accepts 2 electrons and 2 H+’s, but in two stages

Reduced carrier donor + 2 H+’s + Coenzyme Q(ox.) ------> Oxidized carrier + Coenzyme QH2 (red.)

Fig. 20-5, p. 600

ELECTRON CARRIERS (Cont.)3. Cytochromes

a. Conjugated proteins with hemes as prosthetic groups.

b. Three general classes of cytochromes involved in electron transport - a, b, and c.

c. Subclasses exist within each class (slight differences in amino acid sequences of polypeptide chains). d. Cytochromes involved in electron transport: b, c, c1, (a +a3) (also called cytochrome oxidase). e. Heme iron (ferric) serves as a single electron acceptor. It is reduced to ferrous iron.

f. In cytochrome oxidase (a + a3) cupric copper also accepts a single electron, becoming reduced to the cuprous form.

Reduced donor carrier + cyt-Fe+3 or cyt-Cu+2 -----> Oxidized carrier + cyt-Fe+2 or cyt-Cu+1

Fig. 20-10, p. 603

Heme in cytochrome b

Heme in cytochrome c

Heme in cytochrome a

Different classes of cytochromes differ in:

- Exact type of heme - Attachment of heme to polypeptide chain - Amino acid sequence of polypeptide chain

ELECTRON CARRIERS (Cont.)

4. Non-heme iron proteins (NHI’s) or Fe-S Proteins

a. Contain iron, but not as a part of heme. b. Ferric form of iron serves as a single electron acceptor, forming the reduced ferrous form.

Reduced carrier donor + NHI-Fe+3 ------> Oxidized carrier + NHI-Fe+2

ELECTRON CARRIERS (Cont.)

1. Some accept single electrons only

2. Others accept 2 electrons plus 2 H+’s.

3. Carriers are located asymmetrically in the membrane.

NAD

+ H+

heme566L

Cyt-b

heme562H

SEQUENCE OF MAJOR ELECTRON CARRIERS

NADH + H+ → NADH-DH → CoQ → Cyt-b → Cyt-c1 → Cyt-c → Cyt-a+a3 → ½ O2 + 2 e-’s + 2 H+’s → H2O

FADH2 → CoQ → Cyt-b → Cyt-c1 → Cyt-c → Cyt-a+a3 → ½ O2 + 2 e-’s + 2 H+’s

→ H2O

P Side

N Side

Fo

← F1

ELECTRON CARRIERS ORGANIZED INTO FUNCTIONAL COMPLEXES

1. Complex I ( site 1) a. NADH dehydrogenase plus NHI protein. b. Transfers of electrons from NADH to Coenzyme Q. c. Also called NADH-Coenzyme Q reductase.

2. Complex II a. Succinate dehydrogenase plus NHI protein. b. Transfers electrons from FADH2 to Coenzyme Q. c. Also called Succinate-Coenzyme Q reductase.

Fig. 20-6a, p. 601

Fig. 20-7a, p. 602

ELECTRON CARRIERS ORGANIZED INTO FUNCTIONAL COMPLEXES (Cont.)

3. Complex III ( site 2)

a. Cytochrome b (two different hemes attached to the same protein( 566L and 562H)), cytochrome c1 and NHI protein

b. Transfers electrons from Coenzyme Q to Cytochrome .

c. Also called Coenzyme Q-cytochrome c reductase.

Fig. 20-12, p. 605

ELECTRON CARRIERS ORGANIZED INTO FUNCTIONAL COMPLEXES (Cont.)

4. Complex IV (site 3) a. Cytochromes a + a3. b. Contain both Fe +3 and Cu+2 serving as electron acceptors. c. Transfers electrons from cytochrome c to oxygen

d. Cytochrome c- very loosely bound to the outer surface of the inner mitochondrial membrane; Can move from Complex III to Complex IV on inner membrane face. e. Also called Cytochrome c Oxidase.

Fig. 20-16, p. 607

Fig. 20-19, p. 610

NADH Oxidation – Outcome 1. As a pair of electrons from NADH passes through sites 1, 2, and 3, sufficient energy is released to drive the synthesis of ~1 ATP at each site.

2. For every pair of electrons that pass from NADH to oxygen, ~3 ATP (actually 2.5) are formed.

3. At the end of the chain, the pair of electrons is accepted by molecular oxygen, which in the presence of 2 H+’s forms 1 H2O. 4. The ATP molecules are synthesized from ADP and Pi through the action of the enzyme ATPase also called ATP synthase.

5. One H2O is formed for every ATP that is formed.

NADH Oxidation – Outcome (Cont.)

6. ATPase or ATP Synthase

a. Membrane bound enzyme, found in the inner mitochondrial membrane

b. Consists of a “stalk” (hollow) which extends through the inner membrane to which is attached a “knob” which extends into the mitochondrial matrix.

c. The “stalk” is called the Fo portion and the “knob” is called the F1 portion.

d. The “knob” contains the active site (catalytic site) - if the knob is removed, the enzyme loses catalytic activity. e.This membrane bound ATPase is sometimes called Complex V.

FADH2 Oxidation – Outcome

As pair of electrons passes from FADH2 to oxygen, only ~2 ATP (actually 1.5) molecules are synthesized, as site 1 is by-passed.

Coupling of Redox Coenzyme Oxidation to ATP Synthesis (Phosphorylation)

1. Oxidation of NADH and FADH2 is tightly coupled to the synthesis of ATP

(phosphorylation).

2. Electron transport - tightly coupled to phosphorylation.

3. Energy is released gradually during electron transport ( Energy Cascade)

4. At certain sites (1,2, and 3) sufficient energy (more than 30.5 KJ/mole) is released to drive the synthesis of ATP.

5. Coupling of the two processes: a. If electron transport is inhibited or blocked, ATP synthesis will be inhibited. b. If ATP synthesis is inhibited (generally through inhibition of ATPase), electron transport will be inhibited..

Energy Cascade

Fig. 20-28, p. 617

P/O RATIO

1. For every pair of electrons from NADH which enters the electron transport chain and passes to oxygen, ~3 (2.5) ATP are formed.

2. For every pair of electrons from FADH2 that passes down the chain to oxygen, about ~2(1.5) ATP formed.

3. This is expressed in what is commonly called the “P/O Ratio”.

4. Definition of P/O Ratio: The number of molecules of Pi incorporated into ATP (organic form) per gram atom of oxygen consumed or the number of molecules of ATP synthesized for every pair of electrons that passes down the chain to oxygen.

DRIVING FORCE FOR ELECTRON TRANSPORT

1. The need to reoxidize NADH and FADH2.

2. NADH and FADH2 have large negative redox potentials a. Great tendency to give up electrons. b. Unstable in the reduced state; Strong tendency to be oxidized.

3.Oxygen has a large positive redox potential a. Strong tendency to accept electrons; Great tendency to be reduced. B. Oxygen - strong oxidizing agent.

Look at the redox potentials of all of the carriers in the electron transport chain. What do you notice about these values as you proceed down the chain?

2e- ‘s Electron Transport Chain ½ O2 + 2H+ H2O High Energy State Low Energy State

Decreasing Tendency to Donate Electrons --------------------------------------------------------------------------> Decreasing Tendency to be Oxidized

Increasing Tendency to Accept Electrons ---------------------------------------------------------------------------> Increasing Tendency to be Reduced

ELECTRON TRANSPORT CHAINREDOX POTENTIALS OF INDIVIDUAL CARRIERS

Note the magnitudes and signs of the standard redox potentials of carriers as you progress down the chain towards oxygen.

RESPIRATORY INHIBITORS – THREE CLASSES

1 . Those that inhibit the process of electron transport.

2. Those that inhibit phosphorylation or ATP synthesis.

3. Those that "uncouple" electron transport or oxidation from phosphorylation or ATP synthesis; Render electron transport independent of phosphorylation.

ELECTRON TRANSPORT INHIBITORS

Through Complex I

a. Rotenone (a natural plant product which is an insecticide) b. Amytal ( a barbiturate) c. Demerol, a narcotic analgesic( pain killer),

Through Complex III - Antimycin A (an antibiotic)

Through Complex IV

a. Cyanide - binds to Fe+3 of Cytochrome a3, b. Azide - binds to Fe+3 of Cytochrome a3, c. Carbon Monoxide - binds to Fe+2 of cytochrome a3 , as well as to hemoglobin and myoglobin.

RESPIRATORY INHIBITORS - ILLUSTRATION

What will be the state of each of the carriers upstream of the inhibitor? Downstream of the inhibitor?

Fig. 20-28, p. 617

Inhibitors of ATPase (ATP synthase)

Oligomycin

a. Binds to one of the stalk proteins in the Fo fragment of ATPase

b. Inhibits ATP synthesis.. c. Since electron transport and ATP synthesis are tightly coupled, electron transport will also be inhibited.

UNCOUPLORS

1. Render Electron Transport Independent of Phosphorylation a. Electron Transport Occurs b. ATP Synthesis is Inhibited. c. Energy Derived from Electron Transport – released as heat.

2. Some examples a. Dinitrophenol b. Ionophores 1) Nigericin 2) Valinomycin

Respiratory Control (Acceptor Control) of Oxidative Phosphorylation

Rate of Oxidative phosphorylation

1. Not controlled by allosteric mechanisms but by substrate availability. 2. Substrates (raw matreials) required for Oxidative Phosphorylation a. Oxidizable Substrate(s) – freely available in the well nourished organism. b. Oxygen - environment c. ADP - concentration varies, depending upon energy charge of the cell. d. Pi - generally available

3. Bottom line – rate is controlled by the need for ATP, as reflected in the increased availability of ADP. In most aerobic cells, the level of ATP exceeds that of ADP by factors of 4 - 10. We can therefore think of respiratory control as the dependence of respiration on ADP levels. When the cellular concentration of ADP is high, respiration is stimulated; when the cellular concentration of ADP is low, respiration is inhibited. This can also be expressed as the “phosphorylation potential” - [ATP]/[ADP][Pi]. The phosphorylation potential of a cell is normally high, but if its drops, then respiration is stimulated.

Fig. 20-6a, p. 601

Respiratory Complex I

Fig. 20-12, p. 605

Respiratory Complex III (showing both components of Q Cycle)

Fig. 20-16, p. 607

Respiratory Complex IV

Fig. 20-7a, p. 602

Respiratory Complex II

ELECTRON TRANSPORT – PROTON TRANSLOCATION

(Intermembrane Space)

4 H+ 4 H+ 2 H+ NADH + H+ Complex I CoQ Complex III Cyt c Complex IV 2 e-‘s + ½ O2

+ 2H+ H2O 4 H+ 2 H+ 2 H+

(Matrix)

Explanation: Carriers are located asymmetrically in the membrane. Some carriers accepts 2 electrons plus 2 H+’s , whereas others only accept single electrons.

Fig. 20-19, p. 610

ENERGY TALLY – AEROBIC RESPIRATIONAnaerobic Respiratory Phase (Aerobic Glycolysis) 2 ATP 2 NADH + 2H+ -- -> Mitochondrion ---------------------------------------------- 8 ATP ( or 6 ATP) Aerobic Respiratory Phase a. Pyruvate Activation Step 2 NADH + 2H+ ---> 6ATP b. Krebs Cycle (2 turns) 2 GTP 2 ATP 6 NADH + 6H+ 18 ATP 2 FADH2 4 ATP 24 ATP Total ATP = 8 + 6 + 24 = 38

EFFICIENCY – AEROBIC RESPIRATION Exergonic Component 1 Glucose + 6O2 6 CO2 + 6 H2O Go’ = -2870 KJ/Mole

Endergonic Component

38 Pi + 38 ADP 38 ATP + 38 H2O Go’ = +1159 KJ/Mole

Overall Efficiency = 1159/2870 x 100 40%

In a living cell, where conditions are far from standard, efficiency approximates 70%

Fig. 20-31, p. 621

Fig. 20-32, p. 622

CHEMIOSMOTIC HYPOTHESIS (PETER MITCHELL)

Explanation – During Electron Transport, a proton gradient is formed across the inner mitochondrial membrane. This “high energy state” drives the synthesis of ATP.

Experimental evidence in support of hypothesis:

1. Either intact mitochondria or inner mitochondrial membranes are required for oxidative phosphorylation. 2. The inner mitochondrial membrane is impermeable to H+, K+, OH-, Cl-. 3. During oxidative phosphorylation, the pH of the intermembrane space is decreased relative to that of the matrix (by up to 1.14 pH units). 4. Action of uncouplers (see below)

Fig. 20-20, p. 610

FORCES DRIVING PROTON TRANSLOCATION ACROSS INNER MITOCHONDRIAL MEMBRANE

Consider the G of proton translocation a. Sign and Magnitude b. Components 1) Hydrogen (Chemical) or Proton Gradient 2) Membrane Potential

This Driving Force is Often Expressed as the Proton Motive Force (ΔμH+): 1. What is it? a. ΔG/F b. Units (Volts or Millivolts) Proton Motive Force (ΔμH+) = 2.3 RT ΔpH + Δ ψ F Proton Gradient Membrane Potential At 370C: 38% 62%

2). The Proton Motive Force - two components - The H+ or chemical gradient (pH gradient) - pH - The membrane potential or charge gradient -

UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION

1. DNP - will carry protons back across the inner mitochondrial membrane

Intermembrane Space (low pH)

DNP- + H+ → DNPH

Movement Through Inner Mitochondrial Membrane

DNPH (intermembrane space) → DNPH (matrix)

Mitochondrial Matrix (higher pH)

DNPH → DNP → DNP-- + H + H++

Dissipates both the Membrane Potential AND the Proton (pH) Gradient.

UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION (Cont.)

2. Ionophores – transport cations across the inner mitochondrial membrane

a. Valinomycin – carries K+ across the inner mitochondrial membrane into the matrix.

Dissipates the Membrane Potential but NOT the Proton (pH) Gradient.

b. Nigericin - carries H+ from intermembrane space to matrix AND K+ from matrix to intermembrane space.

Dissipates Proton (pH) Gradient but NOT the Membrane Potential.

Fig. 20-20, p. 610

Fig. 20-25a, p. 615

ATP SYNTHASE SUBUNIT COMPOSITIONFo - 3 unique subunits form the “proton channel)

a b c 1 1 (10-15)

- three additional subunits (d, h, OSCP), along with b, form a long “stalk” that connects F0 with F1)

F1 - 5 unique subunits form the “knob or sphere”.

α β γ δ ε 3 3 1 1 1 form a narrower portion connecting the larger part of the knob with the Fo.

β - Catalytic Subunits

c, γ, δ, ε - rotate as protons are being translocated

Other subunits remain stationary (stators).

Table 20-3, p. 612

Fig. 20-21, p. 612

← Rotates

H+ - ATP SYNTHASE FUNCTION

1. ADP + Pi bind to active site of enzyme (located primarily on the β- subunit of F1.

2. Catalysis occurs and ATP is formed, but it cannot yet be released from the active site.

3. Protons pass through the channel in Fo. The proton translocation caused the c subunits, along with the γ, δ, and ε subunits to rotate.

4. This rotation causes a conformational change in the α and β subunits of F1 that enables the newly formed ATP to be released.

5. Now, the catalytic cycle can be repeated.

HOW MANY PROTONS MUST BE TRANSLOCATED THROUGH H+-ATP SYNTHASE TO DRIVE THE

SYNTHESIS AND RELEASE OF 1 ATP?

Most agree on a figure of 3 – 4.

1. For every electron pair from FADH2 that travels down the electron transport chain to oxygen, 6-8 H+’s are translocated from the matrix into the intermembrane space.

#H+’s translocated/#ATP synthesized = 6-8/2 = 3-4 H+’s translocated/1ATP synthesized

2. For every electron pair from NADH that travels down the electron transport chain to oxygen, 10-12 H+’s are pumped into the intermembrane space and 3 ATP and formed.

Again this results in 3-4 H+’s translocated/1ATP formed.

Fig. 20-30, p. 619

Inside out vesicles prepared from inner mitochondrial membranes – Racker Vesicles.

Complete Vesicles –capable of carrying out Oxidative Phosphorylation

If F1 is removed??

GENERATION OF TOXIC FORMS OF OXYGEN

1. O2 + e- O2.- (Superoxide Radical) Long Life; Damages

phospholipids

2. O2.- + e- + 2H+ H2O2 (Hydrogen peroxide) H2O2 + Fe+2 Fe+3 + OH

. + OH-

3. H2O2 + e- + H+ H2O + OH. (Hydroxyl radical) Most potent oxidizing agent known; can damage most organic molecules.

4. OH. + e- + H+ H2O _______________________________________________________

O2 + 4 e-‘s + 4 H+’s 2 H2O

ENZYMES THAT REMOVE TOXIC FORMS OF OXYGEN …

Superoxide Dismutase (SOD) 2 O2

.- + 2 H+ H2O2 + O2

Catalase 2 H2O2 2 H2O + O2

Glutathione Peroxidase 2 GSH + 2 H2O2 GSSG + 2 H2O …OR THEIR PRODUCTS

Glutathione Peroxidase 2 GSH + -ROOH GSSG + ROH + H2O

-ROOH – an organic peroxide which can result from exposure to OH.

BROWN FAT

-Type of adipose tissue found in human infants, mammals born hairless and some hibernating animals.

- Brown color due to presence of large numbers of mitochondria (rich in heme proteins)

- Inner mitochondrial membranes contain protein known as “Thermogenin” or “Uncoupling Protein 1 (UCP1), which foms proton pores or channels through the membrane.

- Electron transport is “uncoupled” from phosphorylation; energy is released as heat.

Note: Certain plants (skunk cabbage and red tomatoes) also generate heat by uncoupling electron transport from phosphorylation.

p. 619

p. 619

Fig. 17-7, p. 519

MITODHONDRIAL PREPARATION ( + Oxidizable Substrate, ADP, Pi and Oxygen)

Additions Electron Transport ATP Formation

None

Rotenone

Oligomycin

DNP

Oligomycin + DNP

Rotenone + DNP

RESPIRATORY CONTROL

Note: Glutamate → α-Ketoglutarate