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Biochemistry. Lecture 12. Energy from Reduced Fuels is Used to Synthesize ATP in Animals. Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell Electrons from reduced fuels used to reduce NAD + to NADH or FAD to FADH 2 .

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Biochemistry

Biochemistry

Lecture 12


Energy from reduced fuels is used to synthesize atp in animals
Energy from Reduced Fuels is Used to Synthesize ATP in Animals

  • Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell

  • Electrons from reduced fuels used to reduce NAD+ to NADH or FAD to FADH2.

  • In oxidative phosphorylation, energy from NADH and FADH2 are used to make ATP


C Animals

H

O

H

2

O

O

H

O

H

O

H

O

H

  • Proteins that mediate e- transport & oxidative phosphorylation are integrally bound to the inner membrane

    • Liver mitochondria  few cristae( respiration)

    • Heart mito.  many cristae ( respiration)

  • TCA cycle enzymes & metabolites are located in the matrix.

+ 6H2O

6CO2 + 24H+ + 24e-

12H2O

24H+ + 24e-+ 6O2


Oxidation of nadh by o 2 is highly exergonic
Oxidation of NADH by O Animals2 is Highly Exergonic

NADH  NAD+ + H+ + 2e- E° = +0.32

½ O2 + 2e + 2H+  H2O E° = +0.82

½ O2 + NADH + H+ H2O + NAD+ E° = +1.14 V

therefore:

∆G° = -nFE° = -2 x 23 kcal/mol/V x 1.14 V = -53 kcal/mol


II Animals


Antimycin a Animals

Rotenone/ amytal

CN

II

FAD

FADH2


The oxidation of nadh or fadh 2 by o 2 is tightly coupled to the phosphorylation of adp
The Oxidation of NADH or FADH Animals2 by O2 is Tightly Coupled to the Phosphorylation of ADP

How many moles of ATP are synthesized from the reduction of 1 mole O2 ?

2 NADH

nADP + nPi

1 O2

nATP

  • Measure the amount of O2 consumed (reduced to 2H2O) for any given amount of ADP added.

  • Experimental Conditions

  • - same as the inhibitor expt (no ADP initially, excess PO4)

  • - isolated mito’s in buffer containing excess phosphate

  • - addition of ADP + an electron donor starts electron transport


“Artificial Respiration”: Experiments that led to Understanding the sequence of Electron Transport Proteins


O 2 consumption as a function of adp p n
O Understanding the sequence of Electron Transport Proteins2 Consumption as a function of ADP P’n

ADP=90 umoles

(90 micromoles)

18 umoles

30 umoles

45 umoles

Conditions: Isolated mitochondria in buffer containing excess PO4. Reaction is initiated by addition of ADP and e- donor.


Interpretation of results
Interpretation of Results Understanding the sequence of Electron Transport Proteins

a) b-OH-butyrate

Conversion of 90 umol ADP (or PO4)  ATP requires 18 umol O2 (36 umol O)

P/O = 90/36 = 2.5

b) Succinate

Conversion of 90 umol ADP (or PO4)  ATP requires 30 umol O2 (60 umol O)

P/O = 90/60 = 1.5

c) TMPD/Ascorbate

P/O = 90/90 = 1


II Understanding the sequence of Electron Transport Proteins


II Understanding the sequence of Electron Transport Proteins


Q Understanding the sequence of Electron Transport Proteins

Complex II


• Oxygen is a bi-radical Understanding the sequence of Electron Transport Proteins

• Can accept e-s only 1 at a time

• ETC starts with e- pairs….

• O2 •


II Understanding the sequence of Electron Transport Proteins


Complex IV (cytochrome oxidase) Understanding the sequence of Electron Transport Proteins


4 Understanding the sequence of Electron Transport Proteins

2

4

II


Evidence that supports the chemiosmotic hypothesis: Understanding the sequence of Electron Transport Proteins

1. e- transport correlates with generation of a proton gradient

2. An artificial pH gradient leads to ATP synthesis in intact mitochondria

3. Complex I,III, and IV are proton pumps

4. A closed compartment is essential

5. Proton carriers (across IMM) “uncouple” oxidation from P’n.


Atp synthase
ATP Synthase Understanding the sequence of Electron Transport Proteins


Coupling H Understanding the sequence of Electron Transport Proteins+ translocation through ATP Synthase with ATP synthesis

Free energy of H+ translocation “forces” an internal cam shaft to rotate, which changes the conformation of each subunit during one complete turn.


Inhibitors of oxidative phosphorylation

H Understanding the sequence of Electron Transport Proteins+

IMS

MATRIX

Inhibitors of Oxidative Phosphorylation

  • Inhibitors of Complexes I, III, & IV.

  • Oligomycin – antibiotic which binds to ATP synthase and blocks H+ translocation.

  • Uncouplers:

    a) Dinitrophenol (DNP).


M Understanding the sequence of Electron Transport Proteins+

M+

H+

M+ = K+ >> Na+

M+ = K+ >> Na+

b) Ionophores

i) Valinomycin – carries charge but not H+’s. - Dissipates electrical gradient.

ii) Nigericin – carries protons but not charge.

- Dissipates chemical gradient. (due to H+)


H Understanding the sequence of Electron Transport Proteins+

H+

+

H+

+

glycerol

FFA’S

HSL

+

TAG

Norepinephrine

c) Thermogenin – active component of brown fat.

- acts as a H+ channel in the IMM of brown fat mitochon. P/O << 1.

Regulation of Thermogenin Conductance

**Uncoupling (and heat generation) occur only if plenty of FFA substrate is available. If not, ATP synthesis prevails.


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