Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Phase II Aerobic Respiratory Phase (in mitochondrion) A. Pyruvate Activation B. Krebs Cycle C. Oxidative Phosphorylation
B. Pyruvate Activation Step transport 2 pyruvate (cytoplasm) ------> 2 pyruvate (mitochondrial matrix)
Pyruvate Acetyl CoA
CH3 O | PDH || 2 C = O + 2 NAD+ + 2 CoA-SH 2 CH3 C S-CoA + 2 CO2 + | Complex COOH 2 NADH + 2 H+
Go = -33.5 KJ/Mole
PDH Pyruvate Dehydrogenase
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 Dehydrogenase 5. 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).
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.
Vitamin B5
CoA - SH
p. 570
LIPOIC ACID
of E2
p. 570
Allosteric: Inhibited by: Acetyl CoA, NADH, ATP, GTP Activated 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. 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 cant, I cant) pain in limbs, muscle weakness, skin sensations, often with enlarged heart and edema.
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
Fatty Acids
-oxidation
ACETYL CoA
Ketone Bodies
Cholesterol
C2
* *
C4
C6
* Secondary Alcohol
C4
Substrate Level Phosphorylation * *
C5
Multienzyme complex
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).
- 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. 575
-Only Krebs Cycle Enzyme which is membrane bound (inner mitochondrial membrane) -Enzyme Marker for Mitochondria - Plant Acid (malonate) is a competitive inhibitor.
p. 576
- ANABOLISM
FATES OF PRODUCTS OF PYRUVATE ACTIVATION STEP AND KREBS CYCLE 1. Acetyl CoA - enters Krebs Cycle
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.
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.
Calculation of the Net Redox Potential for the Reduction of Oxygen by NADH Overall Reaction: O2 + NADH + H+ H2O + NAD+ Eo (net redox potential of complete reaction) = Eo (couple undergoing reduction) Eo = 0.82 V Eo = +1.14 Volts
Couple undergoing oxidation: NAD+ + H+ + 2e- NADH Eo = -0.32V Eo = 0.82 - ( - 0.32) = +1.14 V G o = -nF Eo
ELECTRON CARRIERS
(MOST are proteins)
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.
Heme in cytochrome b Different classes of cytochromes differ in: - Exact type of heme - Attachment of heme to polypeptide chain - Amino acid sequence of polypeptide chain
Heme in cytochrome c
Heme in cytochrome a
NHI-Fe+2
NAD
+ H+
NADH + H+ NADH-DH CoQ Cyt-b Cyt-c1 Cyt-c Cyt-a+a3 O2 + 2 e-s + 2 H+s H2O FADH2 CoQ Cyt-b Cyt-c1 Cyt-c Cyt-a+a3 O2 + 2 e-s + 2 H+s H 2O
P Side
Fo
F1 N Side
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.
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
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.
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?
Note the magnitudes and signs of the standard redox potentials of carriers as you progress down the chain towards oxygen.
What will be the state of each of the carriers upstream of the inhibitor? Downstream of the inhibitor?
.
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
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.
Respiratory Complex I
Respiratory Complex IV
Respiratory Complex II
4 H+ 4 H+ 2 H+ NADH + H+ Complex I CoQ Complex III Cyt c Complex IV 2 e-s + 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.
Total ATP = 8 + 6 + 24 = 38
In a living cell, where conditions are far from standard, efficiency approximates 70%
This Driving Force is Often Expressed as the Proton Motive Force (H+):
1. What is it? a. G/F b. Units (Volts or Millivolts) Proton Motive Force (H+) = 2.3 RT pH F +
At 370C:
Movement Through Inner Mitochondrial Membrane DNPH (intermembrane space) Mitochondrial Matrix (higher pH) DNPH (matrix)
DNPH
DNP-
H+
Dissipates both the Membrane Potential AND 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.
- 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. 3 3 1 1 1
form a narrower portion connecting the larger part of the knob with the Fo.
- Catalytic Subunits
c, , , - rotate as protons are being translocated Other subunits remain stationary (stators).
Rotates
3. Protons pass through the channel in Fo. The proton translocation caused the c subunits, along with the , , and subunits to rotate.
4. This rotation causes a conformational change in the and 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?
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.
Inside out vesicles prepared from inner mitochondrial membranes Racker Vesicles.
1. O2 + e-
2. O2.- + e- + 2H+ H2O2 (Hydrogen peroxide) H2O2 + Fe+2 Fe+3 + OH . + OH3. 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 H2 O 2 2 H 2 O + O 2 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
Oligomycin
DNP Oligomycin + DNP Rotenone + DNP
RESPIRATORY CONTROL