biochem
poorva
PHAR150GElectronTransferChain.pptxPHAR 150G Biochemistry
ETC and Oxidative Phosphorylation
Vicky Mody, PhD
Associate professor of Pharmaceutical Sciences
Office , Rm 3034
Phone: 678-407-7386
1
Objectives
Explain all enzymes and the steps invovled in ETC
Explain the difference between heme based protein
Regulation of ETC and Synthesis of ATP
Uncouplers vs Couplers in ETC
Uncoupling of Electron transport and Adenosine Triphosphate Synthesis (ATP) – Small Molecules
Uncoupling of Electron transport and Adenosine Triphosphate Synthesis (ATP) – Proteins
Case Report: A 28-month-old girl presented to the emergency department with sudden onset unconsciousness and seizure when she was eating apricot seeds with her father. Glasgow coma score was 4. She was transported to the intensive care unit and mechanical ventilation was begun. Gastric lavage was performed and pieces of apricot seeds were observed. On laboratory investigation, blood gas analysis revealed pH 6.8; paO2 80 mmHg; PaCO2 15 mmHg; HCO3 5.5 mmol/L; base excess –29.6 mmol/L; plasma lactate level 10 mmol/L; plasma glucose level 290 mg/dl. With the help of laboratory findings and knowing she had been eating apricot seeds, the patient was diagnosed with acute cyanide poisoning. After collecting a whole blood sample for measurement of cyanide level, cyanide antidote dicobalt edetate (Kelocyanor) was given 10 hours after the patient arrived at the hospital. Repeated arterial blood gas analysis showed that the difference between arterial and venous PO2 levels was 8 mmHg. The result of whole blood cyanide level was more than 3mg/L at hour 4 after presentation. Dicobalt edentate was repeated. The patient died on the 22th day of hospitalization, following supportive care in the intensive care unit.
What is mechanism of cyanide poisoning from seeds?
Am J Case Rep 2011; 12:70-72
Case 1
Glycolysis yields two molecules of NADH and 2 molecules of pyruvate.
Pyruvate Dehydrogenase reaction yields 1 Molecules of NADH per molecule of pyruvate and hence 2 pyruvate molecules from glycolysis will yield 2 molecules of NADH per pyruvate Dehydrogenase Reaction
In Kreb Cycle one molecule of Pyruvate produce 3 molecules of NADH and 1 molecule of FADH2.
However, during glycolysis 2 molecules of pyruvate are released and hence we have 6 molecules of NADH and 2 molecules of FADH2 from Kreb Cycle
Counting NADH and FADH2
We Need NAD+ and FAD+
Must Know
5
NAD+ and FAD+ are converted to NADH and FADH2 during the metabolism of carbs, protein and fats.
They are our major source of energy.
These reducing equivalents (NADH or FADH2 )are passed down a respiratory chain (ETC) directing them to their final reaction with oxygen to form water and release lots and lots of ATP
We need NAD+ and FAD+ for various metabolism
Must Know
Inhibition of Pyruvate Dehydrogenase
If Pyruvate dehydrogenase is inhibited then the metabolism of glucose will not be complete and the lactic acid levels in body will rise.
Must Know
Inhibition of Pyruvate Dehydrogenase via Pyruvate Dehydrogenase Kinase in Cancer Patients
In cancer patients the pyruvate dehydrogenase kinase is active.
It inhibits pyruvate dehydrogenase and hence the metabolism of glucose is not complete in those patients.
So what would be the pH (acidic/basic) of cancer cells.
Must Know
During starvation, Pyruvate Dehydrogenase Kinase increases in amount in most tissues, including skeletal muscle.
The resulting inhibition of Pyruvate Dehydrogenase prevents muscle and other tissues from catabolizing glucose.
Metabolism shifts toward fat utilization and available glucose is spared for use by the brain.
What happens in Fasting
Must Know
Mitochondria
Must Know
Mitochondria
Outer membrane relatively permeable
Compartmentalization
Kreb's and β-oxidation of fatty acids in matrix
Glycolysis in cytosol
Must Know
Most energy from Redox
Glycolysis
In cytosol produces 2 NADH
Pyruvate dehydrogenase reaction
In mitochondrial matrix 2 NADH / glucose
Krebs
In mitochondrial matrix
6 NADH and 2 FADH2 / glucose
Must Know
How to Regenerate NAD+ and FAD+ and get more ATP
Must Know
13
Regeneration of NAD+ and FAD+
14
Electron Transport Chain
FADH2
Must Know
Electron transport to O2 occurs via a series of oxidation–reduction steps in which each successive component of the chain is reduced as it accepts electrons and oxidized as it passes electrons to the next component of the chain.
Electron transport Chain
Must Know
The oxidation–reduction components of the chain include
Complex I -NADH:CoQ oxidoreductase
Complex II - Succinate reductase
Complex III - The cytochrome b-c1 (heme protein, hence Fe)
Complex IV - The cytochrome c oxidase, also called as cytochrome a-a3 complex (heme based Fe). Also contains Cu.
All complexes I, II, III, and IV have Iron–sulfur (Fe–S) centers.
Flavin mononucleotide (FMN) and FAD is present in Complex I and II, respectively. Both are synthesized from the Vitamin B-2 (riboflavin).
MOBILE/freely diffusible component of ETC Ubiquinone or Coenzyme Q (CoQ) and Cytochrome c (heme based Fe).
NOTE: All cytochromes are heme proteins
Electron transport Chain
Must Know Very Important
Coenzyme Q
Only lipid component of the ETC
Electron Transport Chain involving NADH
Must Know
The electron transport chain involving NADH consists of 3 complexes of integral membrane proteins the
NADH:CoQ oxidoreductase or complex (I)
The cytochrome b-c1 or complex (III)
The cytochrome c oxidase or complex (IV)
Two freely-diffusible molecules that shuttle electrons from one complex to the next.
Ubiquinone (UQ) or Coenzyme Q (CoQ) - ONLY non protein component of ETC.
Cytochrome c – NOTE this is different than Cytochrome c oxidase (Complex IV)
Electron Transport Chain involving NADH
Electron Transport Chain involving FADH2
FADH2
Must Know
The electron transport chain involving FADH2 also consists of 3 complexes of integral membrane proteins
It uses Succinate reductase (Complex II) instead of NADH:CoQ oxidoreductase used by NADH
The cytochrome b-c1 or complex (III)
The cytochrome c oxidase or complex (IV)
Two freely-diffusible molecules that shuttle electrons from one complex to the next.
Ubiquinone (UQ) or Coenzyme Q (CoQ) – ONLY non protein component of ETC.
Cytochrome c - NOTE this is different than Cytochrome c oxidase (Complex IV)
Electron Transport Chain involving FADH2
Must Know
Mitochondrial Matrix
Intermembrane Space
Complex I
NADH:CoQ
Oxidoreductase
ATP
Synthase
Complex IV
Cytochrome C
Oxidase
Complex III
Cytochrome b-c1
Oxidase
Complex II
Succinate Dehydrogenase
Cytochrome C
Coenzyme Q/Ubiquinone (CoQ / UQ)
Electron Transfer Chain Mechanism
NADH
Electrons are transferred from NADH to CQ/UQ via flavin mononucleotide (FMN) present on complex I FMN then transfers electrons to FeS which ultimately transferes to UQ
NADH → FMN → FeS → FeS → UQ →UQH2
Cu
Cu
Must Know Very Important
Transfer of Electrons from NADH to UQ
Good to Know
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
FMN - Flavin Mononucleotide
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
Transfer of Electrons from NADH to UQ
Complex I
NADH: UQH2
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
Transfer of Electrons from FADH2 to UQ
Complex I
NADH: UQH2
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
Transfer of Electrons from FADH2 to UQ
Complex I
NADH: UQH2
Oxidoreductase
Complex II
Succinate Ubiquinone
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
Transfer of Electrons from FADH2 to UQ
Complex I
NADH: UQH2
Oxidoreductase
Complex II
Succinate Ubiquinone
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
FAD – Flavin Adenine Dinucleotide
Good to Know
Transfer of Electrons from FADH2 to UQ
Complex I
NADH: UQH2
Oxidoreductase
FADH2
Complex II
Succinate Ubiquinone
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
FAD – Flavin Adenine Dinucleotide
Good to Know
Transfer of Electrons from FADH2 to UQ
Complex I
NADH: UQH2
Oxidoreductase
FADH2
FeS
Complex II
Succinate Ubiquinone
Oxidoreductase
FMN - Flavin Mononucleotide
FeS – Iron Sulfur Protein
FAD – Flavin Adenine Dinucleotide
Mitochondrial Matrix
Intermembrane Space
Complex I
NADH:CoQ
Oxidoreductase
ATP
Synthase
Complex IV
Cytochrome C
Oxidase
Complex III
Cytochrome b-c1
Oxidase
Complex II
Succinate Dehydrogenase
Cytochrome C
Synthesis of ATP from ADP and Pi
NAD+
Electrons
Protons
4H+
2H+
Coenzyme Q/Ubiquinone (CoQ / UQ)
Reduced form of
Cytochrome C
Cu
Cu
4H+
4 Protons from the intermembrane space are used by ADP and Pi to form one ATP Molecule
Must Know Very Important
Mitochondrial Matrix
Intermembrane Space
Complex I
NADH:CoQ
Oxidoreductase
ATP
Synthase
Complex IV
Cytochrome C
Oxidase
Complex III
Cytochrome b-c1
Oxidase
Complex II
Succinate Dehydrogenase
Cytochrome C
Coenzyme Q/Ubiquinone (CoQ / UQ)
NADH
Electrons are transferred from NADH to CQ/UQ via flavin mononucleotide (FMN) present on complex I FMN then transfers electrons to FeS which ultimately transferes to UQ
NADH → FMN → FeS → FeS → UQ →UQH2
Cu
Cu
Complete Oxidative Phosphorylation
Must Know Very Important
Total
Proton gradient created as electrons transferred to oxygen forming water
10 H+ / NADH
6 H+ / FADH2
Must Know Very Important
Components of ETC
39
Electron Transfer Chain
In the electron-transport chain, electrons donated by NADH or FAD(2H) are passed sequentially through a series of electron carriers embedded in the inner mitochondrial membrane.
Each of the components of the electron-transfer chain is reduced as it accepts an electron and then oxidized as it passes the electrons to the next member of the chain.
From NADH, electrons are transferred sequentially through NADH:CoQ oxidoreductase (complex I), coenzyme Q (CoQ), the cytochrome b–c1 complex (complex III), cytochrome c, and finally, cytochrome c oxidase (complex IV).
Must Know Very Important
NADH:CoQ oxidoreductase, the cytochrome b–c1 complex, and cytochrome c oxidase are each multisubunit protein complexes that span the inner mitochondrial membrane.
CoQ is a lipid-soluble quinone that is not protein bound and is free to diffuse in the lipid membrane.
CoQ transports electrons from complex I to complex III and is an intrinsic part of the proton pump for each of these complexes.
Cytochrome c is a small protein in the intermembrane space that transfers electrons from the b–c1 complex to cytochrome c oxidase.
Components of Electron Transfer Chain
Must Know Very Important
Electron Transfer Chain
The terminal complex, cytochrome c oxidase, contains the binding site for O2. As O2 accepts electrons from the chain, it is reduced to H2O.
Must Know Very Important
NADH:COQ OXIDOREDUCTASE or Complex I
NADH:CoQ oxidoreductase (also named NADH dehydrogenase) is an enormous 42-subunit complex that contains a binding site for NADH, several FMN and Fe–S center-binding proteins, and binding sites for CoQ.
An FMN accepts two electrons from NADH and is able to pass single electrons to the Fe–S centers.
Fe–S centers transfer electrons to and from CoQ.
Components of Electron Transfer Chain
Must Know Very Important
Components of Electron Transfer Chain
Coenzyme Q
COENZYME Q (CoQ /UQ)
CoQ is the lipid only component of the electron-transport chain that is not protein bound.
When the oxidized quinone form accepts a electron to forms a free radical (a semiquinone).
Formation of radical makes it potent site for the generation of toxic oxygen free radicals in the body
Components of Electron Transfer Chain
Must Know Very Important
Components of Electron Transfer Chain
COENZYME Q (CoQ /UQ)
The mobility of CoQ in the membrane, its ability to accept one or two electrons, and its ability to form CoQH2 and donate protons enable it to participate in the proton pumps for both complexes I and III as it shuttles electrons between them.
CoQ is also called ubiquinone (the ubiquitous quinone) because quinones with similar structures are found in all plants and animals.
Must Know Very Important
CYTOCHROMES
The remaining components in the electron-transport chain are cytochromes.
Each cytochrome is a protein that contains a bound heme (i.e., an Fe atom bound to a porphyrin nucleus similar in structure to the heme in hemoglobin), however these cytochromes, except, cytochrome c,(mobile carrier) also contain non heme Fe i.e. Fe-S
Because of differences in the protein component of the cytochromes and small differences in the heme structure, each heme has a different reduction potential.
Components of Electron Transfer Chain
Must Know Very Important
COPPER (CU) AND THE REDUCTION OF OXYGEN
The last cytochrome complex is cytochrome oxidase a-a3, which passes electrons to O2.
It contains cytochromes a and a3 and the oxygen-binding site.
A whole oxygen molecule, O2, must accept four electrons to be reduced to two H2O molecules.
Bound copper (Cu) ions in the cytochrome oxidase a-a3 complex facilitate the collection of the four electrons and the reduction of O2 to H2O.
Components of Electron Transfer Chain
Must Know Very Important
The Electrochemical Potential Gradient
49
The electron transfer around the complexes is accompanied by proton pumping across the membrane.
The membrane is impermeable to protons so they cannot diffuse through the lipid bilayer back into the matrix.
Thus, in actively respiring mitochondria, the intermembrane space and cytosol may be approximately 0.75 pH unit lower than the matrix.
The Electrochemical Potential Gradient
Must Know Very Important
The transmembrane movement of protons generates an ELECTROCHEMICAL GRADIENT with two components:
The membrane potential
The proton gradient
The electrochemical gradient is sometimes called the proton motive force because it is the energy that pushes the protons to reenter the matrix to equilibrate on both sides of the membrane.
The protons are attracted to the more negatively charged matrix side of the membrane, where the pH is more alkaline.
The Electrochemical Potential Gradient
Must Know Very Important
ATP Yield from ETC
52
Overall, each NADH donates two electrons, equivalent to the reduction of one-half of an O2 molecule.
A generally (but not universally) accepted estimate of the stoichiometry of ATP synthesis is that four protons are pumped at complex I, two protons at complex III, and four protons at complex IV.
With four protons translocated (used) for the synthesis of ATP synthesized, an estimated 2.5 ATPs are formed for each NADH oxidized,
Similarly, 1.5 ATPs are formed for each of the other FAD(2H)-
ATP Yield from ETC
Must Know Very Important
What Happens in Absence of Oxygen
54
In the absence of O2, no ATP is generated from oxidative phosphorylation because electrons backs up in the chain.
Even complex I cannot pump protons to generate the electrochemical gradient because every molecule of CoQ already has electrons that it cannot pass down the chain without an O2 to accept them at the end.
What Happens in Absence of Oxygen
Must Know Very Important
CO and Cyanide Toxicity
56
How does CN and CO act
Heme in cytochromes
Must Know Very Important
The action of the respiratory chain inhibitor cyanide, which binds to heme in cytochrome oxidase (a3),
It prevents proton pumping by all three complexes.
Although complete inhibition of any one complex inhibits proton pumping at all of the complexes, partial inhibition of proton pumping can occur when only a fraction of the molecules of a complex contains bound inhibitor.
The partial inhibition results in a partial decrease of the maximal rate of ATP synthesis.
How does CN and CO act
Must Know Very Important
Cyanide binds to the Fe3+ in the heme of the cytochrome aa3 component of cytochrome c oxidase and prevents electron transport to O2.
Mitochondrial respiration and energy production cease, and cell death rapidly occurs.
The central nervous system is the primary target for cyanide toxicity.
How does CN and CO act
Must Know Very Important
Acute inhalation of high concentrations of cyanide (e.g., smoke inhalation during a fire) provokes a brief central nervous system stimulation followed rapidly by convulsion, coma, and death.
Acute exposure to lower amounts can cause light-headedness, breathlessness, dizziness, numbness, and headaches.
Cyanide Toxicity
Must Know Very Important
60
Cyanoglycosides such as amygdalin are present in edible plants such as almonds, pits from stone fruits (e.g., apricots, peaches, plums, cherries),sorghum, cassava, soybeans, spinach, lima beans, sweet potatoes, maize, millet, sugar cane, and bamboo shoots.
HCN is released from cyanoglycosides by beta-glucosidases present in the plant or in intestinal bacteria.
Small amounts are inactivated in the liver principally by rhodanase, which converts it to thiocyanate.
Cyanide Toxicity
Must Know Very Important
Case Report: A 28-month-old girl presented to the emergency department with sudden onset unconsciousness and seizure when she was eating apricot seeds with her father. Glasgow coma score was 4. She was transported to the intensive care unit and mechanical ventilation was begun. Gastric lavage was performed and pieces of apricot seeds were observed. On laboratory investigation, blood gas analysis revealed pH 6.8; paO2 80 mmHg; PaCO2 15 mmHg; HCO3 5.5 mmol/L; base excess –29.6 mmol/L; plasma lactate level 10 mmol/L; plasma glucose level 290 mg/dl. With the help of laboratory findings and knowing she had been eating apricot seeds, the patient was diagnosed with acute cyanide poisoning. After collecting a whole blood sample for measurement of cyanide level, cyanide antidote dicobalt edetate (Kelocyanor) was given 10 hours after the patient arrived at the hospital. Repeated arterial blood gas analysis showed that the difference between arterial and venous PO2 levels was 8 mmHg. The result of whole blood cyanide level was more than 3mg/L at hour 4 after presentation. Dicobalt edentate was repeated. The patient died on the 22th day of hospitalization, following supportive care in the intensive care unit.
What is mechanism of cyanide poisoning from seeds?
Am J Case Rep 2011; 12:70-72
Case 1
Cyanide for Cancer treatment
Cyanide for Cancer treatment
In the United States, toxic amounts of cyanoglycosides have been ingested as ground apricot pits, either as a result of their promotion as a health food or as a treatment for cancer. The drug Laetrile (amygdalin) was used as a cancer therapeutic agent, although it was banned in the United States because it was ineffective and potentially toxic.
Commercial fruit juices made from unpitted fruit could provide toxic amounts of cyanide, particularly in infants or children.
In countries in which cassava is a dietary staple, improper processing results in retention of its high cyanide content at potentially toxic levels.
Cyanide for Cancer treatment
Regulation of Electron transport and Adenosine Triphosphate Synthesis (ATP)
66
Increased Consumption of Adenosine Triphosphate Synthesis (ATP)
67
What Happens During Exercise
68
What Happens During Exercise
69
During exercise we use more ATP for muscle contraction, consume more oxygen, oxidize more fuel (which means burn more calories), and generate more heat from the electron-transport chain
What Happens During Exercise
Must Know Very Important
70
In skeletal muscles, the rates of ATP hydrolysis change dramatically as the muscle goes from rest to rapid contraction.
Even under these circumstances, ATP concentration decreases by only approximately 20% because it is so rapidly regenerated.
In the heart, TCA cycle enzymes provides an extra push to NADH generation, so that neither ATP nor NADH levels fall as ATP demand is increased.
The electron-transport chain has a very high capacity and can respond very rapidly to any increase in ATP use.
How do Skeletal and Cardiac Muscles react to Exercise
Must Know Very Important
71
What Happens During Exercise
72
To understand the regulation of ETC and ATP synthesis it is essential to understand the coupling between the electrochemical gradient and ATP synthesis.
Small Molecule Uncouplers
Protein Uncouplers
Electron Transport Chain and ATP Synthesis
Must Know Very Important
73
What is Coupling of Electron transport and Adenosine Triphosphate Synthesis (ATP)
74
Mitochondrial Matrix
Intermembrane Space
Complex I
NADH:CoQ
Oxidoreductase
ATP
Synthase
Complex IV
Cytochrome C
Oxidase
Complex III
Cytochrome b-c1
Oxidase
Complex II
Succinate Dehydrogenase
Cytochrome C
Synthesis of ATP from ADP and Pi
NAD+
Electrons
Protons
4H+
2H+
Coenzyme Q/Ubiquinone (CoQ / UQ)
Reduced form of
Cytochrome C
Cu
Cu
4H+
4 Protons from the intermembrane space are used by ADP and Pi to form one ATP Molecule
Must Know Very Important
75
The electrochemical gradient couples the rate of the electron-transport chain to the rate of ATP synthesis.
Because electron flow requires proton pumping, electron flow cannot occur faster than protons are used for ATP synthesis (coupled oxidative phosphorylation) or returned to the matrix by a mechanism that short-circuits the ATP synthase pore (uncoupling).
What is Coupling of Electron Transport Chain and ATP Synthesis
Must Know Very Important
76
As ATP chemical bond energy is used by energy-requiring reactions, ADP and Pi concentrations increase.
The more ADP is present to bind to the ATP synthase, the greater will be proton flow through the ATP synthase pore, from the intermembrane space to the matrix.
Thus, as ADP levels rise, proton influx increases, and the electrochemical gradient decreases .
What is Coupling of Electron Transport Chain and ATP Synthesis
Must Know Very Important
77
The Electron transport chain respond with increased proton pumping and electron flow to maintain the electrochemical gradient. The result is increased O2 consumption rapidly to any increase in ATP use.
The increased oxidation of NADH in the electron-transport chain and the increased concentration of ADP stimulate the pathways of fuel oxidation, such as the TCA cycle, to supply more NADH and FAD(2H) to the electron-transport chain.
What is Coupling of Electron Transport Chain and ATP Synthesis
Must Know Very Important
78
Uncoupling of Electron transport and Adenosine Triphosphate Synthesis (ATP)
79
Uncoupling of Electron transport and Adenosine Triphosphate Synthesis (ATP) – Small Molecule Uncoupler
Must Know Very Important
80
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
81
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
82
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
83
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
84
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
85
Uncoupling of Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
86
Which of these Drugs can cause uncoupling of Oxidative Phosphorylation
COOH groups have pKa ~3 -5
Must Know Very Important
87
When protons leak back into the matrix without going through the ATP synthase pore, they dissipate the electrochemical gradient across the membrane without generating ATP. This phenomenon is called uncoupling oxidative phosphorylation.
It occurs with chemical compounds known as uncouplers.
Uncoupling the ETS from oxidative phosphorylation generates heat as the due to the movement of proton, this ensures the consumption of oxygen and proton pumping attempt to maintain the electrochemical gradient which speeds up metabolism ?
Uncoupling Adenosine Triphosphate Synthesis from Electron Transport
Must Know Very Important
88
Uncoupling Mechanism and Weight Loss
89
Uncoupling of Electron transport and Adenosine Triphosphate Synthesis (ATP) – Protein Uncoupler
Must Know Very Important
90
Protein as Uncouplers
Must Know Very Important
91