Oxidative phosphorylation by Dr. Ashok KUmar J

for students and teachers

1. OXIDATIVE PHOSPHORYLATION Dr. Ashok Kumar J International Medical School Management and Science University Malaysia

2. Oxidation is defined as the removal of electrons Reduction as the gain of electrons Oxidation is always accompanied by reduction of an electron acceptor Fe 2+ Fe 3+ e - (Electron) Cu + Cu 2+ e - (Electron) Fe 2+ + Cu 2+ Fe 3 + + Cu + Dr. Ashok Kumar J; Professor; Biochemistry

3. Entire process is known as cell respiration Oxidation of metabolic fuels is essential to life Higher organisms dietary fuels are metabolized to: Carbon dioxide (CO2 ; Fully oxidized form of carbon) Water fully reduced form of oxygen Fuels Oxidized Electrons are transferred to coenzymes ETC ATP ADP CO 2 ½ O 2 H 2 O Dr. Ashok Kumar J; Professor; Biochemistry

4. Glycolysis Cytoplasm Pyruvic acid Krebs Cycle Electrons carried in NADH and FADH 2 Electron Transport Chain Mitochondrion Dr. Ashok Kumar J; Professor; Biochemistry Substrate level phosphorylation Oxidative phosphorylation

5. ELECTRON TRANSPORT CHAIN Dr. Ashok Kumar J; Professor; Biochemistry

6. Dr. Ashok Kumar J; Professor; Biochemistry Matrix A Cell One of Its Mitochondria A Crista Outer & Inner Membranes Intermembrane Compartment a b c MITOCHONDRIA

7. ELECTRON TRANSPORT CHAIN There are four different complexes Complex I, II, III, IV, Dr. Ashok Kumar J; Professor; Biochemistry

8. Dr. Ashok Kumar J; Professor; Biochemistry

9. Dr. Ashok Kumar J; Professor; Biochemistry Peter D. Mitchell United Kingdom Glynn Research Laboratories Bodmin , United Kingdom b. 1920 d. 1992 Mitchell’s hypothesis explains Mechanism of Oxidative phosphorylation

10. Dr. Ashok Kumar J; Professor; Biochemistry Energy from oxidation of components in the respiratory chain is coupled to the translocation of hydrogen ions (protons, H+) from the inside to the outside of the inner mitochondrial membrane . Complexes I, III, and IV acts as a proton pump Mitchell’s chemiosmotic theory

11. Dr. Ashok Kumar J; Professor; Biochemistry Complex I and complex III pumps 4 protons each Complex IV pumps 2 protons To i nter-membranous space 10 protons are pumped by the electron transport chain The transfer of two electrons from NADH+ H + to O 2 is accompanied by the outward pumping of 10 H + 10 protons are pumped out per NADH 4 must flow in to produce 1 ATP The proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate

12. Dr. Ashok Kumar J; Professor; Biochemistry ATP synthase ATP Synthesizing enzyme complex Two major components (F and F 1 ) F 1 in all aerobic organisms consists of - α 3 β 3 γδε subunits - binding sites for ATP and ADP - Catalytic site for ATP synthesis

13. Dr. Ashok Kumar J; Professor; Biochemistry P:O Ratio Number of moles of inorganic phosphate consumed per atom of oxygen utilized Phosphate group esterified P:O ratio = Electron pairs transferred Number of moles of inorganic phosphate consumed per atom of oxygen utilized

14. Dr. Ashok Kumar J; Professor; Biochemistry Paul Boyer (1964) proposed that a conformational change in the mitochondrial membrane protein leads to synthesis of ATP Paul Boyer hypothesis is now considered as - rotatory motor - engine driving model - binding change model To explain generation of ATP

15. Dr. Ashok Kumar J; Professor; Biochemistry In response to proton influx ‘ γ ’ subunit physically rotates This induces conformational change in the β 3 subunit β Subunit can adopt different conformations Loose ( L) conformation - ( ADP and Pi bind ) Tight ( T) conformation - (Tightly bound ATP) Open (O) conformation - (Release ATP)

16. [ATP] Dr. Ashok Kumar J; Professor; Biochemistry [ADP] [Pi] [NADH] [NAD + ] Increases rate of mitochondrial respiration Enhanced oxidative phosphorylation Increased ATP synthesis Cellular activity ATP is used ADP concentration increased Increased rate of catabolism of nutrients Mitochondrial ADP concentration has important role in regulating oxidative phosphorylation RESPIRATORY CONTROL ADP transport across the inner mitochondrial membrane can be inhibited by Atractyloside It can inhibit oxidative phosphorylation

17. Uncouplers Dr. Ashok Kumar J; Professor; Biochemistry Uncouplers is to dissociate oxidation in the respiratory chain from phosphorylation Compounds are toxic in vivo, causing respiration to become uncontrolled, since the rate is no longer limited by the concentration of ADP or Pi Uncouplers 2,4 dinitro phenol Dinitrocresol Pentachlorophenol CCCP (m- chlorocarbonyl - cyanide phenyl hydrazine)

18. Dr. Ashok Kumar J; Professor; Biochemistry Physiological uncouplers Thyroxine Long chain free fattyacids Unconjugated bilirubin Ionohores Valenomycin ( Ionophore for K+ ions) Nigercin ( Ionophore for K+ ions) Electron transport chain continues; energy is released as heat

19. Dr. Ashok Kumar J; Professor; Biochemistry Brown adipose tissue Characterized by - High content of mitochondria - High content of cytochrome - Well developed blood supply - Relatively rich in carnitine - Has low ATP synthase activity - High oxygen consumption - Involved mainly in metabolism particularly at times when heat generation is required Brown color is due to relatively high content of mitochondria

20. Dr. Ashok Kumar J; Professor; Biochemistry Tissue is extremely active in In some species in arousal from hibernation In animals exposed to cold ( non-shivering thermogenesis ) In heat production in the newborn animal Shown to be active in normal individuals Responsible for diet induced thermogenesis ‘Eat and do not get fat

21. Dr. Ashok Kumar J; Professor; Biochemistry Disorders of mitochondrial function About 100 polypeptides are required for oxidative phosphorylation 13 of these polypeptides are coded by mitochondrial DNA ( mtDNA ) and synthesized in the mitochondria Mitochondrial is maternally inherited (Mitochondria from the sperm do not enter the fertilized ovum) Mitochondrial DNA is about 10 times more susceptible to mutations than nuclear DNA

22. Dr. Ashok Kumar J; Professor; Biochemistry Characterized by - Accumulation of lactic acid - Myopathies - Cardiomyopathies - Encephalopathies Mitochondrial Myopathies Characterized by the presence of mitochondria with abnormal shape and size Typically results in muscle fibers appearing as ragged red fibers ( Muscle biopsy; Gomori trichrome or other stains) May result in cell death Particularly affect skeletal and cardiac muscles

23. Courtesy of Dr Ana Sotrel Gomori trichrome stain. Mitochondrial myopathy. There are several isolated muscle fibers with red granular staining and disruption of fiber structure.

24. MRI of the brain showing atrophy and enhancing low attenuation lesions. B . Gomori trichrome stain showing a ragged red fiber (black arrow) and two severely atrophic denervated fibers (white arrow). Mitochondrial encepha lomyopathy lactic acidosis stroke like episodes (MELAS) - Complex I defect; Lactic acidosis, strokes,myopathy,seizures , dementia

25. Leber's hereditary optic myopathy (LHON) Characterized by blindness in young males Caused by a single base mutation in NADH Coenzyme Q reductase Streptomycin induced deafness is also found to be due to a mutation in the mitochondrial rRNA Myoclonic epilepsy ragged red fiber disease (MERRF) Myoclonic epilepsy, myopathy , dementia Leigh's syndrome Complex I defect; Movement disorders

26. THANK YOU Dr. Ashok Kumar J; Professor; Biochemistry