1. Electron Transport Chain (ETC) & Oxidative Phosphorylation COURSE TITLE: BIOCHEMISTRY 2 COURSE CODE: BCHT 202 PLACEMENT/YEAR/LEVEL: 2nd Year/Level 4, 2nd Semester M.F.Ullah, Ph.D

2. Electron microscopic studies by George Palade and Fritjof Sjöstrand revealed that mitochondria have two membrane systems: 1.An outer membrane 2.An extensive, highly folded inner membrane The inner membrane is folded into a series of internal ridges called cristae. Hence, there are two compartments in mitochondria: (1)the intermembrane space between the outer and the inner membranes and (2) the matrix, which is bound by the inner membrane Oxidative phosphorylation takes place in the inner mitochondrial membrane, in contrast with most of the reactions of the citric acid cycle and fatty acid oxidation, which take place in the matrix. The outer membrane is quite permeable to most small molecules and ions because it contains many copies of mitochondrial porin, a 30 35 kd pore forming protein also known as VDAC, for voltage-dependent anion channel. Structure of Mitochondria

3. The inner membrane is intrinsically impermeable to nearly all ions and polar molecules. A large family of transporters shuttles metabolites such as ATP, pyruvate, and citrate across the inner mitochondrial membrane. Inner membrane is the site of electron transport chain and oxidative phosphorylation. The two faces of this membrane will be referred to as the matrix side and the cytosolic side (the latter because it is freely accessible to most small molecules in the cytosol). In prokaryotes, the electron-driven proton pumps and ATP-synthesizing complex are located in the inner cytoplasmic membrane.

4. The energy derived from the metabolic reactions is conserved through electron transfer to NAD+ and FAD forming NADH and FADH2: Glycolysis In cytosol produces 2 NADH/glucose Pyruvate dehydrogenase reaction In mitochondrial matrix 2 NADH / glucose Kreb’s cycle In mitochondrial matrix 6 NADH and 2 FADH 2 / glucose 1 Glucose= 2 Pyruvate

5. When NADH and FADH2 are re-oxidized to NAD+ and FAD, the electrons released from them are transferred through a chain of electron carrier complexes (redox proteins) by Oxidation-reduction reactions and ultimately delivered to oxygen forming H2O. This chain of electron carriers is known as ETC (Electron Transport Chain) which is located in the inner mitochondrial membrane. During this transfer of electron from one carrier to another energy is conserved (stored) in the form of a proton gradient (proton motive force;PMF) and this energy is utilized in the synthesis of ATP (oxidative phosphorylation). Energy stored in NADH & FADH2 as electrons from the metabolic pathways is used for ATP synthesis by the process of oxidative phosphorylation

6. The Electron Transport Chain or Respiratory Chain Consists of Four Complexes Basically, the electron transport chain contains three large protein complexes (I, III, and IV) that span (Cross) the inner mitochondrial membrane and one small complex (II) that does not span the membrane but remain bound to the inner side. Complex I- NADH dehydrogenase (electron carrier and proton pump) Complex II- succinate dehydrogenase (electron carrier and NOT a proton pump) Complex III- cytochrome c oxidoreductase, (electron carrier and proton pump) Complex IV- cytochrome c oxidase (electron carrier and proton pump) CoQ is a small electron carrier that links and transfers electron from Complex I or Complex II to Complex III (NOT a proton pump) Cytochrome C is a small electron carrier that links and transfers electron from Complex III to Complex IV (NOT a proton pump) As electrons pass through the membrane spanning complexes (I, III and IV) in a series of oxidation–reduction reactions, protons are transferred from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. Thus these complexes are also called as proton pump. Succinate-Q reductase (Complex II) does not pump protons.

7. Path of electrons through ETC: NADH electrons------------ Complex I-------Complex III--------Complex IV------O 2 FADH2 electrons------------ Complex II-------Complex III--------Complex IV------O 2

8. Most of the energy from oxidation of fuels in the TCA cycle and other pathways is conserved in the form of the reduced electron-accepting coenzymes, NADH and FAD(2H). The electron transport chain oxidizes NADH and FAD(2H), and donates the electrons to O2, which is reduced to H2O. Energy from electron transfer from NADH/FADH2 to O2 generates proton gradient (PMF) which is used for phosphorylation of adenosine diphosphate (ADP) to ATP by ATP synthase (F0/F1 ATPase). The process is called oxidative phosphorylation as oxygen is required for such phosphorylation generating ATP. The net yield of oxidative phosphorylation is approximately 2.5 moles of ATP per mole of NADH oxidized, or 1.5 moles of ATP per mole of FAD(2H) oxidized. Oxidative phosphorylation 1 ATP = 4 H+ pumped by ETC 1NADH pumps 10 H+ Therefore 10/4= 2.5 1NADH=2.5 ATP 1FADH2 pumps 6 H+ Therefore 6/4= 1.5 1 FADH2= 1.5 ATP

10. The process of oxidative phosphorylation is based on the chemiosmotic hypothesis. Chemiosmotic hypothesis proposes that the energy for ATP synthesis is provided by an electrochemical gradient across the inner mitochondrial membrane. This electrochemical gradient is generated by the components of the electron transport chain, which pump protons across the inner mitochondrial membrane as they sequentially accept and donate electrons. The final acceptor is O2, which is reduced to H2O. See the animation at: http://www.youtube.com/watch?v=kN5MtqAB_Ychttp://www.youtube.com/watch?v=kN5MtqAB_Yc Chemiosmosis