Electron Transport and oxidative phosphorylation (ATP Synthesis) Dr. Howaida Nounou Biochemistry department Sciences college

Electron Transport and oxidative phosphorylation (ATP Synthesis) Dr. Howaida Nounou Biochemistry department Sciences college

The Metabolic Pathway of Cellular Respiration All of the reactions involved in cellular respiration can be grouped into three main stages Glycolysis occurs in cytoplasm The Krebs cycle occurs in matrix of mitochondria Electron transport occurs across the mitochondrial membrane

Cytosol High-energy electrons carried by NADH Mitochondrion High-energy electrons carried mainly by NADH Glucose Glycolysis 2 Pyruvic acid Krebs Cycle Electron Transport A Road Map for Cellular Respiration

RedoxReactions Chemical reactions that transfer electrons from one substance to another are called oxidation-reduction reactions REDOX short for oxidation-reduction reactions

REDOX FACTS Reduction-oxidation (redox) couple: pair of molecules of which one is reduced and the other is oxidized (e.g., lactate-pyruvate, NADH-NAD +, and FADH 2-FAD) each pair constitutes a half reaction the reductantof one pair donateselectrons and the oxidant of the other pair accepts the electrons Red1 + Ox2 Ox1 + Red2

REDOX FACTS A:H A Reductant Oxidant + e- B B:H Oxidant + e- Reductant (acceptor) (donor) both oxidation and reduction must occur simultaneously ²

Electrons can move through a chain of donors and acceptors In the electron transport chain, electrons flow down a gradient Electrons move from a carrier with low reduction potential (high tendency to donate electrons) toward carriers with higher reduction potential (high tendency to accept electrons)

Oxidative process Phosphorylation process inner membrane O 2 e - H 2 O H + ADP+ Pi H + ATP outer membrane matrix intermembrane space Figure: Essential features of oxidative phosphorylation Redox reactions of respiratory chain use electrons to reduce oxygen to water Energy generated moves protons from matrix to intermembrane space Inward movement of protons recovers this energy to promote formation of ATP in the matrix.

Succinate II I NADH Coenzyme Q III Cytochrome C IV ½ O 2 electron flow The components of the RC are arranged in order of increasing redox potential

RC exists as four large, multisubunit protein complexes The respiratory electron transport chain complex Iis a NADHubiquinone reductase complex IIis succinate dehydrogenase (part of the TCA cycle) complex IIIis the ubiquinone -cytochrome c reductase complex IVis cytochrome oxidase

Figure: Complex Iof the respiratory chain that links NADH and coenzyme Q. NADH Dehydrogenase accepts 2e--from NADH and transfers them to ubiquinone (coenzyme Q), an electron carrier Uses two bound cofactors to accomplish this: FMN(Flavin mononucleotide) and 6 iron-sulfur (Fe-S) protein Pumps 4 H + out to inter-membrane space

Complex II: Succinate-CoQ reductase Prosthetic groups: FAD; Fe-S Succinate FAD SDH Fumarate FADH 2 CoQ SDHis succinic dehydrogenase an enzyme of the citric acid cycle (associated with membrane) 2 e-transferred from succinate to CoQ 1 mole FADH 2 produced

Electrons from complex I or II Complex III: cytochrome reductase Prosthetic groups: heme b; heme c 1 ; Fe-S CoQ cyt b/cyt c 1 cyt c Figure:Complex IIIof the respiratory chain linking CoQ and cytochrome C. Is composed of cytochome b, cytochrom C1 and iron sulphur proteins Accepts e-from coenzyme Q and transfers e-to cytochrome c coupled with the transfer of protons from the matrix to the intermembrane space

Figure:Complex IV-cytochrome oxidase- reducing oxygen to water Contains cytochromes a/a3 and 2 Cu ions involved in e-transfers Cytochrome oxidase passes electrons from cytochrome c through a series of heme groups and Cu ions to O 2, reducing it to H 2 O (end product) and pumping one proton into the intermembrane space for each e-

Coenzymes and cytochromes in the complexes act as e-donors & acceptors

Flavin MonoNucleotide(FMN),in Complex I, functions like FAD (Flavin adenine dinucleotide, which is an electron acceptor that helps electron transfer during Krebs Cycle and Electron Transport Chain in cellular respiration). iron-sulfur (Fe-S proteins): Fe-S centers transfer e-in Complexes I, II and III Coenzyme Q(ubiquinone), lipid soluble, floats in the membrane and doesn t require protein Cytochromes (b, c 1, c, a, a 3 ; contain heme): transfer e - in Complexes III and IV, Cytc is the only soluble cytochrome NAD+, FMN, CQ are carriers of e- and hydrogen while cytochromes are carriers of electrons only.

Summary of redox complexes of the electron transport chain Complex designation Functional groups Function Poisons I NADH-Q reductase FMN (flavin mono- nucleotide); Fe-S oxidizes NADH to NAD + ; transfers electrons to coenzyme Q Rotenone II Succinate- Q reductase FAD; Fe-S oxidizes succinate to fumarate with reduction of FAD to FADH 2 ; electron transfer to CoQ III - Cytochrome reductase heme b; heme c 1 ; Fe-S transfers electrons between coenzyme Q and cytochrome C (C becomes reduced) Antimycin A IV - Cytochrome C oxidase heme a-a 3 ; Cu oxidizes cytochrome C; reduces ½O to H 2 2 O Carbon monoxide Cyanide

Overall Reaction. NADH + H + + ½ O 2 NAD + + H 2 O Taking into account the protons pumped out: complex I 4 H + complex III 4 H + complex IV 2 H + The equation for electron transfer is: NADH + 11 H + matr + ½ O 2 NAD + + 10 H + intermem + H 2 O

Oxidative Phosphorylation

Definition Process in which ATP is formed as a result of transfer of electrons from NADH or FADH 2 by a series of electron carriers The electron transport chain generates no ATP directly. Rather, its function is to break the large free energy drop from food to oxygen into a series of smaller steps that release energy.

ATP yield Only 4 of 38 ATP ultimately produced by respiration of glucose are derived from substrate-level phosphorylation (2 from glycolysis and 2 from TCA) The vast majority of the ATP (90%) comes from the energy in the electrons carried by NADH and FADH 2

ATP-synthase (complex V),present in the inner mitochondrial membrane,actually actually makes ATPfrom ADP and P i. ATP used the energy of an existing proton gradient to power ATP synthesis. This proton gradientdevelops develops between the intermembrane space and the matrix. This concentration of H + is the proton-motive force. 22

The ATP synthase molecules are the only place that will allow H + to diffuse back to the matrix (exergonic flow of H + ). This flow of H + is used by the enzyme to generate ATP a process called chemiosmosis. Chemiosmosis:(osmos = push) is the oxidative phosphorylation that results in ATP production in the inner membrane of mitochondria.

Properties of ATP Synthase Multisubunit transmembrane protein Molecular mass = ~450 kda Functional units F 0 : water-insoluble transmembraneprotein (up to 8 different subunits) F 1 : water-soluble peripheral membrane protein (5 subunits),contains the catalytic site for ATP synthesis Flow of 3 protons through ATP synthase leads to phosphorylation of 1 ADP

During respiration, most energy flows from glucose -> NADH-> electron transport chain -> proton-motive force -> ATP. One six-carbon glucose molecule is oxidized to 6 CO 2 molecules. Some ATPis produced by substrate-level phosphorylationduring glycolysis and the Krebs cycle, but most ATPcomes from oxidative phosphorylation (through electron transport chain). Each NADHfrom the Krebs cycle and the conversion of pyruvate contributes enough energy to generate a maximum of 3 ATP. Each FADH 2 from the Krebs cycle can be used to generate about 2ATP. Energy produced in electron transport chain gives a maximum yield of 34 ATP by oxidative phosphorylation via ATP-synthase.

These compounds prevent the passage of e-by binding a component of the ETC blocking the oxidation/reduction reaction

Inhibitors of ElectronTransport Chain and oxidative phosphorylation Be familiar with the actions of inhibitors in the red boxes

Uncouplers: Compounds that increase the permeability of the inner mitochondrial membrane to protons. Protons renters the matrix at sites other than ATP synthase through holes made by these compounds. These compounds have no effect on electron transport chain, but they uncouple oxidative phosphorylation. The energy produced by the transport of electron is released as heat rather than being used to synthesis ATP.

Examples: 2,4 dinitrophenol, dinitrocresol, pentachlorophenol, thyroxine, calcium, mchlorocarbonylcyanide phenyl hydrazone (cccp).

Cytosol Adding Up the ATP Mitochondrion Glycolysis 2 Glucose Pyruvic acid 2 Acetyl- CoA Krebs Cycle Electron Transport Maximum per glucose: by direct synthesis by direct synthesis by ATP synthase

Protein complex Electron carrier Inner mitochondrial membrane Electron flow Electron transport chain ATP synthase 31

Food Polysaccharides Fats Proteins Sugars Glycerol Fatty acids Amino acids Amino groups Glycolysis Acetyl- CoA Krebs Cycle Electron Transport 32