The respiratory chain and Oxidative phosphorylation By Dr. Manal Louis Assistant professor of Biochemistry

By the end of this lecture, the student should be able to: 1. Explain the chemiosmotic theory. 2. Determine the inhibitors of the resp. chain.

Electron Transport Chain

How is oxidation coupled to production of ATP? Chemiosmotic Model How is oxidation coupled to production of ATP? Peter Mitchell Proposed chemiosmotic hypothesis 1920-1992

Chemiosmotic Model The theory postulates that the energy obtained from oxidation is coupled to the translocation of protons H+ from the mitochondrial matrix to the inter- mitochondrial space. Complexes I, III, IV act as proton pumps.

So an electrochemical gradient is created across the inner mitochondrial membrane. (outside is +ve, inside is negative)

F0 F1 Scattered on the surface of the inner mitochondrial membrane are units of the ATP-synthase.

Step wise production of Energy in the form of ATP is a physiological behavior

Chemiosmotic Model Several findings support the chemiosmotic theory: Addition of protons (acid) to the external medium of mitochondria stimulates ATP production. Oxidative Phosphorylation does not occur in case of solubilizing mitochondrial membranes. Uncouplers.

P:O Ratio If we start from NADH = 3:1 If we start from FADH2 = 2:1 No. of ATP molecules produced No. of Oxygen atoms consumed If we start from NADH = 3:1 If we start from FADH2 = 2:1 Uncoupler = zero

Respiratory control Mainly by availability of ADP. (ATP/ADP transporters may be rate limiting at certain times). Availability of electrons: NADH/NAD ratio or FADH2/FAD. 3. Availability of O2.

Inhibitors of the respiratory chain These compounds abolished the coupling between oxidation and phosphorylation through increasing the permeability of the IMM Failure of formation of the electrochemical gradient ATP formation stops while oxidation proceeds. A- Uncouplers

What is the mechanism of uncouplers? They act by transporting H+ to inside the mitochondria without passing through F₀F1 And energy is released as heat. What is the mechanism of uncouplers?

Some endogenous compounds when increased: Bilirubin and Thyroxine. A- Uncouplers 2,4 dinitrophenol Dinitrocresol Snake venoms phospholipases Some endogenous compounds when increased: Bilirubin and Thyroxine.

5-Thermogenine It is considered as a physiological uncoupling protein. It is present in the brown adipose tissue of newly born, some people and hibernating animals. It allows protons to pass the mitochondrial matrix without passing F0-F1 complex. No ATP is formed and energy is released in the form of heat.

Inhibitors of the respiratory chain B- inhibitors of ETC proper: These inhibitors act on the three sites that produce the electrochemical gradient: Complex I is inhibited by barbiturates and the insecticide rotenone. Complex III is inhibited by the antibiotic, antimycin A. Complex IV is inhibited by H2S, CO and cyanide ( ATP synthesis especially in the CNS causing Death).

Inhibitors of the respiratory chain C-Inhibitors of phosphorylation: Completely blocks F₀ so it inhibits ATP synthesis Example: oligomycin (antibiotic) D- ATP/ADP transporters inhibitors This is achieved by the compound atractyloside

Derived from mother’s ovumA Mitochondrial DNA Circular DNA Derived from mother’s ovumA It contains 37 genes 13 proteins of ETC 22 tRNA 2 rRNA

Mitochondrial abnormalities Mutations in mitochondrial DNA Mitochondrial Myopathies It usually occurs in mitochondria of muscles resulting in myopathies characterized by muscular weakness, cramping and wasting. Inborn errors of mitochondrial DNA is only inherited from mothers.

MCQ Electron transport and phosphorylation can be uncoupled by compounds that increase the permeability of the inner mitochondrial membrane to (A) Electrons (B) Protons (C) Uncouplers (D) O2 (E) ATP

An uncoupler of oxidative phosphorylation such as dinitrophenol (A) Inhibits electron transport and ATP synthesis. (B) Allow electron transport to proceed without ATP synthesis. (C) Inhibits electron transport without impairment of ATP synthesis. (D) Specially inhibits cytochrome b. (E) Specially inhibits cytochrome c.

Reduced NADH generated in the cytoplasm is able to pass its electrons to oxygen through the mitochondrial electron transport system by which of the following mechanisms? A) A specific transport system enables NADH to pass through the mitochondrial membrane. B) NADH reduces FAD to FADH2, which then passes through the mitochondrial membrane. C) NADH reduces dihydroxyacetone phosphate to glycerol phosphate, which can pass through the mitochondrial membrane. D) NADH reduces membrane-bound NADP to NADPH, which passes electrons to coenzyme Q. E) NADH oxidizes aspartate to oxaloacetate, which can enter the mitochondria.

Which of the following statements about the electron transport chain is correct? a) The electron transport chain is made up of a chain of electron carriers with decreasing electron affinity. b) The electron transport chain is made up of a chain of electron carriers with increasing redox potential. c) The electron transport chain is made up of a chain of electron carriers with decreasing oxidising power. d) The electrons transferred from carrier to carrier in the electron transport chain gain heat.

How does atractyloside affect mitochondrial respiration? ... If you isolate mitochondria and place them in buffer with a low pH, they begin to manufacture ATP. Why?

Loss of electrons from one substance is called: a-Oxidation b-Reduction c-Dehydrogenation d-Phosphorylation e-Condensation

ATP- Synthase (Which is a membrane protein complex) is powered by: a- The flow of hydrogen ions b- ATP c- ADP d- Pi E- O2

Which of the following is oxidized by the respiratory chain? a. NADPH b. Acetyl CoA. c. Oxygen. d. NAD+. e. NADH

Coenzyme Q is correctly described by all except: Reduced by complex I & II Transfers electrons to complex III Is lipid soluble Carry proton & electron Accepts reducing equivalent from cytochrome aa3

The rate of Respiratory chain is mainly controlled by the concentration of: a. ADP b. NAD+ c. Inorganic phosphate. d. NADH. e. FADH2

When dinitrophenol is added to mitochondria, it: decreases the flow of electrons through cytochrome oxidase. B. results in an increase in the intra-mitochondrial ratio of ATP/ADP. C. uncouples oxidative phosphorylation. D. increases the rate of ATP formation.

Cyanide toxicity result from inhibition of: NADH: Q reductase Cytochrome oxidase Cytochrome reductase PDH Malate dehydrogenase