Oxidation of glucose and fatty acids to CO 2 Respiration involves the oxidation of glucose and other compounds to produce energy. C 6 H 12 O P i + 36 ADP + 36 H + 6 CO ATP + 42 H 2 0

Step I: glycolysis Glucose + 2ADP 2 pyruvate + 2ATP

key enzyme: 1. phosphofructokinase (step 3 in Figure 1). stimulated by high ADP levels inhibited by high ATP and citrate. 2. glyceraldehyde 3-phosphate dehydrogenase (step 5). can be inhibited by NEM.

Step II: Mitochondria in respiration Transport of pyruvate into the mitochondria and its subsequent oxidization by O 2 to produce CO 2, generating 34 of the 36 ATP molecules produced during respiration. 1. Structure and function of mitochondria

(1). Generation of acetyl CoA. Ethanol, a substrate that you will be testing in today’s lab, can also be converted to acetyl CoA.

(2). Kreb’s cycle/citric acid cycle. mitochondria matrix Acetyl CoA is oxidized to yield CO 2 and reduced coenzymes. Three reactions in the Krebs cycle reduce the coenzyme NAD + to NADH, and one reduces the coenzyme FAD to FADH 2. The reduced coenzymes (NADH and FADH 2 ) store the energy released in glucose oxidation. succinate, malate

3. Oxidative phosphorylation Oxidative phosphorylation is the process by which the energy stored in NADH and FADH 2 is used to produce ATP. A. Oxidation step----electron transport chain B. Phosphorylation step NADH + H + + O 2 NAD + + H 2 O 1 2 FADH + O FAD + H 2 O

(1). Generation of acetyl CoA. Ethanol, a substrate that you will be testing in today’s lab, can also be converted to acetyl CoA.

Electrons are moved from molecules with low reduction potential (low affinity for electrons) to molecules with successively higher reduction potential (higher electron affinity).

Electrons are transferred through four large protein complexes and two smaller proteins, each of which has an electron carrier group. At complex I, III and IV, the energy released during electron transfer is used to move H + ions from the matrix to the inner mitochondrial space, generating a gradient of protons across the membrane.

B. Phosphorylation step Peter Mitchell’s chemiosmotic hypothesis: The energy stored in an electrochemical gradient across the inner mitochondrial membrane could be coupled to ATP synthesis. F 0 F 1 ATP synthase Movement of protons down the electrochemical gradient through a channel between the a and c subunits releases energy that is coupled to the rotation of the c,  and  subunits. This rotation causes a conformational change in the  and  subunits that promotes synthesis of ATP from ADP and P i.

Other factors involved in oxidative phosphorylation Transporters and shuttles move small molecules across the inner mitochondrial membrane. Transporters include the ATP/ADP antiporter, which transports ADP into the matrix and ATP out of the matrix. In addition there are separate transporters for P i and pyruvate.

(1). Generation of acetyl CoA. Ethanol, a substrate that you will be testing in today’s lab, can also be converted to acetyl CoA.

Inhibitors and substrates Rotenone (also amytal): inhibits complex I. Antimycin A:inhibits cytochrome c. Sodium azide:inhibits complex IV. Oligomycin: inhibits the F 0 F 1 ATP synthase. Atractyloside: inactivates the ATP/ADP antiporter. glutamate malate: reduces NAD + to NADH Succinate: reduces FAD to FADH 2 ascorbate/TMPD enters the electron transport chain in complex IV. Inhibitors: Substrates:

Respiratory Control The rate of oxidative phosphorylation depends on the levels ADP/ATP in mitochondria– ie. oxidation of NADH and FADH 2 only occurs if there is a there is ADP and P i available to generate ATP. Oxidation of NADH and FADH 2 H + gradient ADP/ATP levels

Examining the phenomenon of respiratory control: Inhibitors: Atractyloside, Oligomycin CaCl 2 : stimulates oxidative phosphorylation and ATP production. Uncouplers--DNP DNP acts to shuttle H + across the inner mitochondrial membrane, causing a dissipation of the H + gradient, thus uncouples the process of oxidation and phosphorylation. The uncouplers overcome respiratory control, but do not stop oxidation/the electron transport chain. The energy released from the oxidation of NADH/FADH 2 is dissipated as heat. An uncoupler called thermogenin occurs naturally in brown-fat tissue and functions to uncouple oxidation and phosphorylation, enhancing heat generation.

This week’s experiments: measuring O 2 consumption 1. Yeast respiration lab: Goal: learn how to measure O 2 consumption. Compare O 2 consumption by normal and starved yeast. 2. Mitochondria respiration lab: Examine the effects of various inhibitors and substrates on the rate of respiration. Determine the identity of your unknown (think what substrates you need to add and in what order together with the unknown).