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 6 + 6 0 2.

Oxidation of glucose and fatty acids to CO2

Respiration involves the oxidation of glucose and other compounds to produce energy.

C6H12O6 + 6 02 + 36 Pi + 36 ADP + 36 H+

6 CO2 + 36 ATP + 42 H20

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 O2 to produce CO2, 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 CO2 and reduced coenzymes.

Three reactions in the Krebs cycle reduce the coenzyme NAD+ to NADH, and one reduces the coenzyme FAD to FADH2.

The reduced coenzymes (NADH and FADH2) 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 FADH2 is used to produce ATP.

A. Oxidation step----electron transport chain

B. Phosphorylation step

NADH + H+ + O2NAD+ + H2O

12

FADH + O212

FAD + H2O

(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.

F0F1 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 Pi.

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 Pi 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 F0F1 ATP synthase.

Atractyloside: inactivates the ATP/ADP antiporter.

glutamate malate: reduces NAD+ to NADHSuccinate: reduces FAD to FADH2

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 FADH2 only occurs if there is a there is ADP and Pi available to generate ATP.

Oxidation of NADH and FADH2

H+ gradient ADP/ATP levels

Examining the phenomenon of respiratory control:

Inhibitors: Atractyloside, Oligomycin

CaCl2: 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/FADH2 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 O2 consumption

1. Yeast respiration lab: Goal: learn how to measure O2 consumption.

Compare O2 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).