Objectives: Respiratory Metabolism and Free Radical Detoxification Exercises 5-5, 5-6, 5-7

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1

Indicate the function of cytochrome c oxidase in a bacterial cell

It is the last enzyme in the chain, Complex IV, and is so named because it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm, to the oxygen inside the cell.

2

Understand why all aerobic bacteria are not oxidase positive

Some bacteria are capable of aerobic respiration but have a different terminal oxidase system and give a negative result for the oxidase test.

3

reduction

the gain of electrons or a decrease in oxidation state by a molecule, atom, or ion.

4

oxidation

the loss of electrons or an increase in oxidation state by a molecule, atom, or ion.

5

oxidase

any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen (O2) as the electron acceptor. In these reactions, oxygen is reduced to water (H2O) or hydrogen peroxide (H2O2).

6

catalase

Enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen.

7

peroxidase

Peroxidases are the plant enzymes that use peroxide to break down bacteria and other harmful material. They are located in the granules of neutrophils.

8

Understand why aerotolerants would not use catalase. Identify an example of this characteristic.

They do not use oxygen, so they have no need to use catalase to convert hydrogen peroxide into water and oxygen.

9

Identify sources of error that might produce false positives in the oxidase test

Once the vial has been opened, oxidase reagent will turn purple over time due to oxygen in the air. To avoid false positives, be sure to: 1) use oxidase reagent that is still clear and 2) to read your test quickly once you have performed it.

10

Explain the function of zinc in the test for nitrate reduction

The addition of zinc dust is used to determine if the nitrates were reduced past nitrite stage. If a red color develops after the addition of zinc, the nitrates were not reduced to nitrites. If there is no color after the addition of zinc, the nitrates in the media were reduced beyond nitrites to ammonia or nitrogen gas.

11

Understand the purpose of a durham tube in the nitrate test

Tests for the presence of gas

12

Understand why the presence of a bubble in the durham tube of a nitrate broth is not definitive for nitrate reduction when working with an organism capable of fermentation

The source of the gas is unknown

13

Understand the relationship between the enzyme nitrate reductase and anaerobic respiration

anaerobic respiration involves the reduction of an inorganic molecule, something other than oxygen, as a final electron acceptor in the ETC. In this case, it is nitrogen that gets reduced and the nitrate reductase is what reduces nitrate to nitrite.

14

Describe how the toxins hydrogen peroxide and superoxide radicals may be formed and how bacterial cells convert them to none toxic substances

Hydrogen peroxide and superoxides are produced when aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway during degradation of carbohydrates for energy production. Accumulation of these substances will result in death of the organism unless they can be enzymatically degraded. Catalase or peroxidase rapidly degrades H2O2 into H2O and O2.

All aerobes and some facultative anaerobes are catalase positive. Superoxide dimutase is the enzyme responsible for degradation of the superoxides in catalase negative aerobic organisms. Strict anaerobes are not able to produce these enzymes. Therefore, they cannot be cultivated in the presence of oxygen.

15

Theory behind the nitrate reduction test

Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate. In this test, if the organism reduces he nitrate to nitrite, the nitrite reacts with the reagents sulfanilic acid and alpha-naphthylamine to produce a red color. No color after addition of these reagents indicates nitrates are not reduced or nitrites were reduced further. If the organism possesses a strong nitrate reductase it can further break down nitrites to ammonia or molecular nitrogen. The addition of zinc dust is used to determine if the nitrates were reduced past nitrite stage. If a red color develops after the addition of zinc, the nitrates were not reduced to nitrites. If there is no color after the addition of zinc, the nitrates in the media were reduced beyond nitrites to ammonia or nitrogen gas.