Oxidative phosphorylation

Question 1

Give a brief outline of oxidative phosphorylation

Answer 1

The process by which the free energy derived from glucose and stored in other molecules such as NADH and FADH2 is used to generate ATP from ADP and inorganic phosphate.

Question 2

How are NADH and FADH2 used in the TCA cycle

Answer 2

The catabolism of glucose to pyruvate in glycolysis, and the further breakdown of pyruvate to CO2 and H20 in the Krebs cycle,
the electrons obtained from this oxidation of carbohydrates are transferred to the coenzymes NAD+ and FAD, forming NADH and FAD2

Question 3

How do we make sure NAD+ and FADH2 are continually available as electron acceptors?

Answer 3

For NADH and FADH2 to be readily available as electron acceptors, we need to make sure they are re-oxidized by 02.
This is where oxidative phosphorylation comes in.

Question 4

How are electrons transferred from reduced coenzymes to O2?

Answer 4

The stepwise flow of electrons through a chain of intermediate electron carriers, situated in the mitochondrial inner membrane

The electrons pass along the electron transport chain to cytochrome oxidase (complex 1V) where the final acceptor, O2, is reduced to water

Question 5

What happens to protons as electrons pass along the electron transport chain?

Answer 5

As electrons pass along the electron transport chain, protons are translocated out of the mitochondrial matrix across the inner membrane into the intermembrane space

Question 6

proton electrochemical gradient

Answer 6

3 of the 4 protein complexes in the electron transport chain use the energy released by electron flow to pump protons out of the inner mitochondrial matrix.
The resulting unequal distribution of protons generates a pH gradient and a proton electrochemical gradient across the mitochondrial inner membrane

Question 7

ATP synthase

Answer 7

the flow of electrons back across the membrane through ATP synthase (complex V), drives the synthesis of ATP from ADP and inorganic phosphate.

Question 8

what is the ATP produced as a result of ATP synthesis used for?

Answer 8

Used to do work such as maintaining ion-gradients, transporting substances and proteins across membranes, protein synthesis, DNA synthesis, and muscle contraction.

Question 9

what is an oxygen electrode used for?

Answer 9

Electron flow and the resulting reduction of oxygen by suspensions of isolated mitochondria can be demonstrated in a laboratory using an oxygen electrode

Question 10

what is the trace on an oxygen electrode a measure of

Answer 10

the oxygen content of the reaction mixture

Question 11

what are the first steps in analyzing 02 consumption by mitochondria in the presence of an excess of ADP

Answer 11

1. Pipette reaction medium and inorganic phosphate into the oxygen electrode cham ber and stir.

Question 12

what do stirring of the reaction medium and inorganic phosphate ensure

Answer 12

that the mixture is fully oxygenated.

Question 13

what does the right hand side of the trace indicate

Answer 13

maximum dissolved oxygen

Question 14

what was added to the Oxygen electrode after reaction medium and inorganic phosphate, and what happened

Answer 14

Mitochondria were added after one minute. The pen moved to the left indicating oxygen being used up.
This is the blank rate and represents
respiration and ATP synthesis using substrates and ADP that were present in the
mitochondria when they were isolated.
After a further minute succinate is added
to the chamber.

Question 15

what happens after the additions of succinate to the mixture

Answer 15

rate of oxygen uptake increases, as electrons from the added succinate, move down the electron transport chain to O2

Question 16

what happens after the addition of 100ML ADP to the mixture

Answer 16

One minute
later the addition of ADP to the chamber causes a sudden burst of oxygen
uptake as the ADP is converted into ATP.
The mitochondria are now actively
respiring i.e. using O2 and making ATP.

Question 17

what is state 3 respiration

Answer 17

The mitochondria are now actively
respiring i.e. using O2 and making ATP. This actively respiring state is called
“State 3” respiration.

Question 18

why does the trace curve upwards after about 2.5 min

Answer 18

the rate of oxygen consumption is slowing down as oxygen becomes limiting

Question 19

how do we know respiration is coupled to ATP synthesis

Answer 19

we added succinate to the oxygen electrode chamber as before, however this time we added a limiting amount of ADP, 25 microlitres instead of 100 microlitres.
The mitochondria are now actively
respiring i.e. using O2 and making ATP. This actively respiring state is called
“State 3” respiration.

Question 20

Respiratory control ratio

Answer 20

The ratio [State 3 rate]:[State 4
rate] is called the Respiratory Control Ratio (RCR) and indicates the tightness
of the coupling between respiration and phosphorylation.

It is the rate of oxygen consumption in state 3 divided by the rate of oxygen consumption in state 4.

Question 21

Consider the change from State 3 to State 4 in Figure 5. Why does
oxygen consumption decrease when ADP becomes limiting?

Answer 21

In the absence of ADP, the flow of protons through the ATP synthase (Complex V) cannot occur because the movement of H+ through the complex into the matrix is obligatorily coupled to the synthesis of ATP from ADP and phosphate.

Question 22

What happens to the proton electrochemical gradient in the absence of ADP

Answer 22

The gradient quickly reaches and is maintained at maximum value
This value corresponds to the free energy required to make ATP from ADP and phosphate.

Question 23

Under these conditions what happens to
electron flow through the chain and hence oxygen consumption by the
mitochondria?

Answer 23

Since electron flow through the chain is obligatorily coupled to pumping protons from the matrix across the inner membrane into the intermembrane space, however, if the proton electrochemical gradient is already at a maximal value the system is already fully charged, and further proton pumping cannot occur.

Effectively under these conditions the proton electrochemical gradient
applies an energetic back pressure preventing further H+ pumping by the
electron transport chain.

This in turn decreases the flow of electrons
through the chain which of course leads to a decrease in oxygen
consumption since all electron flow is ultimately transferred to molecular
oxygen which is reduced to water

Question 24

chemiosmotic theory

Answer 24

The chemiosmotic theory explains how ATP is generated in the mitochondria via the electron transfer chain (ETC).

Question 25

chemical uncouplers

Answer 25

ie results in protons going back into the mitochondrial matric without forming ATP

molecules that can ferry protons
across the mitochondrial inner membrane from the intermembrane space into
the matrix. Thus an uncoupler facilitates protons moving back into the matrix
without going through the ATP synthase
The result of an uncoupler is to collapse the proton electrochemical gradient, which means H+ pumping can occur without any back pressure from the gradient.

Question 26

what happens to the free energy available when an uncoupler is added

Answer 26

Free energy available is not linked to the production of ATP but is lost as heat.

Question 27

where do electrons from succinate enter?

Answer 27

Complex II

Question 28

Where do electrons from NADH enter

Answer 28

Complex I

Question 29

sources of NADH used in the experiment

Answer 29

glutamate plus malate

Question 30

Rotenone blocks electron transport when NADH is the source of electrons
but not when succinate is the source of electrons.
Explain

Answer 30

Rotenone blocks electron transport between Complex I and Complex II and
electrons from succinate by-pass the block.

Question 31

When antimycin is added to mitochondria in State 3 respiration
with succinate as substrate respiration stops and cannot be started by
the addition of either succinate or glutamate plus malate. Where in the
electron transport chain does antimycin block?

Answer 31

If succinate cannot by-pass the antimycin block it must be beyond
Complex II (in fact the block is at Complex III)