Oxidative phosphorylation Flashcards
Give a brief outline of oxidative phosphorylation
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.
How are NADH and FADH2 used in the TCA cycle
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
How do we make sure NAD+ and FADH2 are continually available as electron acceptors?
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.
How are electrons transferred from reduced coenzymes to O2?
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
What happens to protons as electrons pass along the electron transport chain?
As electrons pass along the electron transport chain, protons are translocated out of the mitochondrial matrix across the inner membrane into the intermembrane space
proton electrochemical gradient
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
ATP synthase
the flow of electrons back across the membrane through ATP synthase (complex V), drives the synthesis of ATP from ADP and inorganic phosphate.
what is the ATP produced as a result of ATP synthesis used for?
Used to do work such as maintaining ion-gradients, transporting substances and proteins across membranes, protein synthesis, DNA synthesis, and muscle contraction.
what is an oxygen electrode used for?
Electron flow and the resulting reduction of oxygen by suspensions of isolated mitochondria can be demonstrated in a laboratory using an oxygen electrode
what is the trace on an oxygen electrode a measure of
the oxygen content of the reaction mixture
what are the first steps in analyzing 02 consumption by mitochondria in the presence of an excess of ADP
- Pipette reaction medium and inorganic phosphate into the oxygen electrode cham ber and stir.
what do stirring of the reaction medium and inorganic phosphate ensure
that the mixture is fully oxygenated.
what does the right hand side of the trace indicate
maximum dissolved oxygen
what was added to the Oxygen electrode after reaction medium and inorganic phosphate, and what happened
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.
what happens after the additions of succinate to the mixture
rate of oxygen uptake increases, as electrons from the added succinate, move down the electron transport chain to O2
what happens after the addition of 100ML ADP to the mixture
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.
what is state 3 respiration
The mitochondria are now actively
respiring i.e. using O2 and making ATP. This actively respiring state is called
“State 3” respiration.
why does the trace curve upwards after about 2.5 min
the rate of oxygen consumption is slowing down as oxygen becomes limiting
how do we know respiration is coupled to ATP synthesis
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.
Respiratory control ratio
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.
Consider the change from State 3 to State 4 in Figure 5. Why does
oxygen consumption decrease when ADP becomes limiting?
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.
What happens to the proton electrochemical gradient in the absence of ADP
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.
Under these conditions what happens to
electron flow through the chain and hence oxygen consumption by the
mitochondria?
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
chemiosmotic theory
The chemiosmotic theory explains how ATP is generated in the mitochondria via the electron transfer chain (ETC).
chemical uncouplers
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.
what happens to the free energy available when an uncoupler is added
Free energy available is not linked to the production of ATP but is lost as heat.
where do electrons from succinate enter?
Complex II
Where do electrons from NADH enter
Complex I
sources of NADH used in the experiment
glutamate plus malate
Rotenone blocks electron transport when NADH is the source of electrons
but not when succinate is the source of electrons.
Explain
Rotenone blocks electron transport between Complex I and Complex II and
electrons from succinate by-pass the block.
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?
If succinate cannot by-pass the antimycin block it must be beyond Complex II (in fact the block is at Complex III)
