chemiosmotic synthesis of atp in the mitochondria

Chemiosmotic Synthesis of ATP in the Mitochondria

Dr. Carol Hardy & Dr. Tyson Sacco Cornell University

This video is designed to help you understand how ATP is produced within the cell.

Chemiosmotic Synthesis of ATP in the Mitochondria

Dr. Carol Hardy & Dr. Tyson Sacco Cornell University

In particular. it will help you to understand the process of chemiosmosis.

ATP can be synthesized by two processes:

1.  Substrate-level phosphorylation §  Substrate-P + ADP ⇒ Substrate + ATP §  Glycolysis: net gain 2 ATP §  Krebs: net gain 2 ATP

•  There are two ways that ATP can be produced within the cell. •  One is by the process known as substrate level phosphorylation. The name gives you a clue as to what

is happening. When something is phosphorylated, a phosphate group is added and, in this case, the substrate is involved.

•  A substrate group which has a phosphate group bound to it will transfer that phosphate group to an ADP molecule to form ATP.

•  There are two places where substrate-level phosphorylation occurs within the cell: 1) in the process of glycolysis, where there is a net gain of 2 ATP; and 2) in the Krebs cycle where a molecule of glucose will produce an additional 2 ATP by substrate-level phosphorylation.

ATP can be synthesized by two processes:

1.  Substrate-level phosphorylation 2.  Chemiosmosis (oxidative phosphorylation)

•  If we assume that the complete metabolism of a molecule of glucose will yield a net gain of 30 ATP, where does the bulk of the ATP come from? (remember, 4 were produced by substrate-level phosphorylation)

•  Chemiosmosis, or oxidative phosphorylation, is the major source of ATP (26/30 ATP per glucose molecule).

Metabolism of Glucose

I.  Glycolysis I.  Takes place in cytosol. II.  Yields 2 pyruvate (pyruvic acid),

2 ATP (substrate-level), and 2 reduced carrier NADH molecules.

III.  Does not require oxygen but can take place in aerobic conditions.

•  Before looking at the details of chemiosmosis, let’s

review the basics of glucose metabolism. •  Shown here are the three stages in which glucose

is metabolized into carbon dioxide and water. •  The first stage is Glycolysis. •  Glycolysis takes place in the cytoplasm of the cell

and the 2 pyruvate molecules produced by glycolysis will move into the mitochondrion where metabolism will be completed.

•  Redox reactions in glycolysis reduce NAD+ to NADH molecules that will be used in chemiosmosis.

•  Of course, 2 molecules of ATP (net) are produced by substrate-level phosphorylation during glycolysis as well.


I.  Glycolysis II.  Pyruvate ⇒Acetyl-CoA

I.  Takes place in mitochondrion. II.  2 pyruvate are oxidized, yielding

2 Acetyl-CoA molecules. III.  This redox reaction produces 2

more NADHs.

•  Stage 2 begins with the pyruvate from glycolysis moving into the mitochondrion.

•  This stage is essentially a redox reaction in which 2 pyruvate are oxidized and NAD+ molecules are reduced, yielding 2 molecules of Acetyl Coenzyme A and 2 NADHs.

•  2 molecules of CO2 are also released when the pyruvate molecules enter the mitochondrion and gives up their carboxyl groups.


I.  Glycolysis II.  Pyruvate ⇒Acetyl-CoA III.  Krebs cycle

1.  Occurs in mitochondrion. 2.  2 Acetyl-CoA molecules lead to

2 turns of the cycle. 3.  Each turn produces:

•  1 ATP (substrate-level) •  3 NADH’s •  1 FADH2

•  Stage 3 is the Krebs (or citric acid) cycle. •  This stage uses each of the 2 acetyl-CoA

molecules produced earlier to convert a 4-carbon into the 6-carbon compound citrate.

•  Citrate is oxidized over the course of the cycle to produce a number of reduced carriers that will be used in chemiosmosis.

•  In addition, 2 ATP are produced by substrate-level phosphorylation during the Krebs cycle.

•  4 molecules of CO2 are also released in the course of the oxidation of the 2 citrate molecules that pass through the cycle for each glucose molecule.


Summary: I.  Glycolysis

§  2 ATP §  2 NADH

II.  Pyruvate ⇒Acetyl-CoA §  2 NADH

III.  Krebs cycle §  2 ATP §  6 NADH §  2 FADH2

•  In summary, our main products (so far) are: 4 ATP produced by substrate-level phosphorylation and 12 reduced carrier molecules.

•  The reduced carrier molecules store a great deal of energy in them (in a sense) and it is this energy that will be used (indirectly) to make ATP via chemiosmosis.

The Mitochondrion

•  Where does chemiosmotic ATP production take place? In the mitochondrion. •  Recall that the mitochondrion has a double membrane - an outer membrane that

is relatively permeable and an inner, highly folded membrane that has very selective permeability. These membranes separate the mitochondrion into outer and inner compartments.

•  The reactions of stages II and III (shown previously) take place in the inner compartment and chemiosmosis occurs on the inner mitochondrial membrane.

Chemiosmotic Synthesis of ATP

Note the two mitochondrial membranes and the two compartments they form. The cytosol (where glycolysis has taken place) is at the top of the diagram, beyond the outer membrane.

Cytosol


Both mitochondrial membranes are normal lipid bilayers with proteins imbedded in and on the membranes.


Focus on the protein complexes labeled I, II, III, and IV on the inner membrane. They are groups of proteins that are anchored together in the membrane. These complexes are linked by mobile carriers.


The mobile carrier ubiquinone, labeled Q, links complexes I, II, and III. It can move back and forth through the plane of the membrane. Cytochrome C moves back and forth on the outer surface of the inner membrane, connecting complexes III and IV.


Collectively, the 4 protein complexes and their mobile carriers make up the Electron Transport Chain.

Electron Transport Chain


Each element in the chain is electronegative, meaning that it has a strong attraction for electrons. A gradient of increasing electronegativity exists within the electron transport chain. In terms of their electronegativities: I < Q < II < III and so on.

Low High

Electronegativity


The final electron acceptor in the series (and the most electronegative component of the chain) is oxygen.

O2


Electrons transferred from NADH and FADH2 will be passed along the chain, finally reaching oxygen. When oxygen receives those extra electrons it takes on a very strong negative charge and attracts protons. This results in the formation of water.


Remember that as they move down the chain (towards O2), the electrons release (lose) energy. That energy will be used indirectly to create ATP. We’ll now look at the details of this indirect exchange of energy…


Molecules labeled in red above are electron transport molecules that must accept protons (hydrogen ions) along with the electrons they pick up. For example, ubiquinone (Q) can accept 2 electrons and must also accept two protons, so it becomes QH2


The molecules labeled in orange above (Complex III and Cytochrome C) are electron only carriers. They do not accept protons with the electrons they transport.


Notice that Complex I is going to take electrons away from NADH. When it accepts the electrons it will pick up hydrogen ions as well. However, some of the molecules that make up Complex I are electron-only carriers, so something has to happen to the hydrogen ions that have been taken up.


Hydrogen ions picked up by Complex I are released into the outer compartment of the mitochondrion and electrons are passed along the electron transport chain to Q. Notice that one effect of Complex I has been the movement of H+ across the mitochondrial membrane from the inner to the outer compartment.


Like Complex I, ubiquinone (Q) is a hydrogen and electron carrier, when it picks up the electrons from Complex I it also picks up a hydrogen ion from the inner compartment. It then becomes QH2 and moves through the membrane until it finds complex III.


Complex III is an electron-only carrier, so the H+ ions carried by Q are again released into the outer compartment.


Next, Cytochrome C moves across the inner membrane, carrying electrons from Complex III to Complex IV. Like Complex I, Complex IV is a hydrogen and electron carrier, so as it accepts electrons from Cytochrome C it also picks up H+ ions from the inner compartment and deposits them in the outer compartment.


To review, there are three sites where hydrogen ions are moved from the inner to the outer compartment of the mitochondrion. The movement of these ions creates a very strong concentration gradient of hydrogen ions across the inner membrane.

1 2 3


Note that the movement of hydrogen ions from the inner to the outer compartment is active transport because the ions are being moved against their concentration gradient.

1 2 3


Recall that the electrons picked up by Complex IV are finally passed to oxygen, forming water.


The net movement of positively charged hydrogen ions across the inner mitochondrial membrane produces an electrochemical gradient - chemical because there is a gradient of hydrogen concentration and electrical because there is a gradient of charge (more positive in the outer and more negative in the inner compartment).

Hi [H+], ++positive charge++

Low [H+], --negative charge--


Keep in mind that the positively-charged hydrogen ions will “want” to move down this concentration gradient (from the outer to the inner compartment). The inner membrane is impermeable to H+ ions, maintaining the electrochemical gradient.


The tendency of H+ ions to move down the electrochemical gradient can be thought of as “potential energy” that will be harnessed to produce ATP.

An electrochemical gradient provides the power to make ATP.

Imagine the H+ ions as water trapped behind a tall dam. The fact that the water will tend to move down can be used to do work - as in a hydroelectric power plant. The inner mitochondrial membrane is like the wall of the dam with many positively charged hydrogen ions trapped behind it.

Hi [H+], ++positive charge++

Low [H+], --negative charge--

Glen Canyon Dam outside Page, AZ is 710’ tall and over 300’ thick at its thickest point. It holds back enough water to supply

over 26 million families of 4 with water for a year!


There is one place in the membrane that will allow hydrogen ions to pass through and move down the electrochemical gradient - the ATP synthase complex.


As the H+ ions flow down the gradient, the energy stored in the gradient is used to make ATP.

ATP

ATP synthase is like a turbine in a dam.

Returning to our dam metaphor, you can think of the ATP synthase as a turbine inside a channel cut through the dam. Just as a turbine converts the mechanical energy of water turning its blades to electricity, the ATP synthase uses the H+ ions passing through it to power ATP synthesis.


We’ve already seen how NADH from the Krebs cycle passes electrons into the electron transport chain and how hydrogen ions are moved across the inner membrane in three places to create an electrochemical gradient. Now let’s look at how FADH2 plays a role in chemiosmosis.


FADH2 produced in the Krebs cycle transfers its electrons and hydrogen ions to a different carrier than NADH. FADH2 exchanges with Complex II in the electron transport chain. Complex II is a hydrogen and electron carrier and passes both to Q, which passes them on to Complex III where (as before) electrons are passed on and H+ are moved into the outer compartment.


The electrons from FADH2 miss the first step in the electron transport chain entirely. Because of this, FADH2 is responsible for fewer H+ ions being pumped across the membrane, creating less “potential energy”, and resulting in fewer ATP being produced per FADH2 (compared to NADH).


How much ATP is produced by each NADH or FADH2?

ATP


On average, each NADH produced by the conversion of pyruvate to acetyl-CoA or by the Krebs cycle results in the production of 2.5 ATP.

ATP


In contrast, each FADH2 results in the production of (on average) 1.5 ATP.

ATP

X


Since the steps in glucose metabolism preceding chemiosmosis produce a number of NADH and FADH2 molecules. a large number of ATP can be produced by chemiosmosis. How many?

ATP


Before we do the final accounting for ATP production by chemiosmosis there is one last aspect that needs to be addressed - the NADH molecules produced by glycolysis. How are they involved in chemiosmosis?

ATP

Chemiosmotic Synthesis of ATP Cytosol

NADH molecules produced by glycolysis are different than those produced in the inner compartment in that they are out in the cytoplasm. NADH is too large and too polar to pass through the outer mitochondrial membrane so the hydrogen ions and electrons from the NADHs in the cytoplasm are passed through the membrane by a shuttle molecule. (Note: An alternate shuttle molecule, shown unshaded, is used to transport the NADH produced in glycolysis in highly metabolic active tissues which results in the electrons entering the chain at complex I and therefore resulting in the production of 2.5 ATP.)

ATP


This shuttle molecule will oxidize NADH, taking away electrons and a hydrogen ion and carrying them across to molecule Q in the inner membrane. Q accepts both the H+ and the electrons and carries them to Complex III where (as usual) the electrons are passed along to IV and the H+ is released.

ATP


As you can see, the electrons and H+ ions from the NADHs produced by glycolysis enter the electron transport chain at essentially the same point as electrons and H+ ions from FADH2. As a result, each NADH from glycolysis results in the production (on average) of 1.5 ATP, just like FADH2.

ATP

X


ATP

Chemiosmosis ATP accounting: Glycolysis •  2 NADH/glucose •  1.5 ATP/NADH •  = 3 ATP


ATP


Stage II (pyruvate to acetyl-CoA) •  2 NADH/glucose •  2.5 ATP/NADH •  = 5 ATP


ATP


Stage II (pyruvate to acetyl-CoA) •  2 NADH/glucose •  2.5 ATP/NADH •  = 5 ATP Krebs cycle •  6 NADH/glucose •  2.5 ATP/NADH •  = 15 ATP

& •  2 FADH2/glucose •  1.5 ATP/FADH2 •  = 3 ATP

Final ATP accounting:

Glycolysis •  2 NADH/glucose •  1.5 ATP/NADH •  = 3 ATP

Stage II (pyruvate to acetyl-CoA) •  2 NADH/glucose •  2.5 ATP/NADH •  = 5 ATP Krebs cycle •  6 NADH/glucose •  2.5 ATP/NADH •  = 15 ATP •  2 FADH2/glucose •  1.5 ATP/FADH2 •  = 3 ATP

SubTotal = 26 ATP/glucose

•  Adding up the numbers at left (3 + 5 + 15 + 3) we find that 26 ATP are produced via chemiosmosis for each glucose molecule metabolized.






•  Recall that an additional 4 ATP (net) per glucose molecule were produced by substrate-level phosphorylation during glycolysis and the Krebs cycle.







•  This gives us a final net production of 30 ATP per glucose molecule (on average).







•  This gives us a final net production of 30 ATP per glucose molecule (on average).

•  Grand Total = 30 ATP/glucose

Closing Notes:

•  Keep in mind that ATP production described here assumes that aerobic respiration is taking place.

•  As you’ll learn, the number of ATP produced differs dramatically in anaerobic situations.

•  It is also important to realize that the exact number of ATP produced per glucose molecule may actually differ depending on the exact cellular conditions.

•  Total numbers, whether 30 or 32 represent average ATP production. •  The most important concept is that chemiosmosis produces many more ATP

than substrate-level phosphorylation.

Need More Help?

•  Talk to a TA or Dr. Campbell, they’ll be happy to answer your questions.

ATP

chemiosmotic synthesis of atp in the mitochondria

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