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DENF 1521 Biochemistry

Dental Biochemistry

Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

5.2 An overview of the reactions of the
oxidative phosphorylation pathway

5.2A Lesson objectives

The objectives of this lesson are to understand that:

1. The respiratory chain (electron-transport chain) is responsible for transfering the electrons from NADH and FADH2 to molecular oxygen
2. The respiratory chain is found in the inner mitochondrial membrane
3. Four enzyme complexes and 2 mobile carriers comprise the respiratory chain
4. Pumping of protons by the respiratory chain complexes is the primary energy conserving event of oxidative phosphorylation
5. The chemiosmotic hypothesis states that a proton-motive force was responsible for driving the synthesis of ATP
6. A precise arrangement of the respiratory enzymes is required for electron transport

5.2B The Respiratory Chain

The respiratory chain, also known as the electron-transport chain, is a chain of electron carriers that transfer electrons from NADH and FADH₂ to molecular oxygen (O₂). The respiratory chain is found in the inner mitochondrial membrane and is composed of 3 enzyme complexes and 2 mobile carriers. As will be discussed later in this lesson, the 3 enzyme complexes are the sites of the proton pumps.

Enzyme Complexes	Mobile carriers
NADH-Q reductase	Q (ubiquinone)
Cytochrome reductase	Cytochrome c
Cytochrome oxidase	.

The respiratory chain is arranged in a defined sequence as shown. The enzyme complexes are indicated in green and the mobile carriers in purple. Note that NADH enters the chain through the first enzyme complex, NAQDH-Q reductase. FADH2 in contrast enters the chain at the point of the mobile carrier Q. As will be discussed later, this has implications for the number of protons pumped and the number of ATP molecules derived from NADH or FADH2. Energy transfer through the respiratory chain leads to the pumping of protons by the 3 enzyme complexes. The protons are pumped from the matrix to the cytosolic side of the inner mitochondrial membrane. This pumping of protons is the primary energy conserving event of oxidative phosphorylation.

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

Resporatory chain
Practice Exercise 1:	The electrons from NADH enter the respiratory chain through which of the following complexes? No Response NADH oxidase Cytochrome oxidase NADH-Q reductase Succinate-Q reductase
Practice Exercise 2:	The primary energy conserving event of oxidative phosphorylation is the No Response Pumping of protons from the cytosolic to the matrix side of the inner mitochondrial membrane Pumping of protons from the matrix to the cytosolic side of the inner mitochondrial membrane Pumping of protons out of the mitochondrial to the cytosol Entry of electrons from FADH2
Practice Exercise 3:	The respiratory chain is found in the outer mitochondrial membrane. No Response True False
Practice Exercise 4:	The electrons from NADH and FADH2 enter the respiratory chain through the same complex. No Response True False

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

5.2C Details of the respiratory chain components

The mitochondrial respiratory chain is composed of 4 enzyme complexes. The 3 mentioned above, NADH-Q reductase, cytochrome reductase, and cytochrome oxidase, all span the inner mitochondrial membrane and are proton pumps. The fourth complex succinate-Q reductase is positioned between NADH-Q reductase and cytochrome reductase. This complex is found on the matrix side of the inner mitochondrial membrane and does not span the membrane. Succinate-Q reductase is the point of entry into the respiratory chain for electrons from FADH₂. As indicated in the following table, all of these enzymes are large, multisubunit complexes containing several prosthetic groups. The prosthetic groups are the molecules that actually carry the electrons within the complex.

Complex number	Enzyme complex	Molecular weight (x 10-3)	Number of subunits
I	NADH-Q reductase	880,000	>34
II	Succinate-Q reductase	140,000	4
III	Cytochrome reductase	250,000	10
IV	Cytochrome oxidase	160,000	10

Various groups are involved in the actual transfer of electrons within each complex and from one complex to another. These prosthetic groups can be classified as

1. Iron sulfur proteins (Fe-S)
2. Hemes
3. Copper
4. Flavins

All of these serve to carry electrons but each enzyme complex is associated with specifc prostheric groups. It is important to know the function of these groups but it is not necessary to know which prosthetic groups is specifically associated with each enzyme complex.

Complex number	Enzyme complex	Prosthetic groups
I	NADH-Q reductase	FMN, Fe-S
II	Succinate-Q reductase	FAD, Fe-S
III	Cytochrome reductase	Heme b-562, Heme b-566, Heme c1, Fe-S
IV	Cytochrome oxidase	Heme a, Heme a3, CuA, CuB

The following schematic diagram illustrates the position of the 4 enzyme complexes in the inner mitochondrial membrane and shows the flow of electrons within this respiratory chain.

An animated version of electron transport in the mitochondria can be found at this University of Connecticut site. For more inforamtion on the cytochrome oxidase enzyme (complex IV) see clicking here.

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

Respiratory chain components
Practice Exercise 5:	Which of the following compounds is NOT involved in the transfer of electrons with respiratory chain components? No Response Hemes Flavins Magnesium Copper
Practice Exercise 6:	All four (4) of the respiratory chain enzymes are transmembrane proteins. No Response True False
Practice Exercise 7:	The reduction of molecular oxygen to water occurson the matrix side of the inner mitochondrail membrane. No Response True False
Practice Exercise 8:	Which of the following enzymes complexes does NOT pump protons? No Response NADH oxidase Cytochrome oxidase Succinate-Q reductase NADH-Q reductase

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

If you would like to do a matching exercise involving the order of the components in the respiratory chain click the button below.

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

5.2E The Chemiosmotic Hypothesis

The chemiosmotic hypothesis was proposed by Peter Mitchell. This hypothesis stated that a proton-motive force was responsible for driving the synthesis of ATP. In this hypothesis, protons would be pumped across the inner mitochondrial membrane as electrons went through the respiratory chain. This would result in a proton gradient with an elevated pH in the intermembrane space and a lower pH in the matrix of the mitochondria. An intact inner mitochondrial membrane, impermeable to protons, is a requirement of such a model. The proton gradient and membrane potential are the proton-motive force that is used to drive ATP synthesis. In effect, the pH gradient acts as a "battery" which stores energy to produce ATP. Over the past several years, Michell's chemiosmotic hypothesis has been widely as the mechanism of coupling of electron transport and ATP synthesis. He was awarded the Nobel Prize in Chemistry in 1978. This acceptance by the scientific community is a result of accumulating experimental evidence supporting the hypothesis.

The evidence supporting Michell's chemiosmotic hypothesis is as follows.

1. Electron transport generates a proton gradient. The pH measured on the outside is lower than that measured inside the mitochndria.
   
2. Only a proton gradient is needed to synthesize ATP. Electron transport is not required as long as there is another mechanism for generating a pH gradient.
   
3. A reconstitution experiment carried out by Racker and Stoeckenius showed that the generation of a proton gradient resulted in ATP synthesis in a totally artificial system. In their experiment, a mitochondrial ATPase complex from beef heart was inserted into an artificial lipid bilayer. Also inserted in this bilayer was a membrane fragment containing bacteriorhodopsin from the purple bacteria Halobacterium, so called because the bacteriorhodopsin gives the membrane a purple color. Bacteriorhodopsin is a light-driven proton pump. Therefore, shining light on this artificial "purple membrane" formed a proton gradient which was used by the mitochindrial ATPase to synthesize ATP.
   

4. The respiratory chain and the ATPase are assymetrically oriented in the membrane. This was determined in submitochondrial particles using various probes such as specific antobodies or lectins. These experiments showed that some complexes span the membrane while others had an assymetric orientation. This assymetric orientation is a requirement to establish a pH gradient. A random arrangement would not result in a net gradient of protons and therefore, no proton-motive force for the synthesis of ATP.
   
5. A closed compartment is required for reasons similar to those stated above. Free movement of protons would not establish a proton gradient.
   
6. Compounds called uncouplers were found to collapse the pH gradient by shuttling protons back across the membrane. One such uncoupler, dinitophenol is shown below. In the presence of the uncoupler electron transport continues but no ATP synthesis occurs. Can uncoupling of electron transport and ATP synthesis ever be useful to the organism? The answer is "Yes." Such uncoupling will generate heat. This occurs in hibernating animals, in newborn humans, and in mammals adapted to the cold. It occurs in the brown adipose tissue. An uncoupling protein called thermogenin can accomplish this uncoupling and thus allow heat to be generated.

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

Chemiosmotic hypothesis
Practice Exercise 9:	Which of the following is required for the coupling of electron transport and ATP synthesis? No Response The inner mitochondrial membrane is permeable to protons The electron transport complexes are randomly inserted in the inner mitochondrial membrane. An intact mitochondrial membrane Uncouplers must be present to activate the ATP synthase
Practice Exercise 10:	Only proton gradients generated by electron transport can be used to synthesize ATP. No Response True False
Practice Exercise 11:	In the presence of an uncoupler such as dinitophenol, electron transport can continue but no ATP synthesis occurs. No Response True False

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

5.2F The sequence of electron carriers

The components of the electron transport chain are arranged in a precise sequence as shown below. As previously discussed, protons are pumped at 3 sites in the chain. Thermodynamic estimates show that each site generates a proton-motive force sufficient to drive ATP synthesis. Inhibitors have been identified which inhibit electron transport at specific sites (indicated by red bars in figure).

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

Electron carriers
Practice Exercise 12:	Which of the following compounds blocks the transfer of electrons from cytochrome oxidase to O2. No Response Cyanide NADH Rotenone Antimycin A
Practice Exercise 13:	Amytal blocks the transfer of electrons from cytochrome c to cytochrome oxidase. No Response True False
Practice Exercise 14:	In order for electron transport to occur properly, the enzyme complexes must be arranged in a precise order.. No Response True False

Dental Biochemistry

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Lesson 5.2 Respiratory chain & chemiosmotic hypothesis

5.2G Summary

After completing this lesson you should understand the following about the respiratory chain and the chemiosmotic hypothesis.

1. Electrons from NADH and FADH₂ are passed through a series of electron transport complexes
2. These electrons are passed to molecular oxygen as the ultimate acceptor
3. Four enzyme complexes and 2 mobile carriers comprise the respiratory chain
4. As the electrons are transfered, protons are pumped into the intermembrane space of the mitochondria resulting in the formation of a proton gradient.
5. The chemiosmotic hypothesis of Peter Mitchell states that a proton-motive force was responsible for driving the synthesis of ATP
6. Much experimental evidence supports the pumping of protons by the respiratory chain complexes as the primary energy conserving event of oxidative phosphorylation

Final Instructions

Press Button below for your score. After completing Lesson 5.2, including all practice exercises, press the "Submit... " button below for Lesson 5.2 research participation credit. After you press "Submit..." it is possible Netscape may tell you it is unable to connect because of unusually high system demands. If you receive no error message upon submission you're OK. But, if Netscape gives you an error message after you press the "Submit..." button, wait a moment and resubmit or consult the attendant. Finally, press the "Table of Contents..." button below to correctly end Lesson 5.2 and return to the Table of Contents so you may continue with Lesson 5.3. End Lesson 5.2 Overview of oxidative phosphorylation