Oxidative phosphorylation

File:Etc2.png

The Electron Transport Chain. Not all structures represent current knowledge of electron transport chains -- see talk page for more details.

Oxidative phosphorylation is the terminal process of cellular respiration. During oxidative phosphorylation electrons are transferred from NADH or FADH₂ — created in glycolysis, fatty acid metabolism and the Krebs cycle — to molecular oxygen. In eukaryotes this is carried out by a series of protein complexes located in the inner mitochondrial membrane called the electron transport chain. Protons are shuttled from the mitochondrial matrix into the intermembrane space as a result of this flow of electrons. This generates a pH gradient and a transmembrane electrical potential across the membrane. This is a form of potential energy, which is referred to as a proton-motive force. The protons flow down their gradient into the mitochondrial matrix via a large protein complex, ATP synthase. ATP synthase catalyses the following reaction:

ADP^3- + H⁺ + P_i ↔ ATP^4- + H₂O

The synthase functions as a rotary mechanical motor, with each NADH molecule contributing enough proton motive force to generate 2.5 ATP. Each FADH₂ molecule is worth 1.5 ATP. All together, the 10 NADH and 2 FADH₂ molecules created during the oxidation of glucose (glycolysis, conversion of pyruvate to acetyl-CoA, and the Krebs cycle) produce 26 of the 30 total ATP molecules yielded from the complete oxidation of that molecule of glucose. This ATP yield is the theoretical maximum value; in reality, there is some proton leakage across the membrane, resulting in somewhat lower ATP yields.

Photophosphorylation is when plants synthesize glucose during photosynthesis and also uses ATP synthase and a proton gradient to generate ATP. The process occurs across the thylakoid membrane when chlorophyll is energized by light and donates an excited electron to an electron transport chain. In prokaryotes, the electron transport chain is found in the cell's inner membrane.

The electron-transport chain

During many catabolic biochemical processes, including those mentioned at the beginning of this article, NADH and FADH₂ are produced. NADH and FADH₂, each contain two electrons which have a high transfer potential; in other words, they are highly reduced and will release energy upon oxidation. This oxidation will release electrons into the electron transport chain. As the electrons are shuttled through this chain, protons are concomitantly shuttled across the inner membrane into the intermembrane space, and an electrochemical potential is generated.

Complex I

Electron transport in Complex I (NADH-Q reductase). The abbreviations are discussed in the text.

Complex I, also known as NADH-Q oxidoreductase, is the first protein in the electron-transport chain. Complex 1 is a giant enzyme with the bovine complex I having 46 subunits and a molecular mass of about 1,000 kilodaltons (kDa). In prokaryotes, the complex is smaller, with 14 subunits. The structure is not known in detail but the complex resembles a boot with a large “ball” poking out past the membrane. The genes that encode the individual proteins are contained in the cells nucleus and the mitochondrion; this is the case for many enzymes present in the mitochondrion.

The overall reaction which is catalyzed by this enzyme is:

${\mbox{NADH + Q + 5H}}_{matrix}^{+}\rightarrow {\mbox{NAD}}^{+}+{\mbox{QH}}_{2}+{\mbox{4H}}_{cytosol}^{+}$

The start of the reaction, and indeed the entire electron chain, is the binding of a NADH molecule to complex 1 and the donation of the cofactor's two high transfer potential electrons. The electrons enter complex 1 via a prosthetic group attached to the complex, flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH₂. The reduction of the FMN molecule, due to the acceptance of two electrons, causes it to bind two protons. The resulting FMNH₂ molecule is subsequently oxidized back to FMN and donates the two electrons which originally reduced it.

Here are the equations of the reactions:

${\mbox{NADH + H}}^{+}+{\mbox{FMN}}\rightarrow {\mbox{NAD}}^{+}+{\mbox{FMNH}}_{2}$

${\mbox{FMNH}}_{2}\rightarrow {\mbox{FMN}}+{\mbox{2e}}^{-}+{\mbox{2H}}^{+}$

The electrons formed from the oxidation FMNH₂ are transferred through a series of iron-sulphur clusters; the second kind of prosthetic group present in the complex. Iron-sulphur clusters are often present in enzymes which involve a transfer of electrons. There are several types of iron-sulphur clusters. The simplest kind consists of a single iron ion is tetrahedrally coordinated to the thiol groups of four cysteines in the polypeptide chain. The second kind, denoted by 2Fe – 2S, contains 2 iron ions and two inorganic sulfides. Several others are shown in this image. Iron-sulphur clusters usually undergo redox reactions without binding or releasing protons, so serve solely to transport electrons through the protein, rather than contribute to the production of the pH gradient.

The electrons that pass through the iron-sulphur clusters are transferred to a coenzyme Q (ubiquinone or UQ) group present in the complex. There are three 4Fe-4S clusters present between the FMNH₂ and Q groups. The electrons flow through all three on their way from FMNH₂ to Q. The basic mechanism involves the uptake of two protons as Q is reduced to QH₂ due to the transfer of 2 electrons from the aforementioned iron-sulphur cluster. Here is the equation of the reaction (the two electrons are the ones which were released during the oxidation of FMNH₂. They are passed to a Q molecule from the third iron-sulphur cluster):

${\mbox{Q}}+{\mbox{2H}}^{+}+{\mbox{2e}}^{-}\rightarrow {\mbox{QH}}_{2}$

The two high transfer potential electrons present in the QH₂ molecule are transferred to another 4Fe-4S complex and the two protons are released into the cytosolic side; which contributes to the production of the pH gradient. Finally, these two electrons are transferred to a mobile Q molecule in the membrane which accepts the electrons and is reduced to QH₂ (as before), but is then released from the protein complex into the hydrophobic core of the membrane. This QH₂ molecule reattaches to complex III where it is oxidized to release its two high transfer potential electrons.

Complex II

File:Complex2.gif

Complex II

Complex II, also known as succinate-Q(ubiquinione) reductase, is an alternative entry point to the electron-transport chain. It consists of 4 polypeptides and is made up of coenzyme Q (ubiquinone) reductase. It oxidises succinate and reduces coenzyme Q.

Complex III

File:Complex3p1.gif

Complex III

Complex III, also known as Q-cytochrome c oxidoreductase or simply cytochrome reductase, is the third protein in the electron-transport chain. A cytochrome is a kind of electron-transferring protein which contains at least one heme group. The iron ion inside complex II’s heme groups alternate between a reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein.

The overall reaction catalyzed by complex III is:

${\mbox{QH}}_{2}+{\mbox{2Cyt c}}_{ox}+{\mbox{2H}}_{matrix}^{+}\rightarrow {\mbox{Q}}+{\mbox{2Cyt c}}_{red}+{\mbox{4H}}_{cytosol}^{+}$

The enzyme contains 3 heme groups which participate in transferring the electron through it. The electron originates from the mobile coenzyme Q which was released from complex I. It terminates at cytochrome c; an electron carrying protein containing a heme group. Unlike coenzyme Q, which carriers 2 electrons, cytochrome c only carries 1.

The reduced coenzyme Q binds to a site inside complex III. The reduced coenzyme Q molecule transfers its electrons one at a time. The electron travels first through a 2Fe-2S cluster and then to a heme group held inside complex III. The electron travels from this immobile heme molecule to the heme group inside the cytochrome c protein bound to complex III. The heme group within the cytochrome c protein is now in its reduced form and the enzyme is free to diffuse away from complex III. The hydrogen ions released during the oxidation of QH₂ are transferred to the cytosolic side of the membrane and hence serve to create the proton gradient.

Complex IV

File:Complex4.gif

Complex IV

Complex IV, also known as cytochrome c oxidase, is the fourth and final protein in the electron transport chain. It mediates the final reaction and transports electrons to oxygen. The final electron acceptor oxygen is reduced to water in this step.

Proton gradient across the membrane

Further information: [[:Chemiosmosis]]

The electron transport chain is the flow of electrons from NADH to O₂. This is an exergonic process (work is done during the process). The process of oxidative phosphorylation terminates at the synthesis of ATP from ADP and orthophosphate; an endogonic process (work is required to drive the reaction). The work that drives the reaction is the net flow of protons across the membrane in which the ATP synthase is embedded in.

The reactions and the work released are shown below:

File:Png equation 2.PNG

The first reaction creates a net difference of proton concentration across the membrane and hence an increase in entropy. The decrease in entropy across the membrane drives the endogonic process of ATP synthesis. This reaction is carried out by one single protein complex known originally as the mitochondrial ATPase, which is misleading since it implies the reaction is carried out the other way round (which it can be, though this is not its function in this case); today it most often known as ATP synthase.

ATP Synthase

ATP synthase is the final protein in the metabolic pathway. Most of the ATP in any prokaryote or eukaryote comes from the process the ATP synthase catalyses. ATP is synthesized from ADP and orthophosphate:

File:Png equation 1.PNG

ATP synthase is a massive protein complex which resembles a ball on a stick. The “ball” shaped head contains six proteins of two different kinds (three α subunits and three β subunits). The “stick” consists of one protein: the γ subunit, which is long and extends throughout the α and β subunits. Both the α and β subunits bind nucleotides, but only the β subunits participate in the ADP phosphorylation reaction. ATP synthase is part of the P-loop NTPase family.

The mechanism of action of ATP synthase

As protons cross the membrane through the base of ATP synthase the central stem (the subunit) rotates inside the α and β subunits. This movement of the α and β subunits cause the movement of their active sites and thus the production of ATP.

Reactive oxygen species

Further information: [[:Oxidative stress and Antioxidant]]

Molecular oxygen is ideal as a terminal electron acceptor due to its high affinity for electrons. However, the reduction of molecular oxygen yields potentially harmful intermediates. The transfer of four electrons results in the production of water, which is not harmful. The transfer of one or two electrons produces superoxide anion and peroxide as shown below:

${\begin{matrix}\quad &{e^{-}}&\quad &{e^{-}}\\{{\mbox{O}}_{2}}&\longrightarrow &{\mbox{O}}_{2}^{\underline {\bullet }}&\longrightarrow &{\mbox{O}}_{2}^{2-}\\\quad &\quad &{\mbox{Superoxide}}&\quad &{\mbox{Peroxide}}\\\quad &\quad &{\mbox{anion}}&\quad &\quad \end{matrix}}$

These compounds and, particularly, their reaction products such as the hydroxyl radical are very harmful to cellular components.

Due to the efficiency of the cytochrome c oxidase complex very few partly reduced intermediates are released, however small amounts of superoxide anion and peroxide are produced. Particularly important is the reduction of coenzyme Q in complex III, since a highly reactive free radical is formed as an intermediate (Q·⁻). This unstable intermediate can lead to electron "leakage" when electrons jump directly to molecular oxygen and form the superoxide anion, instead of moving through the series of well-controlled reactions of the electron transport chain. Species which can be produced from these such as Hydrogen peroxide (H₂O₂) and the hydroxyl radical (•OH) are collectively referred to as reactive oxygen species or ROS. There are many enzymes whose task is to convert ROS into less reactive species. These include superoxide dismutase, which converts superoxide radicals into molecular oxygen and hydrogen peroxide as shown below:

${\begin{matrix}\quad &\quad &\quad &{\mbox{Superoxide}}&\quad &\quad &\quad \\\quad &\quad &\quad &{\mbox{dismutase}}&\quad &\quad &\quad \\{\mbox{2O}}_{2}^{\underline {\bullet }}&+&{\mbox{2H}}^{+}&\longleftarrow \!\longrightarrow &{\mbox{O}}_{2}&+&2{\mbox{H}}_{2}{\mbox{O}}_{2}\\\end{matrix}}$

There are two varieties of the enzyme within eukaryotes. The hydrogen peroxide released from the cytochrome c oxidase complex and by superoxide dismutase is scavenged by the enzyme catalase. Catalase converts hydrogen peroxide into molecular oxygen and water as shown below:

${\begin{matrix}\quad &{\mbox{Catalase}}&\quad &\quad &\quad \\2{\mbox{H}}_{2}{\mbox{O}}_{2}&\longleftarrow \!\longrightarrow &{\mbox{O}}_{2}&+&2{\mbox{H}}_{2}{\mbox{O}}\\\end{matrix}}$

Superoxide dismutase and catalase are both remarkably efficient and carry out their reactions at or near the diffusion-limited rate.

Antioxidant vitamins C and E also serve to convert ROS to less harmful compounds. Vitamin E cannot exist in the mitochondrial matrix since it will not mix with water; it is hydrophobic. It instead exists in the inner membrane of the mitochondrion and protects the membrane from being harmed by ROS. Vitamin C is hydrophilic and carries out its reactions in the mitochondrial matrix.

Inhibitors

There are a few well-known toxins that affect the process of oxidative phosphorylation and can lead to breakdown of the chain:

1. Cyanide interrupts the electron transport chain in the inner membrane of the mitochondrion because it binds more strongly than oxygen to the Fe³⁺ (ferric iron ion) in cytochrome a₃, preventing this cytochrome from combining electrons with oxygen.
2. Oligomycin inhibits ATP synthase by preventing it to pump protons, thus preventing it to generate ATP from ADP and Pi with the usage of energy of the proton gradient.
3. CCCP (m-chloro-carbonylcyanide-phenylhydrazine) destroys the proton gradient by allowing the protons to flow back into the matrix. Without the gradient, the ATP synthase cannot function and ATP synthesis breaks down.
4. A detergent, or substance that destroys cellular membranes by breaking apart their lipid bilayers, will destroy the membrane used in the process and prevent a proton gradient.
5. Rotenone prevents the transfer of electrons from Fe-S centers in Complex I (most notably) to ubiquinone. The electrons entering into Complex I are those derived from NADH, and provide the bulk of the reducing potential to the electron transport chain.
6. Antimycin prevents tranfer of electrons on the complex III, between cytocromes b and c1.

Although these toxins only inhibit one step of the electron transport chain, inhibition of any one part of this process will stop the rest of the process. For example, if oligomycin is added, protons cannot pass back into the mitochondrion. As a result, the H⁺ pumps are unable to pump protons as the gradient becomes too strong for them to overcome. NADH and FADH₂ are then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD⁺ and FAD coenzymes falls to a level these enzymes cannot use.

See also

• Metabolism
• Mitochondrion
• Cellular respiration

References

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Further reading

• Nelson DL (2005). Lehninger Principles of Biochemistry (4th ed ed.). W. H. Freeman. ISBN 978-0716743392. {{cite book}}: |edition= has extra text (help); Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)

External links

• Interactive Molecular model of NADH dehydrogenase
• Interactive Molecular model of succinate dehydrogenase
• Interactive Molecular model of Coenzyme Q - cytochrome c reductase
• Interactive Molecular model of cytochrome c oxidase
• Template:McGrawHillAnimation