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Energy Generation Using a Membrane

©2000 Timothy Paustian, University of Wisconsin-Madison

So far in catabolism we have extracted a large number of electrons and a bit of ATP from our substrate and ended up oxidizing it completely to CO2 and water. What does the cell do with all these high energy electrons?....... Convert them to a usable form of energy, namely more ATP. The conversion requires a membrane system and involves pumping of ions out of the cell and allowing them to fall back in. The process is called Electron Transport Level Phosphorylation (ETLP)

Energy generation in plants (and other organisms) was a vexing question in the late 50s and Peter Mitchell came up with the chemiosmotic theory in 1961 to explain it. His idea applies to a vast number of biological systems and because of its broad impact he won the Nobel Prize in Chemistry in 1978. Below is an updated version of that hypothesis as it applies to bacteria. The illustrations hilite electron transport in common bacteria, but the general concept applies to all ETLP.

Generalized Structure of the Electron Transport System (ETS)

All ETS systems so far discovered involve some type of membrane. The membrane serves two important functions in energy generation. First, it allows charge separation to build up between the cytoplasm and the outside of the cell. Remember that charged molecules cannot penetrate the membrane and are only allowed entry and exit by special proteins designed to carry them. Second, the membrane holds many of the components involved in electron transport in the a exact confirmation necessary to enable them to perform their duties correctly.

The spacial arrangement of the electron transport system in the membrane is vital to its function. The successive electron carriers are located in close proximity so that it is easy for the electrons to pass from one complex to the next, with a minimum of delay between transfers. Each protein complex that spans the membrane also has a sidedness. One part of the protein has to interact with the outside environment and the other with the inside cytoplasm. Flipping the protein in the membrane would make it nonfunctional. Having this sidedness also is critical for the pumping of protons across the membrane as discussed below.

ETmem picture

Figure 1 - Arrangement of the ETS in the membrane. This is a common arrangement, but different species of bacteria will have different enzymes. The general principles discussed here still hold.

The ETS in Action.

An overview

The proteins are arranged, the electron carriers are loaded up and ready to donate. Lets go! Electron transport (ET) is a series of oxidation-reduction reactions where the electrons flow from high potential electron carriers to low potential electron carriers. Electrons on NADH have a potential of - 0.32 volts. (Remember that negative electron potential is high energy. Go back and look at the table of electron carriers in Basic Energy Concepts to refresh your memory.) The final electron accepter, oxygen has a potential of +0.8 volts. As the electrons flow, protons end up on the outside of the membrane and hydroxide ions (OH-) end up on the inside. These protons want to get back inside and join their friends the hydroxide ions. ATP synthase is more than happy to arrange the reunion and in the process synthesize ATP. Two major questions are of interest here.

  1. How is electron flow down the respiratory chain of electron carriers coupled to proton pumping?

  2. How does proton movement through ATP synthase cause ATP to be synthesized?

Proton pumping

Electrons and protons extracted from catabolism are present on electron carriers such as NADH. The electrons have a high potential (are willing to reduce many other compounds) and the ETS uses this to perform work. NADH first donates it's electrons to NADH reductase. A protein that resides on the cytoplasmic side of the membrane. This protein accepts both protons and electrons. NADH reductase then transfers its protons and electrons to a flavoprotein that is more deeply integrated in the membrane. This flavoprotein then reduces a non-heme iron protein (NH-Fe) that is deep within the membrane. NH-Fe only accepts electrons and not protons. During reduction of NH-Fe, the protons on the flavoprotein are released, and since the reaction takes place near the outside of the membrane, the protons end up being released outside the cell. In effect the three electron transfers pumped the protons across the membrane. Note that the arrangement of the proteins in the membrane dictate the path that the protons and electrons follow and where the protons eventually end up.

The free electrons are now donated to the quinone pool. Since reduction of quinone requires both protons and electrons, protons are picked up from the cytoplasm. Quinones are oxidized by the cytochrome b/c1 in a complex reaction called the Q cycle. This complex is the best understood in electron transport and a description of its function is useful when trying to get your head around the process.

Bc1protonPumping picture

Figure 2 - A figure of the proton pumping action of the cytochrome b/c1 complex. To view an animation of the process, click on the image. The animation is a shockwave movie and is 72K in size.

Each quinone donates its electrons and protons to cytochrome b. The first electron/proton pair takes a high potential path, passing through the ISP protein and onto cytochrome c1. The first reduction causes a conformational shift in the protein such that the second electron takes a low potential path ending up on the second heme group of cytochrome b. Oxidation of a second quinone results in a fully reduced heme that can then donate both pairs of electrons, and two protons from the cytoplasm back to one of the quinones. The net result is, as each electron passes to cytochrome c1, 2 protons are pumped across the membrane. View the animation to get a clear picture of what is going on.

Cytochrome c1 passes its electrons to the soluble cytochrome c that is present in the periplasm. Cytochrome c then reduces cytochrome oxidase, also know as the cytochrome a/a3 complex. Cytochrome oxidase takes electrons from cytochrome c and reduces oxygen to water; the final step in this electron transport chain. This protein also pumps protons across the membrane, but its method is completely different than the cytochrome b/c1 complex. Proton pumping is not directly associated with the reduction of electron carriers but is indirectly coupled to the process by as yet unknown mechanism.

cytooxidase picture

Summary of electron transport

A consequence of this electron flow is that protons move from the inside of the membrane to the outside and OH- accumulates in the cytoplasm. Despite their small size, neither H+ nor OH- can pass through the membrane to create an equilibrium and they accumulate on opposite sides, creating a pH and a charge gradient. The pH and charge gradient is analogous to a charged battery and like a battery has the potential to do work. This build up of protons is called the proton motive force and is used by the cell for many tasks including; transport, flagella movement and ATP synthesis.

ATP synthesis

To synthesize ATP, the protons on the outside of the membrane are allowed entry to the inside of the cell by falling through the protein ATP synthase. As ATP synthase moves protons into the cell, ATP is synthesized from ADP and inorganic phosphate. The mechanism of ATP synthesis until recently was unclear, but recent research has determined the crystal structure of ATP synthase. With the molecular structure of the enzyme, the black art of converting moving protons into ATP has become clearer.

ATP synthase structure

A cartoon of ATP synthase

Figure 3 - The structure of ATP synthase

ATP synthase is composed of two major regions, F0, a membrane bound protein complex of subunits a, b, and c, and F1 located on the cytoplasmic side of the membrane that is composed of subunits a, b, g, d and e in a ratio of 3:3:1:1:1. (see figure). A dodecamer of subunit c (that is 12 subunits) form a complex that acts something similar to a gear or rotor. F1 is connected to subunit c and rotates along with it as the gear moves. Subunit a, a dimer of subunit b and subunit d form a stator arm. This arm limits the rotation of F1 to three discrete positions.

ATP synthase function

ATP synthesis is dependant upon the rotation of F1 relative to F0 and this movement is driven by protons falling between the c subunits. A possible model suggested by Wolgang Junge is that the a-subunit provides a port for entry of protons from outside and also interacts with one of the twelve c subunits. This interaction deprotonates a conserved acidic amino acid residue on the c subunit. When a proton enters from the outside, it neutralizes the conserved acidic residue in the c-subunit. Only in this neutral form can the c-subunit now rotate away from association with the a-subunit. Rotation now brings the next neutral c-subunit to the exit port, allowing it to lose its proton (which migrates to the inside of the membrane) and associate with the a-subunit complex. Successive protonations allow the c-subunit complex to rotate. About nine to twelve protons are needed for a full rotation of ATP synthase. The number is dependent upon the number of c subunits in the complex.

ATPmem picture

Figure 4 - ATP synthase in action. To view an animation of the process, click on the image above. A quicktime movie of 640 k will be downloaded.

Rotation drives synthesis of ATP at the three b subunits of F1. The active site (The site that synthesizes ATP) on the b subunit can exist in three states, loose, tight and open. Each b subunit on F1 is in a different state. To better understand how the catalysis works, we will follow an ADP molecule through one rotation of ATP synthase. One of the b subunits is in the loose state. In this state one ADP and one inorganic phosphate bind to the active site. A one-third rotation of F1 changes the conformation of the b subunits. The subunit in the loose state, now switches to tight, bringing the ADP and phosphate in close proximity. ADP and phosphate then react to form ATP. More protons falling through subunit c cause another rotation and the b subunit in the tight state now switches to the open state. In this state, the active site does not bind ATP well and it diffuses into the cytoplasm. Another rotation changes the state back to loose and the b subunit is ready for another round. The below animation illustrates this process.

In 1997 Paul Boyer and John Walker were awarded a Nobel Prize for their contributions to understanding the novel mechanism used for ATP synthesis.

Putting it all together

To summarize, the high potential electrons on NADH and FADH are passed into the electron transport system. The energy of the electrons is used to pump protons from the inside of the membrane to the outside. This creates a proton gradient or proton motive force and that is converted to ATP by ATP synthase. Below is an animation of the whole process.

ETSFinal picture

Figure 5 - Respiration in total. Click on the above image to view an animation of the process. The animation is a shockwave movie and is 164 k in size.

For more information on bioenergetics in mitochondria (and most of this information applies to bacterial cells), read this review article in "Science Magazine"

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