The Electron Transport System of Mitochondria

Embedded in the inner membrane are proteins and complexes of molecules that are involved in the process called electron transport. The electron transport system (ETS), as it is called, accepts energy from carriers in the matrix and stores it to a form that can be used to phosphorylate ADP. Two energy carriers are known to donate energy to the ETS, namely nicotine adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). Reduced NAD carries energy to complex I (NADH-Coenzyme Q Reductase) of the electron transport chain. FAD is a bound part of the succinate dehydrogenase complex (complex II).

It is reduced when the substrate succinate binds the complex.

What happens when NADH binds to complex I? It binds to a prosthetic group called flavin mononucleotide (FMN), and is immediately re-oxidized to NAD. NAD is"recycled," acting as an energy shuttle. What happens to the hydrogen atom that comes off the NADH? FMN receives the hydrogen from the NADH and two electrons. It also picks up a proton from the matrix. In this reduced form, it passes the electrons to iron-sulfur clusters that are part of the complex, and forces two protons into the intermembrane space.

The obligatory forcing of protons into the intermembrane space is a key concept. Electrons cannot pass through complex I without accomplishing proton translocation. If you prevent the proton translocation, you prevent electron transport. If you prevent electron transport, you prevent proton translocation. The events must happen together or not at all.
Electron transport carriers are specific, in that each carrier accepts electrons (and associated free energy) from a specific type of preceeding carrier. Electrons pass from complex I to a carrier (Coenzyme Q) embedded by itself in the membrane. From Coenzyme Q electrons are passed to a complex III which is associated with another proton translocation event. Note that the path of electrons is from Complex I to Coenzyme Q to Complex III. Complex II, the succinate dehydrogenase complex, is a separate starting point, and is not a part of the NADH pathway.
From Complex III the pathway is to cytochrome c then to a Complex IV (cytochrome oxidase complex). More protons are translocated by Complex IV, and it is at this site that oxygen binds, along with protons, and using the electron pair and remaining free energy, oxygen is reduced to water. Since molecular oxygen is diatomic, it actually takes two electron pairs and two cytochrome oxidase complexes to complete the reaction sequence for the reduction of oxygen. This last step in electron transport serves the critical function of removing electrons from the system so that electron transport can operate continuously.
The reduction of oxygen is not an end in itself. Oxygen serves as an electron acceptor, clearing the way for carriers in the sequence to be reoxidized so that electron transport can continue. In your mitochondria, in the absence of oxygen, or in the presence of a poison such as cyanide, there is no outlet for electrons. All carriers remain reduced and Krebs products become out of balance because some Krebs reactions require NAD or FAD and some do not. However, you don't really care about that because you are already dead. The purpose of electron transport is to conserve energy in the form of a chemiosmotic gradient. The gradient, in turn, can be exploited for the phosphorylation of ADP as well as for other purposes. With the cessation of aerobic metabolism cell damage is immediate and irreversible.
From succinate, the sequence is Complex II to Coenzyme Q to Complex III to cytochrome c to Complex IV. Thus there is a common electron transport pathway beyond the entry point, either Complex I or Complex II. Protons are not translocated at Complex II. There isn't sufficient free energy available from the succinate dehydrogenase reaction to reduce NAD or to pump protons at more than two sites.

Is the ETS a sequence?

Before the development of the fluid mosaic model of membranes, the ETS was pictured as a chain, in which each complex was fixed in position relative to the next. Now it is accepted that while the complexes form 'islands' in the fluid membrane, they move independently of one another, and exchange electrons when they are in mutual proximity. Textbooks necessarily show the ETS as a physical sequence of complexes and carriers. This has the unintentional effect of implying that they are all locked in place. The fluid nature of membranes allows electron exchange to take place in a test tube containing membrane fragments.
The location of ETS complexes on the inner membrane has two major consequences. By floating in two-dimensional space, the likelihood of carriers making an exchange is much higher than if they were in solution in the three dimensional space of the matrix. They are exposed to the matrix side of the membrane, of course, for access to succinate and NADH, but have limited mobility. Second, the location of the ETS on the inner membrane enables them to establish a chemiosmotic gradient.

Electron pathways and inhibition

Electron transport inhibitors act by binding one or more electron carriers, preventing electron transport directly. Changes in the rate of dissipation of the chemiosmotic gradient have no effect on the rate of electron transport with such inhibition. In fact, if electron transport is blocked the chemiosmotic gradient cannot be maintained. No matter what substrate is used to fuel electron transport, only two entry points into the electron transport system are known to be used by mitochondria. A consequence of having separate pathways for entry of electrons is that an ETS inhibitor can affect one part of a pathway without interfering with another part. Respiration can still occur depending on choice of substrate.

An inhibitor may competely block electron transport by irreversibly binding to a binding site. For example, cyanide binds cytochrome oxidase so as to prevent the binding of oxygen. Electron transport is reduced to zero. Breathe all you want - you can't use any of the oxygen you take in. Rotenone, on the other hand, binds competitively, so that a trickle of electron flow is permitted. However, the rate of electron transport is too slow for maintenance of a gradient. 

Chemiosmotic Gradient: Generation and Maintenance

Energetics of proton translocation at Compex I

The energy needed to push protons out of the matrix and into the intermembrane space comes from the oxidation of either reduced NAD (NADH) or reduced FAD (FADH2). In the case of NADH, passage of an electron pair to Coenzme Q provides a "pulling" force. That is, NADH is much less electronegative than Coenzyme Q, while the iron-sulfur protein carriers in between are intermediate in electronegativity. The amount of available free energy is 69.5 kJ/mole of NADH (kiloJoules per mole). The efficiency of electron transport can be represented by the standard reduction potential difference, namely the voltage generated by a redox reaction under standard biochemical conditions.

The standard reduction potential of NADH is -0.315V, while that of coenzyme Q is 0.045V (difference of 0.345 V). Therefore there is a strong 'pull' by Coenzyme Q on electrons through the components of Complex I.
Just for the sake of understanding the principles, let Complex I (NADH dehydrogenase complex), embedded in an intact inner membrane, be the only component of an experimental electron transport system. We'll simply take the electrons from Coenzyme Q when they reach it, so the system can keep going. The removal of protons from the matrix and deposition of protons in the intermembrane space creates a concentration difference of protons across the inner membrane. This is called the chemiosmotic gradient. As the gradient builds up, more and more energy is required to push protons across. When the amount of energy required to push protons reaches 69.5 kJ/mole, electron transport has to stop. In fact, the second law of thermodynamics requires that electron transport stop before the gradient builds up to that point. 

If there was no way of draining energy from the system, electron transport could not continue despite the presence of adequate substrate. However, a mitochondrion is always in a steady state of respiration, in which the energy lost by processes that dissipate the gradient is constantly replaced by electron transport.

Respiratory Control

The limitation placed on electron transport by the chemisosmotic gradient is termed respiratory control. Mitochondria are said to exercise respiratory control as long as they can restrict electron transport by means of the gradient. If the gradient is destroyed by damaging the membranes, respiratory control is abolished and electron transport can run freely.

Cause and effect relationship

Electron transport is driven by the free energy that is available from the energy carriers, in turn obtained from substrates such as glutamate or Krebs intermediates. It is restricted by the chemiosmotic gradient. The only way electron transport can proceed is to the extent that the energy in the gradient is dissipated. In healthy mitochondria the gradient is maintained. That is, electron transport keeps up with the utilization of the energy stored in the gradient. Even in the presence of ADP, which allows ATP synthetase to exploit the gradient, the chemiosmotic gradient is maintained at a set energy level.

Think of physical causes and effects when you attempt to describe respiratory control. What drives electron transport? It can't be driven by a "need" to maintain the gradient, because that implies a sense of purpose. The electron transport system is just a structure, complex as it is. Electron transport is driven by the increasing affinities of successive carriers for electrons, and by the availability of substrates to provide electrons and free energy. It is restricted by the chemiosmotic gradient - electron transport can only go as fast as energy is lost from the gradient. Anything that increases turnover of energy from the gradient increases the rate of electron transport proportionally.
Here is an analogy that might help with the concept. Suppose that you are trying to blow up a balloon. Blowing with all your might you succeed in filling the balloon so that it is two feet in diameter. You cannot force any more air into it so all you are doing now is holding pressure. Now some joker comes along and pokes a couple of holes in your balloon, so that air leaks out at a controlled rate. You have maintained constant pressure, so now you find yourself moving air into the balloon as it leaks out. The balloon diameter, proportional to internal pressure, remains constant. If you plug one of the holes you move less air. Open up more holes and you move more air. The electron transport system applies constant "pressure," holding the gradient at a constant level. The rate of electron tranport (analagous to air flow in the balloon example) varies as energy is drained from the system at different rates. Adding ADP in vitro, for example, opens up avenues for protons to be forced into the matrix, draining energy from the gradient. Its addition is the equivalent of poking additional holes in your balloon. Electron transport spontaneously increases.

Respiratory Control in Exercise

Sometimes in wading through all of the details, one loses sight of the big picture. Just why is all of this information about electron transport and oxidative phosphorylation so important, anyhow? Suppose you are vegging in front of the TV - in the prone position with a bag of cholesterol chips. You are very relaxed and are hardly aware that you are breathing at all. Suddenly the fire alarm sounds, and a bunch of naked people are running down the hall. This definititely provokes activity on your part, and you run down the hall also. You start breathing heavily (due to the exercise, not the sight of the naked people).

Your demand for energy is rather low when you are relaxed. However running is heavy exercise. Your muscle activity causes an immediate demand for ATP which is met in part by muscle glycogen. When that runs low you need to replace your reserves with aerobic metabolism, that is, your mitochondria need to make more ATP. Electron transport is stimulated when the ratio of ATP to ADP goes down. The rate of binding of ADP to the ATP synthetase automatically increases as more ADP is transported into the matrix. In turn, electron transport is allowed to speed up - consuming more oxygen in the process. Sensors in your cardiovascular system (primarily the carotid bodies of the carotid arteries) detect an increase in carbon dioxide (indicating an increased need for oxygen). The sensors send a signal to the brain via the nervous system that you need to speed up breathing.
So... you breathe primarily to get oxygen into the blood and to the tissues, in order to deliver oxygen to the mitochondria for cellular respiration - to make ATP. In the process, carbon dioxide is returned to the lungs and exhaled. The processes are essential to your well being, in fact, to your being at all. When you think of the effects of some of the poisons that are discussed on these pages, think of the consequences to an intact organism such as yourself.
The big picture, including regulatory pathways such as the very simplified description presented above, are in the realm of integrative disciplines such as physiology. Such disciplines are increasingly ignored in both research and academic curricula in favor of a purely molecular approach to science. Please consider the importance of knowing the consequences of molecular and cellular processes to the whole organism, and the fact that the regulatory pathways controlling those processes are still incompletely understood.

Uncoupling and the basal metabolic rate

Endotherms like ourselves (formerly called homeotherms) maintain a constant body temperature. The setpoint for that temperature is determined by a region the hypothalamus (part of the brain, for you newcomers). But just how is the temperature regulated? That is, how does the body maintain a balance between heat production and heat loss?

The answer to that one is very complex. There are many mechanisms for heat dissipation and retention (sweating, changes in distribution of the circulation, shivering, piloerection - no, it's not a dirty word, etc.). Heat production results from the loss of some of the free energy of every chemical reaction as heat - that is, every chemical reaction results in the loss of some of the total free energy in the universe. That's one of the laws of thermodynamics - I forgot which. A major source of heat production is electron transport in mitochondria.
How do mitochondria produce heat? Much of the energy released by electron transport does no useful work at all (that law of thermodynamics is at work again). That is, each exchange of electrons results in a loss of some energy as heat. Furthermore, dissipation of the chemiosmotic gradient without doing work results in the transformation of a great deal more energy to heat. The mechanism of respiratory control can be exploited to increase heat production by dissipating the gradient at a faster rate, or to slow heat production by making the ETS more efficient. One example of that type of regulation is the hormone thyroxine (thyroid hormone).
Thyroid hormone has many functions, mostly associated with promotion of anabolic activity - synthesis of compounds and cellular growth. However thyroxine is also a mildly effective uncoupling agent. Uncoupling agents dissipate the chemical gradient, usually by creating a mechanism by which protons can escape the intermembrane space (or otherwise 'short out' the gradient), allowing an increase in electron transport. In colder regions of the world a noticible change in thyroid hormone levels takes place circannually, that is, levels are higher in winter and lower in summer.