MCRB 500/BIOL 400 Lectures
Weekly Update
Fall 1995

The information presented here is not meant to be a substitute for class notes taken during lecture. Instead, the information is presented to highlight the material covered in lecture and to provide a source of text to enable you to correct errors you may have made within your lecture notes. If you have any questions about the material, email your question and I will post both your question and my answer to Replies.

Copyright, Jack Brown, Department of Microbiology, University of Kansas. 1995
WEEK FOUR - Sept 11 - Sept 15

The Electron Transport System

We will discuss how electrons removed from glucose (oxidation) and accepted by compounds such as NAD+ and FAD to form the reduced compounds NADH + H+ and FADH2, ultimately reduce molecular oxygen and in the process provide the energy for ATP synthesis. Remember, this system is comprised of proteins, some of which are enzymes, and is located as an integral part of the bacterial cell's plasma membrane (and the inner membrane of eucaryotic mitochondria). It is important to develop a basic understanding of redox reactions and the energy available from such reactions.

We all know that if electrical charges of different sign, e.g., positive/negative are separated from one another - a positive and a negative pole - a true force exists between them - an electrical potential difference exists between the regions of opposite charge (the two poles). In such an arrangement there is a potential for electrons to flow from the negative pole toward the positive pole - from a higher to lower energy level - therefore there exists a tendency to eliminate the gradient and return the system to neutrality (a lower, more thermodynamically-stable energy state). This potential is actually in the form of potential energy, a force similar to the potential energy present if one is standing on top of a hill. This electrical force, named voltage, is analogous to water pressure; consequently, if a conductor is present (a water pipe) and a switch is available (a valve) to provide a connection between the two poles, electrons (water) will flow and the pressure exhibited will be in the form of volts. The rate of flow - the current - depends on the conductor (type of pipe) and voltage (pressure) available.

Chemical reactions can be described in a similar fashion if the reactions involve a flow of electrons from one substance to a different substance - an oxidation/reduction or redox reaction. And, we can use what is known about chemical equilibria to develop an understanding of this potential energy (voltage) in terms of Standard Free Energy in calories. To understand this relationship, the following equation is used:
dG' = - (nFdE') where, n = # of electrons involved; F = Faraday's constant = 23,063 calories/mole/equivalent; and dE' = potential difference in volts. We know that reactions with negative dG' values are thermodynamically favorable as written. Therefore, for a redox reaction to be thermodynamically favorable, and to provide a - dG' in the above equation, the value of (nFdE') must be positive. Since the # of electrons will be positive, and Faraday's constant is positive, then, the value for dE' must also be positive. For practice, let's use a reaction for which we already know the dG' value, e.g., the hydrolysis of ATP to ADP and Pi. For this reaction, dG' = - 7.3 kcal/mole. Now, let's assume transfer of two (2) electrons. Then, substitute these known values in the above equation, and solve for dE'.
Then, - 7,300 cal/mole = - [ (2)(23,063 cal/mole/volt-equivalent)(dE') ]
Then, - 7,300 cal/mole = - [ (46,126 cal/mole/volt-equivalent)(dE') ]
Then, - 7.300 cal/mole//46,126 cal/mole/volt-equivalent = - dE'
Then, - dE' = - 0.158 volts; or, dE' = 0.158 volts
Therefore, if one wished to determine how much energy in calories/mole of volt equivalents would be available for 2 electrons transferred in a reaction and a potential energy difference of 0.158 volts between reactants, we would obtain - 7,300 calories/mole; or, the amount of energy obtained upon hydrolysis of one of the high-energy bonds in ATP. Consequently, if ATP is synthesized from ADP + Pi via redox reactions, we'd better have about 158 mVolts available as potential energy.

In order to see what is going on in such reactions, we must first write only 1/2 of the reaction - 1/2 of the redox couple. Well, which one do we write - the substance accepting or giving electrons? In which direction do we write the reaction - as an oxidation or as a reduction? How do we compare different substances with regard to their tendency to accept or to provide electrons? Lots of people in the past pondered these questions and decided to hold a meeting - a convention. At the convention everyone agreed to agree on some rules. therefore:
1. Standard Reduction Potential ( dE')
By convention the Standard Reduction Potential ( dE') of a given 1/2-reaction relative to the universal standard, hydrogen, is defined as: the electrical potential in volts of a given substance to accept electrons when the 1/2-reaction is written as a reduction; and, the more positive the voltage value, the more likely the substance will accept electrons. We can see how this convention works if we take a look at some familiar compounds and write all of the 1/2-reactions in exactly the same way, e.g., as reduction reactions - their reaction with electrons to produced a reduced substance.

Redox 1/2-reaction		Potential Difference (dE') in Volts
NAD+ + 2e- + 2H+ => NADH + H+             - 0.320
1/2O2 + 2e- + 2H+ => H2O                  + 0.816
If one examines the two reactions, it is clear that NAD+ is substantially less likely to accept electrons relative to O2. Indeed, reversing this reaction would yield a positive dE'; therefore, it is more likely that NADH + H+ would give up electrons to something. Similarly, it is clear that O2 is very likely to accept electrons. With this information, one may now couple these two reactions and observe the result of an overall redox reaction between these two substances. First, we can write each 1/2-reaction in the direction most probable, and then add the two reactions:
NADH + H+ => NAD+ + 2e- + 2H+ and the dE' = + 0.320 volts
1/2O2 + 2e- + 2H+ => H2O and the dE' = + 0.816 volts

Now, we can sum the two reactions:
1/2O2 + 2e- + 2H+ + NADH + H+ => H2O + NAD+ + 2e- + 2H+ and dE' = + 1.136 volts
By eliminating reactants which appear on both sides, we obtain:
1/2O2 + NADH + H+ => H2O + NAD+ and dE' = + 1.136 volts
Consequently, O2 will accept electrons given by NADH + H+ and will become reduced to water as NADH + H+ is oxidized to NAD+; and, this particular redox couple will yield 1.136 volts of energy - which can be applied to the synthesis of substances such as ATP, for example. Therefore, we can see that electrons are more likely to flow from substances with a more negative dE' toward substances with a more positive dE', if the original 1/2 reactions are written as reductions to give Standard Reduction Potentials. Knowing these relationships, we can now examine the integral plasma-membrane components and the 1/2-reactions which are part of the Electron Transport System.

1/2-Reaction                             dE' in Volts
NAD+ + 2e- + 2H+  =>  NADH + H+            - 0.320
FAD  + 2e- + 2H+  =>  FADH2                - 0.060
Cytochrome b (Fe+3) + e- => Cyt b (Fe2+)   + 0.040
Cyt c (Fe+3)  + e-       => Cyt c (Fe+2)   + 0.250
Cyt a (Fe+3)  + e-       => Cyt a (Fe+2)   + 0.290
1/2O2 + 2e- + 2H+ =>  H2O                  + 0.816
Therefore, as electrons and protons are generated by the oxidation of NADH + H+ to NAD+ in the electron transport system, and the released electrons are shuttled through the cytochrome proteins in a step-wise manner to eventually react with O2, O2 is reduced to water - with an overall voltage produced of 1.136 volts. This energy is subseqently used for the synthesis of ATP from ADP and Pi in an elegant reaction catalyzed by the integral plasma membrane enzyme, ATP synthetase. If we calculate the theoretical yield of ATP from 1,136 mV, we simply divide 1,136mV by 160 mV (the amount required for one high-energy bond in ATP) and we obtain: approximately 7.0. However, we know that oxidation of NADH + H+ in aerobic respiration yields only 3 ATP. Well the system isn't perfect! Still, we achieve a 3/7 yield, which is approximately 42% of maximum - an astonishing efficiency of energy conservation! Now, we need to determine exactly how ATP synthesis is accomplished.

2. The Proton Motive Force

The catalytic mechanism through which ATP synthetase functions is not yet completely understood. What is known is that an energy gradient in the form of a voltage difference (potential energy difference) across the semi-permeable plasma membrane is somehow applied to form a covalent bond between a molecule of ADP and a molecule of Pi which are present within the active site of the enzyme. If the voltage gradient is disrupted, ATP synthesis by this mechanism, ceases. How then is this voltage gradient formed? We know that the presence of reduced compounds, an electron transport system and reduction of molecular oxygen in aerobic respiration provide voltage (1.136 volts) for every NADH + H+ available. From years of research of this system, it has been determined that there is a voltage difference across the bacterial cell's plasma membrane. This voltage (energy) gradient is formed as follows:
As the electrons are shuttled "down" the electron transport chain, protons also follow along. However, the protons (H+) do not passage through the chain, but instead are vectored outside of the cell. The proteins of the electron transport system are physically oriented in such a way that the H+ (protons) are always extruded to the outside of the cell. Consequently, there will develop a net positive charge on the external side of the plasma membrane - a pH difference - a pH gradient - a charge difference - a charge gradient. Since the dissipation (elimination) of any kind of gradient, chemical or electrical, is favored thermodynamically, there is a "pressure" generated across the membrane to return the H+ back inside of the cell. As we've said before, this pressure is voltage- potential energy. The pH difference is maintained because only water and gases passively cross the plasma membrane - all other substances, including protons (H+), require specific transport mechanisms.

Too, the reduction of molecular oxygen requires not only the acceptance of the passaged electrons, but also the acceptance of protons to form H2O. Since the protons entering the electron transport system are vectored outside of the cell, the protons which react with oxygen must come from the cytoplasm. Consequently, there is a net loss of available protons (H+) from the cytoplasm, therefore a decrease in positive charge on the inside of the cell as aerobic respiration occurs. Together then, the increase in positive charge outside of the cell, and the decrease in positive charge inside of the cell, generates the charge-difference across the semi-permeable plasma membrane.

3. ATP Synthetase

As previously stated, because of the pH gradient across the plasma membrane, there is a stong "pressure" (voltage) generated for the H+ to re-enter the cell - which would result in dissipation of the gradient - and therefore a highly favorable thermodynamic condition. However, the H+ cannot re-enter the cell without a specific transport mechanism. It is here that ATP synthetase provides the mechanism. The enzyme is structured in a way which provides a conduit for H+ to enter the bacterial cell. Some of the voltage energy that is made available when H+ fires through this enzyme and returns to the cytoplasm is captured by this enzyme and used in a catalytic reaction to generate ATP from ADP and Pi. Thus, for every NADH + H+ which enters the system, 3 ATP are synthesized, and for every FADH2, 2 ATP are synthesized if aerobic respiration occurs.

4. Other Mechanisms which Dissipate the pH Gradient

Remember the discussion about Active Transport. In this instance we said that active transport was the energy-requiring transport of a substance across the plasma membrane which occurred against a concentration gradient. Now, assume we have a symporter which transports an amino acid X against a concentration gradient (more amino acid X in the cytplasm relative to the outside of the cell) and an additional substance. If the additional substance is a proton (H+), the energy released by dissipation of the pH gradient can provide the energy to transport amino acid X. In this way the Proton Motive Force is involved in active transport of chemical substances. Therefore, many symporter and antiporter transport proteins are also conduits which allow H+ to re-enter the cytoplasm - they have two sites, one which specifically transports H+, and one which specifically transports a chemical substance. Now, if the H+'s which form the Proton Motive Force on the outside of the cell enter the cell through transport proteins instead of ATP synthetase, the cell loses some capacity for the synthesis of ATP. It is for this reason that active transport is an energy-requiring process.

Copyright, Jack Brown, Department of Microbiology, University of Kansas, 1995

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