W2018_Bis2A_Lecture14_reading

Substrate-level phosphorylation and Fermentation Revisited:

In Lecture 14, we will be discussing a number of topics covered in the lecture 13 reading assignment. Two concepts that many students found confusing were substrate-level phosphorylation and fermentation. Some of the reading associated with these concepts is repeated below.

Substrate-level phosphorylation (SLP)

The simplest route to synthesize ATP is substrate-level phosphorylation. ATP molecules are generated (that is, regenerated from ADP) as a direct result of a chemical reaction that occurs in catabolic pathways. A phosphate group is removed from an intermediate reactant in the pathway, and the free energy of the reaction is used to add the third phosphate to an available ADP molecule, producing ATP. This very direct method of phosphorylation is called substrate-level phosphorylation. It can be found in a variety of catabolic reactions, most notably in two specific reactions in glycolysis (which we will discuss specifically later). Suffice it to say that what is required is a high-energy intermediate whose oxidation is sufficient to drive the synthesis of ATP.

Figure 5. Here is one example of substrate-level phosphorylation occurring in glycolysis. There is a direct transfer of a phosphate group from the carbon compound onto ADP to form ATP. Attribution: Marc T. Facciotti (own work)

In this reaction, the reactants are a phosphorylated carbon compound called G3P (from step 6 of glycolysis) and an ADP molecule, and the products are 1,3-BPG and ATP. The transfer of the phosphate from G3P to ADP to form ATP in the active site of the enzyme is substrate-level phosphorylation.

Substrate-level phosphorylation occurs twice in glycolysis and once in the TCA cycle.

The "purpose" of fermentation

The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway. Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle NAD⁺ to NADH. You were already asked to figure out what options the cell might reasonably have to reoxidize the NADH to NAD⁺ in order to avoid consuming the available pools of NAD⁺ and to thus avoid stopping glycolysis. Put differently, during glycolysis, cells can generate large amounts of NADH and slowly exhaust their supplies of NAD⁺. If glycolysis is to continue, the cell must find a way to regenerate NAD⁺, either by synthesis or by some form of recycling.

In the absence of any other process—that is, if we consider glycolysis alone—it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped off of the glucose derivatives right back onto the downstream product, pyruvate, or one of its derivatives. We can generalize the process by describing it as the returning of electrons to the molecule that they were once removed, usually to restore pools of an oxidizing agent. This, in short, is fermentation. As we will discuss in a different section, the process of respiration can also regenerate the pools of NAD⁺ from NADH. Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose fermentation as an alternative mechanism for garnering energy from small molecules.

A note on the link between substrate-level phosphorylation and fermentation

Fermentation occurs in the absence of molecular oxygen (O₂). It is an anaerobic process. Notice there is no O₂ in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized to be some of the first energy-generating metabolic reactions to evolve. This makes sense if we consider the following:

1. The early atmosphere was highly reduced, with little molecular oxygen readily available.
2. Small, highly reduced organic molecules were relatively available, arising from a variety of chemical reactions.
3. These types of reactions, pathways, and enzymes are found in many different types of organisms, including bacteria, archaea, and eukaryotes, suggesting these are very ancient reactions.
4. The process evolved long before O₂ was found in the environment.
5. The substrates, highly reduced, small organic molecules, like glucose, were readily available.
6. The end products of many fermentation reactions are small organic acids, produced by the oxidation of the initial substrate.
7. The process is coupled to substrate-level phosphorylation reactions. That is, small, reduced organic molecules are oxidized, and ATP is generated by first a red/ox reaction followed by the substrate-level phosphorylation.
8. This suggests that substrate-level phosphorylation and fermentation reactions coevolved.

Suggested discussion

If the hypothesis is correct that substrate-level phosphorylation and fermentation reactions co-evolved and were the first forms of energy metabolism that cells used to generate ATP, then what would be the consequences of such reactions over time? What if these were the only forms of energy metabolism available over hundreds of thousands of years? What if cells were isolated in a small, closed environment? What if the small, reduced substrates were not being produced at the same rate of consumption during this time?

Introduction to Respiration and Electron Transport Chains

General Overview and Points to Keep In Mind

In the next few modules, we start to learn about the process of respiration and the roles that electron transport chains play in this process. A definition of the word "respiration" that most people are familiar with is "the act of breathing". When we breath, air including molecular oxygen is brought into our lungs from outside of the body, the oxygen then becomes reduced, and waste products, including the reduced oxygen in the form of water, are exhaled. More generically, some reactant comes into the organism and then gets reduced and leaves the body as a waste product.

This generic idea, in a nutshell, can be generally applied across biology. Note that oxygen need not always be the compound that brought in, reduced, and dumped as waste. The compounds onto which the electrons that are "dumped" are more specifically known as "terminal electron acceptors." The molecules from which the electrons originate vary greatly across biology (we have only looked at one possible source - the reduced carbon-based molecule glucose).

In between the original electron source and the terminal electron acceptor are a series of biochemical reactions involving at least one red/ox reaction. These red/ox reactions harvest energy for the cell by coupling exergonic red/ox reaction to an energy-requiring reaction in the cell. In respiration, a special set of enzymes carry out a linked series of red/ox reactions that ultimately transfer electrons to the terminal electron acceptor.

These "chains" of red/ox enzymes and electron carriers are called electron transport chains (ETC). In aerobically respiring eukaryotic cells the ETC is composed of four large, multi-protein complexes embedded in the inner mitochondrial membrane and two small diffusible electron carriers shuttling electrons between them. The electrons are passed from enzyme to enzyme through a series of red/ox reactions. These reactions couple exergonic red/ox reactions to the endergonic transport of hydrogen ions across the inner mitochondrial membrane. This process contributes to the creation of a transmembrane electrochemical gradient. The electrons passing through the ETC gradually lose potential energy up until the point they are deposited on the terminal electron acceptor which is typically removed as waste from the cell. When oxygen acts as the final electron acceptor, the free energy difference of this multi-step red/ox process is ~ -60 kcal/mol when NADH donates electrons or ~ -45 kcal/mol when FADH₂ donates.

Note: Oxygen is not the only, nor most frequently used, terminal electron acceptor in nature

Recall, that we use oxygen as an example of only one of numerous possible terminal electron acceptors that can be found in nature. The free energy differences associated with respiration in anaerobic organisms will be different.

In prior modules we discussed the general concept of red/ox reactions in biology and introduced the Electron Tower, a tool to help you understand red/ox chemistry and to estimate the direction and magnitude of potential energy differences for various red/ox couples. In later modules, substrate level phosphorylation and fermentation were discussed and we saw how exergonic red/ox reactions could be directly coupled by enzymes to the endergonic synthesis of ATP.

These processes are hypothesized to be one of the oldest forms of energy production used by cells. In this section we discuss the next evolutionary advancement in cellular energy metabolism, oxidative phosphorylation. First and foremost recall that, oxidative phosphorylation does not imply the use of oxygen. Rather the term oxidative phosphorylation is used because this process of ATP synthesis relies on red/ox reactions to generate a electrochemical transmembrane potential that can then be used by the cell to do the work of ATP synthesis.

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A Quick Overview of Principles Relevant to Electron Transport Chains

An ETC begins with the addition of electrons, donated from NADH, FADH₂ or other reduced compounds. These electrons move through a series of electron transporters, enzymes that are embedded in a membrane, or other carriers that undergo red/ox reactions. The free energy transferred from these exergonic red/ox reactions is often coupled to the endergonic movement of protons across a membrane. Since the membrane is an effective barrier to charged species, this pumping results in an unequal accumulation of protons on either side of the membrane. This in turn "polarizes" or "charges" the membrane, with a net positive (protons) on one side of the membrane and a negative charge on the other side of the membrane. The separation of charge creates an electrical potential. In addition, the accumulation of protons also causes a pH gradient known as a chemical potential across the membrane. Together these two gradients (electrical and chemical) are called an electro-chemical gradient.

Review: The Electron Tower

Since red/ox chemistry is so central to the topic we begin with a quick review of the table of reduction potential - sometimes called the "red/ox tower" or "electron tower". You may hear your instructors use these terms interchangeably. As we discussed in previous modules, all kinds of compounds can participate in biological red/ox reactions. Making sense of all of this information and ranking potential red/ox pairs can be confusing. A tool has been developed to rate red/ox half reactions based on their reduction potentials or E₀^' values. Whether a particular compound can act as an electron donor (reductant) or electron acceptor (oxidant) depends on what other compound it is interacting with. The red/ox tower ranks a variety of common compounds (their half reactions) from most negative E₀^', compounds that readily get rid of electrons, to the most positive E₀^', compounds most likely to accept electrons. The tower organizes these half reactions based on the ability of electrons to accept electrons. In addition, in many red/ox towers each half reaction is written by convention with the oxidized form on the left followed by the reduced form to its right. The two forms may be either separated by a slash, for example the half reaction for the reduction of NAD⁺ to NADH is written: NAD⁺/NADH + 2e^-, or by separate columns. An electron tower is shown below.

Figure 1. A common biological "red/ox tower"

Note

Use the red/ox tower above as a reference guide to orient you as to the reduction potential of the various compounds in the ETC. Red/ox reactions may be either exergonic or endergonic depending on the relative red/ox potentials of the donor and acceptor. Also remember there are many different ways of looking at this conceptually; this type of red/ox tower is just one way.

Note: Language shortcuts reappear

In the red/ox table above some entries seem to be written in unconventional ways. For instance Cytochrome c_ox/red. There only appears to be one form listed. Why? This is another example of language shortcuts (likely because someone was too lazy to write cytochrome twice) that can be confusing - particularly to students. The notation above could be rewritten as Cytochrome c_ox/Cytochrome c_red to indicate that the cytochrome c protein can exist in either and oxidized state Cytochrome c_ox or reduced state Cytochrome c_red.

Review Red/ox Tower Video

For a short video on how to use the red/ox tower in red/ox problems click here. This video was made by Dr. Easlon for Bis2A students.

Using the red/ox tower: A tool to help understand electron transport chains

By convention the tower half reactions are written with the oxidized form of the compound on the left and the reduced form on the right. Notice that compounds such as glucose and hydrogen gas are excellent electron donors and have very low reduction potentials E₀^'. Compounds, such as oxygen and nitrite, whose half reactions have relatively high positive reduction potentials (E₀^') generally make good electron acceptors are found at the opposite end of the table.

Example: Menaquinone

Let's look at menaquinone_ox/red. This compound sits in the middle of the red/ox tower with an half-reaction E₀^' value of -0.074 eV. Menaquinone_ox can spontaneously (ΔG<0) accept electrons from reduced forms of compounds with lower half-reaction E₀^'. Such transfers form menaquinone_red and the oxidized form of the original electron donor. In the table above, examples of compounds that could act as electron donors to menaquinone include FADH₂, an E₀^' value of -0.22, or NADH, with an E₀^' value of -0.32 eV. Remember the reduced forms are on the right hand side of the red/ox pair.

Once menaquinone has been reduced, it can now spontaneously (ΔG<0) donate electrons to any compound with a higher half-reaction E₀^' value. Possible electron acceptors include cytochrome b_ox with an E₀^' value of 0.035 eV; or ubiquinone_ox with an E₀^' of 0.11 eV. Remember that the oxidized forms lie on the left side of the half reaction.