2020_Spring_Bis2A_Facciotti_Lecture_12
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Metabolism in BIS2A
Cellular metabolism represents roughly 1/3 of the BIS2A curriculum. While this may seem like a lot, we cover very little of what a classic course in metabolism (like BIS103) covers, and a minuscule fraction of the metabolism that occurs on the planet. What we cover, however, is the very important foundational knowledge. You will learn about common chemical reactions that
What have we learned? How will it relate to metabolism?
- We have focused on the identification and chemical properties of common biological functional groups. As we dive into metabolism, this will help you be familiar with and sometimes even predict the chemical nature/reactivity of compounds you have never seen before.
- We have practiced recognizing and classifying molecules into four major functional groups. This will help you as we discuss how to build and break down these molecules.
- We have learned some basic thermodynamics. This gives us a common set of concepts with which to discuss whether a biochemical reaction or process is likely to occur, and if so in which direction and how fast. This will be critical as we consider some key reactions that take place in metabolism.
- We have learned and practiced the energy story rubric. This will allow us to study new biochemical reactions and to discuss them with a common, consistent language and approach
which also reinforces the lessons we learned about thermodynamics.
An overview of this section
- We will introduce an important concept called reduction
potential andyou will be given the opportunity to use a redox tower. There is also a discussion on redox chemistry in your discussion manual. Use both resources. - We will introduce two major players in metabolism, ATP and NADH. We expect you to recognize their structures if shown on an exam.
- We will cover the metabolic pathway glycolysis. Keep in mind that we want you to look at any reaction and tell us an energy story of that reaction. You should not try to memorize these pathways (though it will help to remember some big picture things - we will stress these). Often we will give you the pathway as a figure on the exams. Glycolysis produces 2 ATP via a process called substrate level phosphorylation, 2 NADH and 2 pyruvate compounds.
- We will use the reactions of the TCA cycle to create multiple examples of energy stories. The TCA cycle will also produce more ATP, NADH and oxidize glucose into CO2.
- We will look at an alternative pathway to that of the TCA cycle, fermentation. Here, for the first time, we will see NADH used as a reactant in a metabolic reaction.
- We will follow NADH to the end of its journey, as it donates its electrons to the electron transport chain (ETC). In this module, you will need to use a redox tower. The ETC produces a proton gradient. No ATP
is directly generated in this process. However, the proton gradientis then used by the cell to run an enzyme called ATP synthase, whichcatalyzes the reaction ADP + Pi --> ATP. This method of ATP production (called oxidative phosphorylation) results in more ATP being produced than through substrate level phosphorylation. - And finally, we will go through the process of photosynthesis.
Reduction-Oxidation Reactions
In this class, most of the oxidation/reduction reactions that we discuss occur in metabolic pathways (connected sets of metabolic reactions) where compounds consumed by the cell
Lets start with some generic reactions
Transferring electrons between two compounds results in one of these compounds losing an electron and one compound gaining an electron. For example, look at the figure below. If we use the energy story rubric to look at the overall reaction, we can compare the before and after characteristics of the reactants and products. What happens to the matter (stuff) before and after the reaction? Compound
Put another way, when an electron
In
Remember the Definitions:
The Half Reaction
To formalize our common understanding of red/ox reactions, we introduce the concept of the half reaction. A full red/ox reaction requires two half reactions. We can think each half reaction as a description of what happens to one of the two molecules involved in the full red/ox reaction. We illustrate this below. In this example, compound AH is being oxidized by compound B+; electrons are moving from AH to B+ to generate A+ and BH. Each reaction can
Reduction Potential
By convention we analyze and describe red/ox reactions
What is this intrinsic property to attract electrons?
Different compounds, based on their structure and atomic composition have intrinsic and distinct attractions for electrons.
Possible NB Discussion Point
Rephrase for yourself: How do you describe or think about the difference between the concept of electronegativity and red/ox potential?
The Red/ox Tower
All kinds of compounds can take part in red/
For example, we write the half reaction for the reduction of NAD+ to NADH:
NAD+/NADH. The tower below also lists the number of electrons that
oxidized form |
|
|
Eo´ (volts) |
---|---|---|---|
PS1* (ox) |
PS1* (red) |
- |
-1.20 |
Acetate + CO2 |
pyruvate |
2 |
-0.7 |
ferredoxin (ox) version 1 |
ferredoxin (red) version 1 |
1 |
-0.7 |
succinate + CO2 + 2H+ |
a-ketoglutarate + H2O |
2 |
-0.67 |
PSII* (ox) |
PSII* (red) |
- |
-0.67 |
P840* (ox) |
PS840* (red) |
- |
-0.67 |
acetate |
|
2 |
-0.6 |
|
glyceraldehyde-3-P + H2O |
2 |
-0.55 |
O2 |
O2- |
1 |
-0.45 |
ferredoxin (ox) version 2 |
ferredoxin (red) version 2 |
1 |
-0.43 |
CO2 |
|
24 |
-0.43 |
CO2 |
formate |
2 |
-0.42 |
|
H2 |
2 |
-0.42 (at [H+] = 10-7; Note: at [H+] = 1; |
α-ketoglutarate + CO2 + 2H+ |
isocitrate |
2 |
-0.38 |
acetoacetate |
b-hydroxybutyrate |
2 |
-0.35 |
Cystine |
cysteine |
2 |
-0.34 |
Pyruvate + CO2 |
malate |
2 |
-0.33 |
NAD+ + 2H+ |
NADH + H+ |
2 |
-0.32 |
NADP+ + 2H+ |
NADPH + H+ |
2 |
-0.32 |
Complex I FMN (enzyme bound) |
FMNH2 |
2 |
-0.3 |
Lipoic |
Lipoic acid, (red) |
2 |
-0.29 |
1,3 bisphosphoglycerate + 2H+ |
glyceraldehyde-3-P + Pi |
2 |
-0.29 |
Glutathione, (ox) |
Glutathione, (red) |
2 |
-0.23 |
FAD+ (free) + 2H+ |
FADH2 |
2 |
-0.22 |
Acetaldehyde + 2H+ |
|
2 |
-0.2 |
Pyruvate + 2H+ |
lactate |
2 |
-0.19 |
Oxalacetate + 2H+ |
malate |
2 |
-0.17 |
α-ketoglutarate + NH4+ |
glutamate |
2 |
-0.14 |
FAD+ + 2H+ (bound) |
FADH2 (bound) |
2 |
0.003-0.09 |
Methylene blue, (ox) |
Methylene |
2 |
0.01 |
Fumarate + 2H+ |
succinate |
2 |
0.03 |
CoQ (Ubiquinone - UQ + H+) |
UQH. |
1 |
0.031 |
UQ + 2H+ |
UQH2 |
2 |
0.06 |
Dehydroascorbic acid |
ascorbic acid |
2 |
0.06 |
Plastoquinone; (ox) |
Plastoquinone; (red) |
- |
0.08 |
Ubiquinone; (ox) |
Ubiquinone; (red) |
2 |
0.1 |
Complex III Cytochrome b2; Fe3 |
Cytochrome b2; Fe2 |
1 |
0.12 |
Fe3+ (pH = 7) |
Fe2+ (pH = 7) |
1 |
0.20 |
Complex III Cytochrome c1; Fe3 |
Cytochrome c1; Fe2 |
1 |
0.22 |
Cytochrome c; Fe3 |
Cytochrome c; Fe2 |
1 |
0.25 |
Complex IV Cytochrome |
Cytochrome |
1 |
0.29 |
1/2 O2 + H2O |
H2O2 |
2 |
0.3 |
P840GS (ox) |
PS840GS (red) |
- |
0.33 |
Complex IV Cytochrome a3; Fe3 |
Cytochrome a3; Fe2 |
1 |
0.35 |
Ferricyanide |
ferrocyanide |
2 |
0.36 |
Cytochrome f; Fe3 |
Cytochrome f; Fe2 |
1 |
0.37 |
PSIGS (ox) |
PSIGS (red) |
. |
0.37 |
Nitrate |
nitrite |
1 |
0.42 |
Fe3+ (pH = 2) |
Fe2+ (pH = 2) |
1 |
0.77 |
1/2 O2 + 2H+ |
H2O |
2 |
0.816 |
PSIIGS (ox) |
PSIIGS (red) |
- |
1.10 |
* Excited GS Ground State, PS1: Oxygenic photosystem I P840: Bacterial reaction center containing bacteriochlorophyll (anoxygenic) PSII: Oxygenic photosystem II |
Video on electron tower
For a short video on how to use the electron tower in red/
What is the relationship between ΔE0' and ΔG?
The question now becomes: how do we know if any
Figure 3. Generic red/ox reaction with half reactions written with
The change in ΔE0' correlates to changes in Gibbs free energy, ΔG.
Figure 4. The Nernst equation relates free energy of a red/ox reaction to the difference in reduction potential between the reduced products of the reaction and oxidized reactant.
Attribution:
Where:
n is the number of moles of electrons transferred- F is the Faraday constant of 96.485 kJ/V. Sometimes it
is given in units of kcal/V which is 23.062 kcal/V, which is the amount of energy (in kJ or kcal) released when one mole of electrons passes through a potential drop of 1volt
Note:
What you should notice is that ΔG and ΔE have an inverse relationship: When ΔG is positive, ΔE is negative and when ΔG is negative, ΔE is positive. For additional review see the red/ox discussion in the Bis2A Discussion Manual.
Introduction to Mobile Energy Carriers
Section Summary
Energy moves and transfers within the cell in a variety of ways. One critical mechanism that nature has developed is the use of recyclable molecular energy carriers. While there are several major recyclable energy carriers, they all share some common functional features:
Properties of Key Cellular Molecular Energy Carriers
- We think of the energy carriers as existing in "pools" of
available carriers. One could, by analogy, consider these mobile energy carriers analogous to the delivery vehicles of parcel carriers—the company has a certain "pool" ofavailable vehicles at any one time to pickup and make deliveries. - Each individual carrier in the pool can exist in one of multiple distinct states: it is
either carrying a "load" of energy, a fractional load, or is "empty". The molecule can interconvert between "loaded" and empty and thus canbe recycled . Again by analogy, the delivery vehicles can beeither carrying packages or be empty and switch between these states. - The balance or ratio in the pool between "loaded" and "unloaded" carriers is important for cellular function,
is regulated by the cell, and can often tell us something about the state of a cell. Likewise, a parcel carrier service keeps close tabs on how full or empty their delivery vehicles are—if they are too full, there may be insufficient "empty" trucks to pick up new packages; if they are too empty, business must not be going well or they shut it down. There is an appropriate balance for different situations.
In this course, we will examine two major types of molecular recyclable energy carriers: (1) the adenine nucleotides: nicotinamide adenine dinucleotide (NAD+), a close relative, nicotinamide adenine dinucleotide phosphate (NADP+), and flavin adenine dinucleotide (FAD2+) and (2) nucleotide mono-, di-, and triphosphates, with particular attention paid to adenosine triphosphate (ATP). These two types of molecules
Red/ox chemistry and electron carriers
The oxidation of, or removal of an electron from, a molecule (whether accompanied with the removal of an accompanying proton or not) results in a change of free energy for that molecule—matter, internal energy, and entropy have all changed. Likewise, the reduction of a molecule also changes its free energy. The magnitude of change in free energy and its direction (positive or negative) for a red/ox reaction dictates the spontaneity of the reaction and how much energy it transfers. In biological systems, where a great deal of energy transfer happens via red/ox reactions, it is important to understand how these reactions
Note: BURNING BEAR
Relate the burning of (the full oxidation of the sugar in) a gummy bear with the last paragraph above. What does that demonstration have to do with our upcoming discussion on red/ox carriers? There is some mention above already—can you find it?
Note: DESIGN CHALLENGE
The problem alluded to in the previous discussion question is a great place to bring in the design challenge rubric. If you recall, the first step of the rubric asks that you define a problem or question. Here, let's imagine that there is a problem to define for which the mobile electron carriers below helped Nature solve.
***Remember, evolution DOES NOT forward-engineer solutions to problems, but in retrospect, we can use our imagination and logic to infer that what we see preserved by natural selection provided a selective advantage, because the natural innovation "solved" a problem that limited success.***
Design challenge for red/ox carriers
- What was a problem
( s) that the evolution of mobile electron carriers helped solve? - The next step of the design challenge asks you to identify criteria for successful solutions. What are criteria for success in the problem you've identified?
- Step 3 in the design challenge asks you to identify solutions. Well, here Nature has identified some for us—we consider three in the reading below. It looks like Nature is happy to have multiple solutions to the problem.
- The penultimate step of the design challenge rubric asks you to test the proposed solutions against the criteria for success. This should make you think/discuss why there are multiple different electron carriers. Are there different criteria for success? Are they each solving slightly different problems? What do you think? Be on the lookout as we go through metabolism for clues.
NAD+/H and FADH/H2
In living systems, a small class of compounds function as electron shuttles: they bind and carry electrons between compounds in different metabolic pathways. The principal electron carriers we will consider derive from the B vitamin group and nucleotides. These compounds can both become reduced (that is, they accept electrons) or oxidized (they lose electrons) depending on the reduction potential of a potential electron donor or acceptor that they might transfer electrons to and
We are expecting you to memorize the two forms of NAD+/NADH, know which form
NAD+ can accept electrons from an organic molecule according to the general equation:
Here is some vocabulary review: when electrons
You need to get this down! We will (a) test specifically on your ability to do so (as "easy" questions), and (b) we will use the terms
You will also encounter a second variation of NAD+, NADP+. It is structurally very similar to NAD+, but it contains an extra phosphate group and plays an important role in anabolic reactions, such as photosynthesis. Another nucleotide-based electron carrier that you will also encounter in this course and beyond, flavin adenine dinucleotide (FAD+), derives from vitamin B2, also called riboflavin. Its reduced form is FADH2. Learn to recognize these molecules as electron carriers.
Figure 1. The oxidized form of the electron carrier (NAD+)
The cell uses NAD+ to "pull" electrons off of compounds and to "carry" them to other locations within the cell;
Energy story for a red/ox reaction
***
When NADH is a product and NAD+ is a reactant, we know that NAD+ has become reduced (forming NADH);
Figure 2. This reaction shows the conversion of pyruvate to lactic acid coupled with the conversion of NADH to NAD+. Source: https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/sequential_reactions
In the figure above, we see pyruvate becoming lactic acid, coupled with the conversion of NADH into NAD+. LDH catalyses this reaction. Using our "rule of thumb" above, we categorize this reaction as a red/ox reaction. NADH is the reduced form of the electron carrier, and NADH is converted into NAD+. This half of the reaction results in the
Figure 3. Above are a series of compounds than can
Figure 4. This reaction shows the conversion of G3P, NAD+, and Pi into NADH and 1,3-BPG. This reaction
Energy story for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase:
Lets make an energy story for the reaction above.
First, let us characterize the reactants and products. The reactants are glyceraldehyde-3-phosphate (a carbon compound), Pi (inorganic phosphate), and NAD+. These three reactants enter a chemical reaction to produce two products, NADH and 1,3-
What can we say about this reaction when it
This is a red/ox reaction. We know that because we have produced a reduced electron carrier (NADH) as a product and NAD+ is a reactant. Where did the electron come from to make NADH? The electron must have come from the other reactant (the carbon compound).
ATP
An important chemical compound is adenosine triphospate (ATP). The main cellular role of ATP is as a “short-term” energy transfer device for the cell. The hydrolysis reactions that liberate one or more of ATP's phosphates are exergonic and many, many cellular proteins have evolved to interact with ATP in ways that help facilitate the transfer of energy from hydrolysis to myriad other cellular functions. In this way, ATP is often called the “energy currency” of the cell: it has reasonably fixed values of energy to transfer to or from itself and can exchange that energy between many potential donors and acceptors. We will see many examples of ATP "at work" in the cell, so be looking for them. As you see them, try to think of them as functional examples of Nature's uses for ATP that
ATP structure and function
Figure 1. ATP (adenosine triphosphate) has three phosphate groups that can
The phosphorylation (or condensation of phosphate groups onto AMP) is an endergonic process.
Since Gibbs free energy is a state function, it doesn't matter how the reaction happens; you just consider the beginning and ending states. As an example, let's examine the hydrolysis of ATP. The reactants ATP and water are characterized by their atomic makeup and the kinds of bonds between the constituent atoms. We can associate some free energy with each of the bonds and their possible configurations—likewise for the products. If we examine the reaction from the standpoint of the products and reactants and ask "how can we recombine atoms and bonds in the reactants to get the products?," we find that a phosphoanhydride bond between an oxygen and a phosphorus must be broken in the ATP, a bond between an oxygen and hydrogen must be broken in the water, a bond must be made between the OH (that came from the splitting of water) and the phosphorus (from the freed PO3-2), and a bond must be formed between the H (derived from the splitting of water) and the terminal oxygen on the phosphorylated nucleotide. It is the sum of energies associated with all of those bond rearrangements (including those directly associated with water) that makes this reaction exergonic. We could make a similar analysis with the reverse reaction.
Is there something special about the specific bonds involved in these molecules? Much is made in various texts about the types of bonds between the phosphates of ATP. Certainly, the properties of the bonds in ATP help define the molecule's free energy and reactivity. However, while it is appropriate to apply concepts like charge density and availability of resonance structures to this discussion, trotting these terms out as an "explanation" without a thorough understanding of how these factors influence the free energy of the reactants is a special kind of hand-waving that we shouldn't engage in. Most BIS2A students have not had any college chemistry and those who have are not likely to have discussed those terms in any meaningful way. So, explaining the process using the ideas above only gives a false sense of understanding, assigns some mystical quality to ATP and its "special" bonds that don't exist, and distracts from the real point: the hydrolysis reaction is exergonic because of the properties of ATP and ALSO because of the chemical properties of water and those of the reaction products. For this class, it is sufficient to know that dedicated physical chemists are still studying the process of ATP hydrolysis in solution and in the context of proteins and that they are still trying to account for the key enthalpic and entropic components of the component free energies. We'll just need to accept a certain degree of mechanistic chemical ignorance and be content with a description of gross thermodynamic properties. The latter is perfectly sufficient to have deep discussions about the relevant biology.
"High-Energy" bonds
What about the term "high-energy bonds" that we so often hear associated with ATP? If there is nothing "special" about the bonds in ATP, why do we always hear the term "high-energy bonds" associated with the molecule? The answer is deceptively simple. In biology the term "high-energy bond" is used to describe an exergonic reaction involving the hydrolysis of the bond in question that results in a "large," negative change in free energy. Remember that this change in free energy has not only to do with the bond in question but rather the sum of all bond rearrangements in the reaction. What constitutes a large change? It is a rather arbitrary assignment usually associated with an amount of energy associated with the types of anabolic reactions we typically observe in biology. If there is something special about the bonds in ATP, it is not uniquely tied to the free energy of hydrolysis, as there are plenty of other bonds whose hydrolysis results in greater negative differences in free energy.
Figure 2. The free energy of hydrolysis of different types of bonds can be compared to that of the hydrolysis of ATP. Source: http://bio.libretexts.org/Core/Biochemistry/Oxidation_and_Phosphorylation/ATP_and_Oxidative_Phosphorylation/Properties_of_ATP
Table 1. Table of common cellular phosphorylated molecules and their respective free energies of hydrolysis.
External link discussing the energetics of coupling ATP hydrolysis to other reactions
Possible NB Discussion Point
You have just now read about two important molecules: NADH/NAD+ and ATP. In what biological contexts/process do you expect to see NADH/NAD+? What about ATP? Can you state what you know so far about the relationship between NADH/NAD+ and ATP? Take a moment to identify any gaps in comprehension you might have -- what questions are you left with after reading the text? Help your peers out with their questions/discussions to reinforce your own knowledge!
The cycling of ATP pools
Estimates for the number of ATP molecules in a typical human cell range from ~3x107 (~5x10-17 moles ATP/cell) in a white blood cell to 5x109 (~9x10-15 moles ATP/cell) in an active cancer cell. While these numbers might seem large, and already amazing, consider that it is estimated that this pool of ATP turns over (becomes ADP and then back to ATP) 1.5 x per minute. Extending this analysis yields the estimate that this daily turnover amounts to roughly the equivalent of one body weight of ATP getting turned over per day. That is, if no turnover/recycling of ATP happened, it would take one body weight worth of ATP for the human body to function, hence our previous characterization of ATP as a "short-term" energy transfer device for the cell.
While the pool of ATP/ADP may be recycled, some of the energy that is transferred in the many conversions between ATP, ADP, and other biomolecules is also transferred to the environment. In order to maintain cellular energy pools, energy must transfer in from the environment as well. Where does this energy come from? The answer depends a lot on where energy is available and what mechanisms Nature has evolved to transfer energy from the environment to molecular carriers like ATP. In nearly all cases, however, the mechanism of transfer has evolved to include some form of redox chemistry.
In this and the sections that follow we are concerned with learning some critical examples of energy transfer from the environment, key types of chemistry and biological reactions involved in this process, and key biological reactions and cellular components associated with energy flow between different parts of the living system. We focus first on reactions involved in the (re)generation of ATP in the cell (not those involved in the creation of the nucleotide per se but rather those associated with the transfer of phosphates onto AMP and ADP).
Video link
For another perspective - including places you'll see ATP in Bis2a, take a look at this video (10 minutes) by clicking here.
How do cells generate ATP?
A variety of mechanisms have emerged over the 3.25 billion years of evolution to create ATP from ADP and AMP. The majority of these mechanisms are modifications on two themes: direct synthesis of ATP or indirect synthesis of ATP with two basic mechanisms known respectively as substrate level phosphorylation (SLP) and oxidative phosphorylation. Both mechanisms rely on biochemical reactions that transfer energy from some energy source to ADP or AMP to synthesize ATP. These topics are substantive, so they will be discussed in detail in the next few modules.