I. Biological Energetics: A Brief Primer
    What is energy? A simple definition - energy is the ability to do work (which is the same as moving matter). Organisms need to do a lot of work (i.e., metabolism, membrane transport, movement).  Measured in units of kJ (kilojoule) or kcal (kilocalorie - this is the traditional unit and less preferred than kJ since it is a unit of heat.  It is used since all work can essentially be converted to heat)

1. Forms of energy.
       There are three biologically-important forms of energy: (1) Chemical energy - energy involved in chemical bonds; (2) Electrical energy - energy associated with electron flow; (3) Radiant energy - energy that travels in waves and discrete particles (photons). Each may exist in a potential or kinetic state.
   
   
2. States of energy.
       Potential energy is energy in a stored or inactive form; energy of position (e.g., dynamite, standing on a desk, pulling back a bow); Kinetic energy is energy in action (e.g., burning wood, falling off a cliff, releasing the arrow).
   
   
3. First Law of Thermodynamics.
       Energy can be converted from one form to another, but never created or destroyed. Or, to state it another way, the total amount of energy in the universe is constant. Thus, plants adhere to the First Law when, during photosynthesis, they convert solar (radiant) energy into chemical energy. Similarly, during respiration mitochondria convert glucose (potential chemical energy) into ATP (another form of potential chemical energy) and heat (radiant energy). This process requires a flow of electrons through a series of electron carriers (electrical energy).
   
   
4. Second Law of Thermodynamics.
       No energy conversion is 100% efficient. Or stated another way, all systems tend to run down (because of inefficient energy conversions). Or, all systems tend to a state of minimum energy, which is the most stable state, which is a state of disorganization - called entropy (symbolized by the letter S). It's no surprise that our rooms and offices tend to get messy - because it takes energy to maintain things in an organized state. In fact, all housework can be considered a battle against the Second Law.  Remember the Morowitz article, "Women's Lib and the energy crisis?"
   
   
5. Do organisms violate the laws of thermodynamics, especially the Second Law, considering that organisms are highly organized?
       No way.  Life follows the Rules! To maintain an ordered state requires a constant input of energy. Just like keeping a room tidy requires a constant energy input, life requires a constant energy input. Life is an open system - meaning it exchanges energy with its environment (constantly replenishing energy needs). Life could not persist isolated in a closed system (one that doesn't exchange materials with the environment).  Death can be considered loosing the battle to entropy and metabolism can be considered the process by which life battles the Second Law - the collective chemical processes by which energy is acquired and utilized.  Remember the Morowitz article, "Six Million Dollar Man?"

II. Reducing Potential - ability of a substance to participate in a redox reaction. Living organisms must carry out many redox reactions.

1. Some definitions: 
       (1) Reduction - gain of electrons; (2) Oxidation - loss of electrons; (3) A helpful mnemonic: "oil rig" - oxidation is loss, reduction is gain; (4) Redox reaction - reaction in which one component is oxidized and the other is reduced. Obviously, electrons must come from somewhere and go somewhere.
   
   
2. The reduction sequence of carbon
   
   carbon dioxide (most oxidized form of carbon) � carboxyl (organic acid) � carbonyl (aldehydes, ketones) � hydroxyl (alcohols) � methyl � methane (most reduced form of carbon). 
   
   Note: each step requires the addition (or removal) of two electrons and two protons for reduction (oxidation). Two steps also require the addition of water.
   
   
3. How can you tell if something has been oxidized or reduced? 
       (1) look for a change in valence (i.e., Fe^{2+ �} Fe³⁺ is an oxidation because an electron was lost, increasing the total positive charge on the molecule); (2) In many biological redox reactions, oxidation is accompanied by a loss of protons (hydrogen ions) and reduction is accompanied by a gain of protons. Thus, you can count the number of hydrogen atoms on each side of the equation (the more H, the more reduced), or count the number of oxygen atoms (the more O, the more oxidized).
   
   
4. Biological redox reactions typically require electron donors and/or acceptors. 
       These are usually: (1) NAD⁺ (2) NADP⁺ and (3) FAD. These are coenzymes (organic compounds, other than the substrate, required by an enzyme for activity). The reaction sequence for these coenzymes is given below:

NAD⁺ + 2H⁺ + 2e^- � � NADH + H⁺
NADP⁺ + 2H⁺ + 2e^- � � NADPH + H⁺
FAD + 2H⁺ + 2e^- � � FADH₂

III. Chemical Potential - energy available from bond cleavage. 
    The primary source is ATP (adenosine triphosphate), which is the energy currency of life. If a cell or organism wants to get some "work" done, it "pays" for the work with ATP. It is estimated that we use and cycle approximately a body weight worth of ATP everyday.

1. Hydrolysis of ATP to ADP + P_i releases energy used by cells.

    eqn:  ATP + H₂O � ADP + Pi + energy

2. ATP hydrolysis is exergonic (delta G = -7.3 kcal/mol).
   
   
3. ATP hydrolysis is coupled to endergonic reactions. 
       Remember the hill model - ATP is what pushes the rock up the hill.
   
   
4. ATP synthesis is termed a phosphorylation reaction (because a phosphate group is added to ADP)
   
   
5. ATP formation - substrate level phosphorylation
       Occurs during glycolysis or the Citric Acid cycle. In a substrate level phosphorylation the phosphate used to phosphorylate ATP comes from an organic compound and the process is NOT associated with redox reactions
   
   
6. ATP formation - oxidative phosphorylation
       Is the result of electron transport in the mitochondria and chloroplasts. In contrast to substrate level phosphorylation, oxidative phosphorylation uses inorganic phosphate and the process IS associated with redox reactions via an electron transport chain (ETC).
   

IV.  Equation Review.
    Given that background, let�s review the equations for photosynthesis and respiration that you�ve seen many times:

Now, let's look at some exciting details!

1. Photosynthesis and respiration are redox reactions. For example, during photosynthesis carbon dioxide is reduced to a carbohydrate (which is abbreviated as (CH₂0)_n ) and the water is oxidized to yield oxygen. Thus, the purpose of water in photosynthesis is to supply the electrons for the reduction of carbon dioxide to a carbohydrate. The situation is reversed for respiration.
   
   
2. Photosynthesis is an anabolic and endergonic reaction. Light energy is required for the reduction of carbon dioxide.
   
   
3. Respiration is a catabolic and exergonic reaction. There is a net energy loss during the process. Some of the energy is used to make ATP.
   
   
4. Hill Model Revisited - a diagram of the hill will be provided in class. Some take-home-lessons from the hilltop:

1. Anabolism is analogous to pushing the rock uphill, catabolism is analogous to the rock rolling downhill;
2. Photosynthesis (an anabolic process) is analogous to pushing the rock uphill, respiration (a catabolic process) is analogous to the rock rolling downhill;
3. The energy required to push the rock (glucose) uphill comes from light (radiant energy);
4. The release of energy from glucose rolling downhill is coupled to ATP production (ca. 40% of the energy is trapped in ATP but more than half of the energy is lost as heat).

VI. Rolling metabolic rocks downhill - A look at Glucose Catabolism

1. Glucose catabolism occurs in a series of small, sequential, highly controlled and regulated steps (reactions).
   
   
2. The processes involved are glycolysis, which is the first step of glucose breakdown, and it is followed by either fermentation or cellular respiration (depending on the availability of oxygen).
   
   
3. Why so many steps? Back to our hill model for an answer. There are two kinds of hills - those with a gradual, step-wise slope and those with a steep precipice or overhang. In each case the rock will roll down the hill and release the same total amount of energy, which is equal to the energy difference between the top and bottom of the hill. However, the energy is released so quickly when rolling off a steep cliff that it is difficult to trap much in a usable form (ATP). However, if the energy release occurs in a slow and orderly way, then it is possible to trap a greater percentage of energy as ATP.

VII. Glycolysis - the first steps of glucose catabolism.

1. Translation. Gr: glyco = sugar; Gr: lysis = split or cleave. Thus, the literal translation of glycolysis is the splitting of glucose.
   
   
2. Net reaction - glucose (C6, a sugar with 6 carbon atoms) is cleaved to yield two pyruvic acid molecules (C3).
   
   
3. Glycolysis consists of about 10 different reactions.
   
   
4. Each reaction is catalyzed by a different enzyme.
   
   
5. Organic acids, like pyruvic acid, are usually ionized at cellular pH values. We name the ionized form "ate". Thus, "pyruvic acid" and pyruvate refer to the same compound but in a slightly different form (ionized vs. not). Essentially these terms can be used synonymously.
   
   
6. Glycolysis occurs in the cytoplasm of virtually all cells. This suggests that glycolysis is an evolutionary ancient pathway. In fact, glycolysis likely evolved more than 3 billion years ago. Wow, it's even older than me!
   
   
7. Oxygen is not required. Glycolysis functions with (aerobic conditions) or without (anaerobic conditions) oxygen present.
   
   
8. Two ATP are required to get the "rock" rolling (hmmm, sounds like a beer brand). They "activate" the glucose making it energetically favorable to react. Remember how people who "misbehaved" were "drawn and quartered"? - this is the molecular version.
   
   
9. A total of four ATP are produced during the process at two different reactions. Thus, there is a net yield of 2 ATP.
   
   
10. ATP is produced during glycolysis by substrate level phosphorylation. For example: ADP + phosphoenolpyruvate (PEP) � ATP + pyruvate. (Note that the PEP is an organic phosphate).
    
    
11. There is one redox reaction during glycolysis. The oxidation of glucose begins during glycolysis. NAD⁺ accepts the electrons during the oxidation, and as a result it gets reduced. A total of 2 NADH are produced. Recall that NAD⁺ is a coenzyme (organic compound required by an enzyme for activity) that is used in redox reactions. Enzymes that catalyze redox reactions with the help of coenzymes such as NAD⁺ are called dehydrogenases.
    
    
12. Pyruvic acid is a branch point. It still has much energy and can be further degraded. If oxygen is present (aerobic conditions) pyruvate is metabolized via cellular respiration (i.e., citric acid cycle and ETC. Without oxygen, anaerobic conditions, pyruvate is metabolized via fermentation.

VIII. Fermentation - stepping in an anaerobic environment

IX. Aerobic or Cellular Respiration - stepping in air.

1. This is not the same thing as breathing.
   
   
2. Occurs only in the presence of oxygen.
   
   
3. Occurs In the mitochondrion. 
       (Note, the entire cell of an aerobic bacteria can act like a mitochondrion to produce ATP aerobically). Remember the structure of a mitochondrion? (a) double membrane, inner and outer; (b) matrix - central or inner compartment; (c) inter-membrane space. Simply put, there is a liquid matrix where "biochemical reactions" occur, an inner membrane where electron transfer reactions occur and an area where low pH won�t do any harm (the "space")
   
   
4. Pyruvate Oxidation.
       Pyruvate from glycolysis is shuttled into the mitochondrion.  As it enters, both of the pyruvate are oxidized (another redox reaction involving NAD⁺) and a carbon dioxide is lost from each. Note, that two carbons (one from each of the pyruvate molecules that originated from glucose) have been lost. The leftover two carbon piece is hooked onto a carrier (Acetyl coenzyme A). 
   
   
5. Welcome to the Citric Acid Cycle or Kreb�s cycle or Tricarboxylic Acid Cycle 

1. It is named in honor of Sir Hans Krebs, who owned a bicycle shop in Kent, England. Just kidding - he was a British biochemist who worked out the details of many of the reactions. It is also called "Citric Acid Cycle" because citric acid is one of the important intermediate molecules or called the "Tricarboxylic Acid Cycle"  (TCA) because citric acid has three carboxyl groups.  Take your pick of names.
   
   
2. Occurs in the matrix of the mitochondrion (thus the reactants are water soluble)
   
   
3. There are several different reactions in the Citric Acid cycle, each catalyzed by a different enzyme.
   
   
4. There is one substrate level phosphorylation reaction that produces ATP. [Actually, the first product of the cycle is GTP (which is a nucleotide phosphate carrier like ATP). The GTP then donates a phosphate to ADP to make ATP.]
   
   
5. Carbon dioxide is released during two reactions (alpha-ketoglutarate dehydrogenase and isocitrate dehydrogenase) (or three total reactions if you include the reaction during which pyruvate was shuttled into the mitochondrion). Thus, during the Citric Acid cycle, the breakdown of glucose into carbon dioxide is completed.
   
   
6. There are four redox reactions, three of which yield reduced NADH and one FADH₂. Thus, the oxidation of glucose is completed in the Kreb's cycle. If you count the redox reaction that occurred when shuttling pyruvate into the mitochondrion there is a total of 5 redox reactions in the mitochondrion.

X. Regenerating Coenzymes in aerobic conditions
    Recall that one problem of glycolysis was regenerating oxidized coenzymes. Under anaerobic conditions, this problem was solved by a variety of fermentation reactions or anaerobic respiration. Well, the solution is much more elegant in cells in an aerobic environment. Not only are oxidized coenzymes recovered, but the process is coupled to the production of ATP. Thus, the evolution of an oxygenated atmosphere allowed cells the added bonus of producing additional cellular energy while regenerating oxidized coenzymes. The electron transport chain (ETC) provided the means for this process.

XI. Mitochondrial Electron Transport Chain

1. Occurs in the inner membrane (cristae)
       Thus, the components are more or less lipid-soluble and are embedded in the membrane
   
   
2. Occurs only under aerobic conditions
   
   
3. The mitochondrial ETC consists of a series of electron carriers that alternately accept and pass along an electron - like a hot potato or to use our glass analog - like a glass that is alternately filled (given an electron) and emptied (pass it off to the next carrier). In the process the carriers become reduced (glass full) and then oxidized again (glass empty).

1. There are four major groups, called complexes, of electron carriers in the membrane. This is the reason why the text shows several blobs in the membrane. Each complex has a unique set of carriers. In addition, there are molecules that shuttle electrons from one complex to another.
   
   
2. 2. Among the carriers in the complexes (don't memorize these): Complex I - flavoproteins (FMN) and Fe-S proteins; Complex II - more flavoproteins, Fe-S proteins; Complex III - an assortment of proteins including cytochromes (c1 and b); Complex IV - cytochrome a,a₃.
   
   
3. The sequence of electron flow occurs from complex I to complex IV as depicted: I � III � IV
   
   
4. Since the complexes are physically separate in the membrane, carriers must shuttle electrons from one complex to another. Ubiquinone (coenzyme Q, or simply, Q) shuttles electrons from complex I to complex III (and also between II and III). Cytochrome c shuttles electrons from complex III to complex IV. Thus:

I � Q � III � cyt c � IV

5. NADH from the Kreb's cycle donates its electrons at the end of the chain (to complex I). After donating its electrons, NADH gets oxidized to NAD⁺. Thus, we can modify the reaction chain:

NADH � I � Q � III � cyt c � IV

6. The final acceptor of the electrons in the ETC is oxygen. Oxygen becomes reduced to water. Thus:

NADH � I � Q � III � cyt c � O₂

7. FADH₂ and exogenous NADH (produced outside of the mitochondria, i.e., during glycolysis) donate electrons to Complex II and then on to oxygen. Thus, the electrons bypass Complex I. We can write this as:

FADH₂/NADH_ex � II � Q � III � cyt c � IV � O₂

    D. The passage of electrons along the ETC is associated with the production of ATP.

XII. Chemiosmotic Production of ATP

1. The mechanism of ATP production is the same as for chloroplasts - via chemiosmosis.
   
   
2. A review of chemiosmosis: (a) at intervals along the ETC, protons (hydrogen ions) are moved from the matrix (or stroma) side of the membrane to the space between the inner and outer membranes; (b) the membrane is impermeable to protons which results in; (c) a pH and electrochemical (more positive charges in the intermembrane space than the matrix) gradient is established across the membrane. The pH of the matrix (or stoma) is alkaline (about pH 8) relative to the space (pH 5); (d) this gradient provides the driving force for ATP production; (e) ATP is made at an ATPase - which are stalked "lollipops", protein channels that span the membrane. Protons flow (like a waterwheel) through these.
   
   
3. There are three sites where protons are moved across the mitochondrial membrane "roughly" associated with complexes I, III and IV.
   
   
4. Each site is "roughly" associated with synthesis of 1 ATP. Thus the ATP yields: 3 ATP/NADH (endogenous - those produced inside the mitochondrion such as during the Kreb�s cycle); 2 ATP/NADH (exogenous - those produced in the cytoplasm such as during glycolysis); 2 ATP/FADH₂.
   
   
5. The total yield of ATP from oxidizing glucose is about 36 ATP/glucose. Let's add 'em up - 4 ATP (2 NADH from glycolysis which each yield 2 ATP) plus 24 ATP (NADH is produced in four steps in the mitochondria times 2 for each pyruvic acid times 3 ATP) plus 4 ATP (2 FADH₂ times 2 ATP) plus 2 ATP from glycolysis plus 2 ATP from Kreb's cycle = 36 ATP.
   
   
6. Note there are 18x more ATP produced under aerobic conditions (36) than under anaerobic conditions (2). Which do you choose?

XIII. Function of Aerobic Respiration/Glucose Catabolism

1. Energy production (36 ATP/glucose)
   
   
2. Produce intermediates for many other metabolic reactions. Thus, various molecules can be siphoned off and used for other purposes.
   
   
3. Heat production. Glucose catabolism yields a TOTAL of 38 ATP. 38 ATP x 7.3 kcal/mol ATP = 262 kcal. Glucose has 686 kcal. Thus the efficiency of glucose metabolism is 262/686 x 100 = 38%. Or in other words, about 62% of the energy is lost as heat. This keeps us, shrews, and elephants warm. It also explains why compost piles generate lots of heat (microbial decomposition) and why there are sometimes spontaneous silo fires (improperly dried/prepared silage). But don't worry - there is no such thing as spontaneous human combustion.

XIV. Regulation
    Many of the enzymes of glycolysis and respiration are allosteric enzymes. This allows for tight control of metabolism.

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