Biology C2005 Lecture 9

::Krebs Cycle (TCA cycle, citric acid cycle)::
Last time we followed the metabolism of glucose through the glycolytic pathway, or glycolysis, and at the end we had 2 molecules of pyruvate. We also saw that in the absence of oxygen, pyruvate could be further metabolized by a process called fermentation, two examples of which were conversion to lactate or to ethanol plus carbon dioxide. So now let us turn to the case when oxygen is present. In this case we are headed to the complete oxidation of glucose to 6 molecules of CO₂ and 6 molecules of water:

C₆H₁₂O₆ + 6 O₂ ---> 6 CO₂ + 6 H₂O

The Delta G^o for this reaction is -686 kcal/mole, and we are going to hope to get a lot more ADP --> ATP conversions out of this.

In the presence of oxygen, glycolysis down to pyruvate is the same. But the fate of pyruvate is now different.

THE KREBS CYCLE

Rather than heading toward lactate or ethanol, as shown at the bottom of the glycolysis handout, the pyruvate jumps to another handout: the KREBS CYCLE. Keep in mind that by forgoing the reduction of pyruvate, we have not satisfied our loose end of NADH₂ accumulation from the oxidation step (step 6) in glycolysis.

The fate of pyruvate is now different, it will enter a series of reactions known as the Krebs Cycle, (also TCA cycle, or citric acid cycle) in which all of its carbons will indeed end up as CO₂. However, as we are about to see, its H's will not be converted to water here, and very little ATP will be produced here. [Purves6ed 7.9a], [Purves6ed 7.9b].

Please follow along in the Krebs Cycle diagram as the reaction are discussed. At the top of the Krebs Cycle diagram, we see our pyruvate entering from the top left, in a reaction that is not part of the cycle of reactions seen below. Before entering the cycle, pyruvate undergoes a relatively complicated reaction involving an oxidation, once again using NAD as the oxidizing agent, as well as a DECARBOXYLATION, the splitting off of the carboxyl carbon as CO₂ (as when ethanol was made in yeast) and leaving a 2-carbon acetate group. So here is some CO₂ produced, which is what we expected from the oxidation of glucose. In addition, a new co-factor makes an appearance: Coenzyme A, a sulfur-containing small molecule [pADP-pantothenate-SH] which becomes bound to the acetate in a thioester linkage. A thioester is analogous to an ester except a sulfhydryl is one of the reactants instead of an alcohol, so a sulfur atom takes the place of an oxygen. A thioester contains a high energy bond, and so there should be a SQUIGGLE (see Becker p. 413-414 for the exact structures, if you wish). It is acetyl-CoA, the product of this dehydrogenation of pyruvate, that is the compound that now enters the Krebs Cycle proper. [Purves6ed 7.9b].

Acetyl-CoA condenses with a molecule of oxaloacetate, a 4-carbon dicarboxylic acid, to produce citrate, a 6-carbon tricarboxylic acid (thus the name tricarboxylic acid cycle or TCA cycle as a synonym for the Krebs Cycle). CoA is split off in the course of this reaction. The high energy bond to CoA is utilized to help drive this otherwise endergonic synthesis of a 6C molecule. The free CoA is regenerated and so is not consumed.

Now we have a new loose end, however: OA (oxaloacetate). We have introduced, borrowed, this molecule, much as we borrowed an NAD, so it's now OA plus 2 NADs; no, really 4 NADs and 2 OA's (per glucose molecule). We must pay back these debts by the end of our path (which remember now is going to be glucose going to CO₂).

We have labeled the carbons of acetyl-CoA with an asterisk and a dot so that we can follow them as they go through this set of reactions. In the laboratory, it is also possible to use organic molecules labeled in this way, by using molecules in which particular carbons or hydrogens have been replaced with their radioactive counterparts, or isotopes, for instance ¹⁴C instead of the usual ¹²Cor ³H instead of ¹H. Radioactivity counters can then be used to track and measure the appearance of the isotopes in various purified intermediate compounds.

The citrate is isomerized to isocitrate; note the movement of the hydroxyl group from the middle to one end. The isocitrate is oxidized once again using NAD and it's simultaneously decarboxylated to produce alpha-ketoglutarate and now our second molecule of CO₂. We need to get 3 from our 3-carbon pyruvate molecule. So this is 2. Except the actual carbon is not from our acetyl-CoA, i.e., from our pyruvate, but rather from the OA that we borrowed. Looks like it may be a bad debt: here we've borrowed an OA and now we've blown it into CO₂.......How are we ever going to pay it back? Oh well, let's go on.

Next we again have an oxidative decarboxylation, from the 5-carbon alpha-ketoglutarate to the 4-carbon succinate. This is actually a set of two reactions, as can be seen in the Becker text. Once again the CO₂ comes not from the acetyl-CoA carbons, but from a carbon atom originally in OA. But here we have a new and welcome feature, the production of GTP from GDP. The free energy from this oxidation is coupled to the phosphorylation of GDP with Pi. GDP is a compound, a nucleotide, related to ADP, the structure being the same except for the substitution of the guanine ring for the adenine ring. The production of GTP is energetically equivalent to producing ATP, since:

GTP + ADP --> ATP + GDP, Delta G^o = 0

So we finally get some ATP here, 2 moles per mole of glucose, equal to what we netted in glycolysis. {Q&A}

The mechanism of this coupling is less obvious than those we saw in glycolysis (where the substrates were phosphorylated). Here the inorganic phosphate and GDP are both bound by the enzyme as part of the overall reaction.

[not responsible for this mechanistic detail:
aKG + NAD + CoA --> CO₂ + NADH₂ + Succ~CoA; Succ~CoA + Pi + E --> CoA + E-succ~P --> E~P + Succinate, E~P + GDP -->E + GTP]

::FAD::
Next the succinate is dehydrogenated across its central 2 C's, producing fumaric acid This is the reaction we discussed earlier illustrating enzyme specificity. Here the oxidizing agent is FLAVIN ADENINE DINUCLEOTIDE (FAD), rather than NAD. FAD is a better oxidizing agent (it is more easily reduced) than NAD; the Delta Go for this reaction using NAD would be highly unfavorable, whereas with FAD it is much more favorable; about a 10 kcal/mole difference. So now we have to add 2 FADH2's to our debt LIST. Our debt list now includes: NAD's, FAD's, and OA.

Continuing, we add water across fumarate's C=C double bond to get malate, and then once again dehydrogenate, i.e., oxidize, using NAD to get the 4-carbon dicarboxylic keto acid OA.

So we can pause here, our OA has been regenerated, and is ready to take on another acetyl-CoA. We have utilized one pyruvate and have released 1, 2, 3 CO₂ molecules. We have carried out 5 oxidations per pyruvate, 4 with NAD and 1 with FAD, 4 oxidations in the cycle proper and one oxidation getting into the cycle. We produced one ATP equivalent per pyruvate. And we have accumulated 4 NADH₂'s and 1 FADH₂ per pyruvate (double all these for a per glucose accounting). But we have paid back our OA debt, not with the original OA molecule, but no one will ever know the difference (except perhaps some nosy biochemist with radioactive isotopes).

In Krebs Cycle per pyruvate per glucose

CO2 released 3 6

oxidations: 5 10

NADH₂ produced 4 8

FADH₂ produced 1 2

ATP produced (as GTP) 1 2

O₂ consumed 0 0

Our labeling showed that the CO₂ molecules produced in this turn of the cycle were not derived from the acetyl-CoA. The acetyl carbons have ended up on either the top or the bottom of the OA that was regenerated. We don't know which end because fumarate is a symmetric molecule, and the water addition forming malate could have produced a hydroxyl on either the labeled or the unlabeled end.

So far we've gotten precious little energy out in the form of ATP, and we still have the NAD and FAD to pay back. And oxygen has not been involved yet. The electrons of glucose (entering here as pyruvate) have not been delivered to O₂, but are still on the way-station of NADH₂ and FADH₂.

::electron transport chain::
In the next part of the story, NADH₂ and FADH₂ will pass their electrons on to oxygen AND we will get a lot of ATP from this passage of electrons. This oxidation of NADH₂ and FADH₂ will return them for further action as NAD and FAD.

So let' s pass them on to oxygen:

NADH₂ + 1/2 O₂ --> NAD + H₂O, Delta G^o = -53 kcal/mole

53 kcal/mole released, TOO high. Too much energy released: if it were used in one fell swoop of the usual coupled reaction, we would get only a single ATP's worth, 7 kcal/mole, from this 53 kcal/mole, and we'd release a LOT of heat besides. It would be better if we could break up this 53 kcal/mole into smaller bits to use. The scheme for breaking up the free energy change involved in the reduction of oxygen involves passing the electrons from NADH₂ and FADH₂ not directly to oxygen but rather through a chain of intermediate transfer steps. This chain of steps is called the ELECTRON TRANSPORT CHAIN (E.T.C.).

Let's look at View #2, on the ETC View 2-3 handout: The electrons from NADH₂ are seen to be passed to various ELECTRON CARRIERS of the electron transport chain, in a precise sequence of transfers (here simplified, see Becker p. 423-433, Figs. 14-15 to 14-18).

Some of the participants in this chain are:

- Proteins = Iron-sulfur protein (in which iron as Fe+++ (ferric) accepts the electrons). This the 1st acceptor.

- Coenzyme Q (CoQ) , a small molecule, hydrophobic, lipid soluble (also called ubiquinone).

- CYTOCHROMES b, c, and a; [really cytochromes b, an Fe/S protein, and cytochromes c1, c, a, and a3 (with prosthetic groups containing Fe or Cu)]

- then to O₂ (forming water)

Follow the electrons in this simplified diagram (ETC View 2-3 handout), in which several steps are often condensed into one. The electrons from an NADH₂ are transferred to CoQ, reducing it to CoQH₂. Subsequently, the electrons are passed from CoQH₂: that is, CoQH₂ gets oxidized by passing the electrons to cytochrome B. The cytochromes contain heme as a prosthetic group, in which an iron oxidation-reduction occurs: Fe+++, +1 electron ---> Fe++. So now cytochrome B has the electrons. Cytochrome B gets oxidized by the heme group in cytochrome C. Its iron returns to ferric (Fe+++), while cytochrome B's heme group get reduced from the Fe+++ to the Fe++ state. A similar transfer occurs between cytochromes C and A. Finally, cytochrome A passes the electrons to molecular oxygen, which also picks up 2 protons to go with two electrons hydrogen atoms, so that the product of this reduction is H₂O, which is the final resting place for these travel-weary electrons.

View #1 shows the free energy changes associated with some of these electron transfers. Each transfer is energetically favorable, with some of the changes releasing much more free energy than others. It can be seen here that the 53 kcal per mole for the reaction between NADH2 and oxygen has been broken up into smaller packets of free energy changes. The changes marked with an asterisk are those that are capable of generating a molecule of ATP from ADP. We will get to the mechanism of that generation a little later. You can also see that whereas NADH₂ can generate 3 ATPs, FADH₂ can only produce 2.

View #3 in ETC View 2-3 handout shows that these electron transport proteins of the ETC are organized into 3 groups; these protein complexes, called respiratory complexes are geographically fixed next to each other within a membrane in the cell, as we shall soon see. That is, these are membrane-bound complexes of proteins.

Thus CoQ and the cytochromes C, and NAD and FAD, are constantly shuttling electrons, picking them up originally from glucose-derived molecules and then delivering them elsewhere and then returning to pick up another load.
[Purves6ed 7.11]

In the end, O₂ receives the electrons. All the reduced forms of the oxidative cofactors (NADH₂, FADH₂) return to the oxidized state (NAD, FAD), having gotten rid of these electrons..

So: ..... we have no NAD or FAD loose-end debt any longer.

All debts have been paid. All the glucose carbon atoms have been converted to CO2, and the electrons from glucose have now all been delivered to O₂ to form water.

So where does the ATP come in, after all this bother? If ATP is the energy currency of the cell, show me the money.

::mitochondria::
To understand how ATP is generated in this process of electron transport, we must discuss the special structures in the cell where all this electron transport and ATP generation takes place: the MITOCHONDRIA. Some important features of mitochondrial structure can be seen in the simple diagram of the mitochondrion handout.

There is an OUTER membrane that is permeable to most small molecules (to MW 5000) and which need not concern us much here.

There is an INNER membrane, with cristae, or extensive invaginations to increase membrane surface area, and which does provide a barrier to transport. [Purves6ed photo]

The reactions of the Krebs Cycle as well as the entrance reaction to the cycle take place in the inside of the mitochondrion, which is called the MATRIX.

Glycolysis takes place in the cytoplasm: so the pyruvate produced must get in to the mitochondrial matrix.

The ETC proteins complexes are held within the INNER MEMBRANE (within the cristae).

But where is the energy? the ATP?

::chemiosmotic theory::
The answer lies in the Chemiosmotic Theory first proposed by Peter Mitchell in 1961.

::proton pump::
The energy released at each of the electron transfers is stored in an electro-chemical gradient, established across the mitochondrial inner membrane. Concomitant with electron flow, H⁺'s are being pumped out of the matrix into the inter-membrane space. These hydrogen ions are not clearly from NADH₂ per se, as H⁺'s get pumped out even in later steps in the ETC where no protons are directly involved (just electrons on Fe++ atoms). [Purves6ed 7.13a] So this pumping out of H+ ions must be coupled the binding and release of electrons by the proteins involved (see one proposed mechanism on the handout).

The immediate effect of this H+ pumping is a higher concentration of H+ ions outside the mitochondrion's inner membrane and a lower concentration of H+ ions inside the mitochondrion. Now we allow the pumped-out H⁺'s to flow back. By mass action, the protons will flow from a region of high concentration to a region of lower concentration. A steady-state increment (between the outside and the inside of the inner membrane is thus maintained, resulting in the matrix (inside) being about 1 pH unit higher than the outside). A constant kicking-out, and flowing-back.

The H⁺'s may not get that far, but the outer membrane is no barrier to H⁺ ions, so how far they get is not an important factor.

::ATP synthetase::
The flow-back is through lollipop-like structures that populate the inner surface of the inner membrane. Each lollipop is a complex of proteins; the stem is called Fo and forms a channel through the membrane. The sphere is called F₁ and contains the ATP SYNTHETASE activity; that is, it is in the spheres that the generation of ATP from ADP + Pi takes place. It is the flow-back of H⁺'s through the F1 spheres that generates the phosphorylation of ADP by P_i to form ATP. An analogy would be to use one source of energy to pump water up to a high level behind a dam (the pumping of protons tied to the free energy released in the oxidations of the electron transport chain components) and then letting the water drive turbines to generate electricity as it falls from high level behind the dam (the generation of ATP).

This is one place where reading of both texts can help in the understanding of this very indirect mechanism. Indeed, this theory was doubted for many years after its proposal by Mitchell (it is also known as the Mitchell Hypothesis; Mitchell was known to do experiments in the basement of his mansion, like in the movies).

The mechanism of this reaction, the ATP synthetase, has only become clearer in the last few years, and is still not completely understood. The F1 spheres are organized like a propeller with 3 identical blades. [Purves6ed 7.13b] The blades can be configured in space to provide a binding site for ADP and Pi or for ATP. The protons outside the mitochondrial membrane flow back first through the Fo stem channel and then bind to an F1 propeller blade. This binding produces an allosteric change that affects a binding site for ADP and Pi that is on the face of one of the protein subunits exposed on the inside of the mitochondrion. This distortion forces the ADP and Pi together on one propeller blade, while the ATP that just had been formed on another propeller blade is distorted in the opposite way to release the ATP. The sequence of these 3 events is thus 1) the binding of ADP and Pi, 2) a kind of mechanical force pushing them together, followed by 3) a quick release of the ATP. The formation of these 3 conformations is driven by protons binding to specific amino acids on the protein subunits. What is quite amazing is that this successive shifting of conformations is accompanied by a movement of the F1 sphere relative to the Fo stem, or base. Thus as the protons flow back into the mitochondrion, the F1 spheres are spinning like propellers, (and thus the propeller analogy). This movement has been seen in elegant experiments in which long fluorescent molecules have been attached to the F1 spheres and then seen to rotate, or whip around, when a H+ gradient is applied. Thus we have here perhaps the world's smallest motor.

::oxidative phosphorylation and substrate level phosphorylation::
This process of forming ATP by a proton motive force is called OXIDATIVE PHOSPHORYLATION (OXPHOS). . {Q&A}. And so there are two methods of producing ATP from glucose metabolism: Oxidative phosphorylation and the "regular", direct phosphorylation of ADP from phosphorylated intermediates that was seen during glycolysis, or in the GTP-forming step in the Krebs Cycle. This direct phosphorylation is called SUBSTRATE-LEVEL PHOSPHORYLATION, to distinguish it from OXPHOS.

Some evidence for the validity of the chemiosmotic theory of oxidative phosphorylation is:

1) Adding H⁺ ions (adding acid, in moderation) to closed vesicles (or membrane-bound spheres) that have been formed from membranes containing the F1-Fo protein complexes generates ATP from ADP + Pi in the test tube.

2) Isolated ETC complexes I, III, or IV inserted into artificial membranes are able to pump H+ ions in the predicted directions when provided with the appropriate substrates (reduced electron carriers like NADH₂).

3) DINITROPHENOL, a small partially hydrophobic molecule, can return H⁺'s to the inside of the mitochondria via a short circuit; it ferries them across the inner membrane; the H⁺'s thus avoid the Fo tunnel. This compound uncouples H⁺ transport from ATP generation, so you get electron transport, but no OX-PHOS (since there is no longer a build-up of a proton gradient across the membrane).

What about E. coli, they have no mitochondria (in fact a mitochondria is abonut the size of an E. coli cell). Bacteria simply use their own plasma (cell) membrane, and kick the H⁺'s out into the medium.

OK, so how much ATP do we get after all this?

1 ATP per pair of electrons transferred through EACH of the 3 enzyme complexes (I, II, and IV). The number of protons transferred per pair of electrons is not really known precisely (it'sbeen estimated at 10-12, so 3-4 protons flowing back can produce an ATP).

So 3 ATPs per pair of electrons passing through the full ETC.

So 3 ATPs per 1/2 O₂

So 3 ATPs per NADH₂

But only 2 ATPs per FADH₂, which skips complex I, and delivers its electrons to CoQ via complex II, with little free energy released at that first step.

Overall ATP tally of RESPIRATION (as this oxidative metabolism of glucose is called) (see also OUTLINE of energy metabolism handout):

ATP from substrate-level phosphorylation, SLP (per glucose):

Glycolysis: -2 that have to be invested, then +4 for a net of +2

Krebs Cycle: 2 (as GTP)

Total SLP = 4

ATP from OxPhos (per glucose)

(first per Glyceradhyde-3-P, where the first oxidation takesplace:

1 NADH₂ from glycolysis and 1 from entry into the KC and 3 from the KC proper)

So 5 NADH2 @ 3 ATP/NADH2 = 15

FADH₂: 1 from the KC @ 2 ATP/FADH₂ = 2

Total OXPHOS per molecule of glyceradhyde-3-phosphate = 1

Per glucose molecule, multiply by 2:

17 X 2 = 34

Grand total = 4 + 34 = 38 ATPs per glucose (in E.coli and other prokaryotes)

Now, if we are considering eukaryotic cells, we need to subtract 2 ATPs from this total, as 2 ATPs are used to get electrons from the 2 cytoplasmic NADH₂'s from glycolysis into the mitochondria (by an indirect mechanism), so the net for eukaryotes is 36.

Efficiency: 36 ATPs X 7 kcal/mole ATP hydrolysis = 252 kcal/mole harnessed as ATP.

252/686 available from glucose combustion = 37% efficiency.

Once again, better than most engines (20-25%).

And compare: 36 ATPs per glucose from respiration to 2 per glucose for fermentation. [Purves6ed 7.17a], [Purves6ed 7.17b].
{Q&A}

So with or without air, ATP is no problem............

::You are what you eat::
From glucose, at least ... But do we live from glucose alone?

How about a carbon and energy source OTHER THAN GLUCOSE?

How about fat? More specifically, let's consider the glycerol part of the fat molecule.

Consider GLYCEROL : CH₂OH-CHOH-CH₂OH.

You can follow some of this argument on the glycolysis handout.

Now the first step is: glycerol + ATP --> glycerol-1-P

glycerol-1-P + NAD --> DHAP (= dihydroxyacetone phosphate) + NADH₂

DHAP looks familiar, Yes, it was there in the glycolytic pathway, where it was isomerized to glyceraldehyde-3-P and then down the path to pyruvate.. So then DHAP can continue to be metabolized in glycolysis.

Under aerobic conditions, no problem, the NADH₂'s produced will get re-oxidized in the ET chain.

How about under ANAEROBIC conditions? Consider E. coli trying to grow in glycerol minimal media (with glycerol as the sole organic compound instead of our usual glucose) under anaerobic conditions:

We used two NADs to get down to pyruvate starting from glycerol, one to make DHAP by oxidizing glycerol-1-P and one at the usual place in glycolysis at the oxidation of glyceraldehyde-3-P but we get back only one in going to lactate. If we try to run the lactate fermentation using glycerol as our only carbon and energy source, we will grind to a halt as all our NAD ends up as NADH₂. (You will reach a state where all the NAD is in the form of NADH₂, and there is no pyruvate left, just DHAP waiting for an NAD that is not there). So, although E. coli will grow just fine on glycerol in the presence of air (oxygen), it will NOT, in fact, grow on glycerol in the absence of air (anaerobically).

So these loose-end debts are real.

What about the fatty acids from fats? What about using proteins as a carbon and energy source, or polysaccharides? Most of these can be used, they get hydrolyzed down to small molecules and the small molecules get transformed in a short series of reactions to the same intermediates we have met in glycolysis and the Krebs Cycle. Thus these two energy yielding pathways are the common endpoints for most catabolism, or breakdown of molecules for energy. Similarly, the biosynthetic pathways to the monomers (anabolism) start with glycolytic and Krebs Cycle intermediates, which get transformed in a series of small steps to all the amino acids, the fatty acids, and sugars necessary to build the cell. See the overall scheme on the "You are what you eat" diagram.

[ Although you have a handout for fatty acid oxidation, you will NOT be responsible for this topic. ]

Almost all of these pathways are known, can be summarized on the metabolic map. It is not necessary to read such maps in detail to get the idea that there are a complex series of pathways that boil down to a manageable number that can be put on one sheet, with glycolysis and the Krebs Cycle at its center.

Rather than continue to reveal the beauty of these pathways, we will return to our story of building E. coli. We have seen in the last few lectures the function of proteins as enzymes. Now we must consider the biosynthesis of proteins, how the amino acids are put together in this all-important primary structure. But to understand proteins synthesis, we must first understand the nature of nucleic acids. Our next chapter then will be a consideration of the structure and function of DNA.

(C) Copyright 2001 Lawrence Chasin and Deborah Mowshowitz Department of Biological Sciences Columbia University New York, NY
Clickable pictures are from Purves, et. al., Life, 5th Edition, Sinauer-Freeman's Images of Life 5.0.
A production of the Columbia Center for New Media Teaching and Learning