16.2: Reactions of the Citric Acid Cycle

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Logic and Architecture of the Citric Acid Cycle

• Explain why the citric acid cycle is organized as a cycle rather than a linear pathway, including the roles of anaplerotic reactions in replenishing cycle intermediates that are withdrawn for biosynthesis, and describe the historical context of Krebs's discovery using isotope labeling and minced pigeon muscle tissue.
• Account for the complete stoichiometry of one turn of the cycle — identifying the two carbons entering as acetyl-CoA, the two carbons departing as CO₂, and the three NADH, one FADH₂, and one GTP produced — and explain why the primary value of the cycle lies in reduced electron carriers rather than direct ATP synthesis.

Reaction Mechanisms of the Eight Steps

• Describe the chemical logic connecting sequential steps of the cycle, explaining how the aconitase reaction (step 2) repositions the hydroxyl group to a β-position to enable the oxidative decarboxylation catalyzed by isocitrate dehydrogenase (step 3), and why oxidation must precede decarboxylation in step 3.
• Explain the mechanistic rationale for using the α-ketoglutarate dehydrogenase complex (step 4) — with its TPP, lipoamide, FAD, CoASH, and NAD⁺ cofactors — to achieve the oxidative decarboxylation of an α-keto acid, drawing the explicit mechanistic parallel to the pyruvate dehydrogenase complex described in the preceding chapter.
• Describe how the thermodynamically unfavorable thioester hydrolysis in succinyl-CoA synthetase (step 5) is coupled to GTP synthesis through a covalent phosphohistidine intermediate, and explain the ordered ter-ter kinetic mechanism of the reaction.
• Explain why succinate dehydrogenase (step 6) uses FAD rather than NAD⁺ as its oxidizing agent — connecting this to the relative reduction potentials of the fumarate/succinate and NAD⁺/NADH couples — and describe its dual role as a citric acid cycle enzyme and as Complex II of the mitochondrial electron transport chain.
• Describe the mechanisms of citrate synthase (step 1), fumarase (step 7), and malate dehydrogenase (step 8), identifying the key active-site residues, the chemical transformation accomplished, and — for malate dehydrogenase — why the thermodynamically unfavorable reaction (ΔG°′ = +7.1 kcal/mol) proceeds in vivo.

Introduction

The acetyl-CoA formed by the pyruvate dehydrogenase complex (PDC) then enters a cyclic, non-linear pathway called the citric acid cycle, tricarboxylic acid (TCA) cycle, or Krebs cycle, named after Hans Krebs, who discovered it. The cycle is shown in Figure \(\PageIndex{1}\) in wedge/dash form with stereochemistry included.

Figure \(\PageIndex{1}\): Citric Acid Cycle

Why a cycle?

Why is this pathway cyclic and not linear, as we have seen for glycolysis? A simple answer is that it evolved that way, but why would that be advantageous? Some key "intermediates" in the pathway are pulled away for the biosynthesis of other biomolecules. If the citric acid cycle were linear and intermediates were pulled off for other reactions, the pathway would taper off, which would not be optimal for a key energy-production pathway. Of course, removing intermediates from a cyclic pathway would also slow down the process. Still, when this happens, enzymes outside the cycle produce key reaction intermediates that sustain the cycle. The replenishing reactions are called anaplerotic.

Look at Figure 1.  2 Cs enter at the top as CH₃(C=O)-SCoA.  At the bottom, 2 Cs depart as CO₂s.  Also, 2 Hs leave as 2 NADHs, which are generated on the right-hand side of the cycle along with some protons.  Those have to be put back into the cycle for the next round.  Krebs thought the input was from pyruvate, CH₃(C=O)CO₂^-, the end product of glycolysis. Pyruvate also comes from the metabolism of some amino acids.  

In his detailed analysis of the enzymes involved in "intermediary" metabolism, Krebs used radioisotope-labeled reactants to trace carbon atoms in respiring tissue. He found that when radiolabeled pyruvate and oxaloacetate were added to muscle tissue in vitro, radiolabeled citrate was formed.

Pyruvate + Oxaloacetate → citrate + CO₂

His first version of the cycle had pyruvate as the incoming 2C molecule. But pyruvate is a 3C molecule, so something was amiss. Another CO₂ likely was released. But from what? 

Left out of this equation is the initial reaction of pyruvate with CoASH to form acetyl-CoA (an oxidative decarboxylation reaction, discovered by Fritz Lipmann in 1947), which then enters the pathways at the top by reacting with oxaloacetate to form citrate.   Oxaloacetate, the pathway's end product if it were linear, reacts with another incoming acetyl-CoA to reform citrate, restarting the cycle.   He correctly surmised that the pathway was circular.  

Krebs used minced pigeon flight muscles for his experiments.  It was a good choice, as these have a very high metabolic rate, allowing him to detect changes in the system after adding specific metabolites.  Note that the last four species in the cycle are 4 C carboxylic acids, as shown in Figure \(\PageIndex{2}\) below.  Carboxylic acids are inherently not very reactive.  

Figure \(\PageIndex{2}\):  Structure of the 4 C carboxylic acids of the citric acid cycle

Exercise \(\PageIndex{1}\)

In separate experiments, Krebs and others added four different 4-carbon carboxylic acids to minced pigeon muscle.  Each sped up CO₂ generation separately, but, interestingly, none of the added carboxylic acids were further metabolized, so their concentrations remained the same.  Explain how this could occur.

Answer

In a linear pathway, added reactants would be consumed unless all steps are equally reversible.  The latter seems unlikely in actively respiring flight muscle.  The only reasonable explanation is that the pathway was not linear but cyclic, so each species was regenerated in the next turn of the cycle.  By definition, each of the 4 C organic acids acted as a general catalyst - their addition sped up the reaction, but they were not themselves consumed! Each increased the rate not just of the reaction in which it was a substrate, but of all the reactions. Krebs showed that each species acted not as a "hydrogen carriers" but as a reaction intermediate.

In Figure 2, each successive intermediate differs by 2 Hs.  

Exercise \(\PageIndex{2}\)

Account for the changes in Hs at each step, as succinate is converted to oxaloacetate.

Answer

Each step, except the second, involves an oxidation reaction.  In the first step (succ → fum), FAD goes to FADH₂.  In step 3 (mal → oaa), NAD⁺ → NADH + H⁺.  In step 2 (fum → mal), H₂O is added in a hydration (addition) reaction across the double bond.

It may seem unimportant to focus on Hs, but they are a key to the cycle.  Let's revisit glucose and its metabolism.  The empirical formula of glucose is C₆H₁₂O₆ or C₆(H₂O)₆. If the 4 C carboxylic acids were just shuttling Hs, removing 6 H₂ equivalents would also mean removing 6 CO₂s from the complete respiration of glucose.  That is what happens in the presence of oxygen when the cycle is running full tilt, and those Hs are passed to Os to form water.  If the 4 C acids were just carriers for Hs (and not intermediate in chemical reactions), then if you added lots more of the 4 C acids, even in the absence of oxygen, all of the C atoms in glucose would be fully oxidized to CO₂.  That doesn't happen.  Oxygen is required, so these 4 C carboxylic acids are not just passive shuttles but active intermediates.

Why are there so many steps involved in simply oxidizing 2 C atoms in pyruvate?

The next obvious question is this: Why are eight reactions required to oxidize just 2 Cs in pyruvate? Partly, this is a matter of evolution again, as the early evolutionary pathway likely served both oxidative (clockwise) and reductive (counterclockwise) reactions, based on the cell's needs. Part of the cycle's chemistry produces β-keto acids, which are readily oxidatively decarboxylated.  The other part converts an α-ketoacid to a molecule with a better electron sink at the carbon β to the departing CO₂. After the two carbon atoms from acetyl-CoA enter the cycle, the cycle regenerates oxaloacetate, allowing it to continue.

Of course, the ultimate goal of an energy-extractive oxidative pathway is not just to form CO₂ but to form ATP or its equivalent (i.e., GTP). Notice that 3 NAD⁺s are used and converted to 3 NADH. In addition, a new, more potent oxidizing agent, FAD, is used and is converted to FADH₂. NAD⁺ and FAD are replenished by reoxidation of NADH and FADH₂ (the reduced forms) back to NAD⁺ and FAD, through mitochondrial electron transport (oxidation) reactions, in which electrons are passed to stronger and stronger oxidizing agents, the last being O₂. In this thermodynamically favored process, many ATPs are produced. We will explore those reactions in the next section.

We will go through each step of the citric acid cycle separately and show how the pathway is regulated (section 16.3). Why such detail? There are only eight steps. Given that the citric acid cycle is a hub that, along with glycolysis, controls metabolic flow through many interconnected pathways, we will examine each in turn. 

Other issues

The PDC and citric acid cycle reactions occur in the mitochondrial matrix. Cytoplasmic pyruvate and NAD⁺ must be transported from the cytoplasm into the matrix. Additionally, some enzymes in the citric acid cycle have both cytoplasmic and mitochondrial variants. Some homologous pairs are differentiated by whether NAD⁺ or NADP⁺ is used as the oxidizing agent. The cytoplasmic ones are not part of the cycle. You would expect these enzyme pairs to have similar tertiary structures and active site chemistry. Prokaryotic forms of these enzymes are structurally similar to their eukaryotic counterparts, so the interactive molecular models shown below depict enzymes from different organisms. Finally, many variants, shunts, and bypasses of the citric acid pathway are used in different organisms. We will explore this topic in section 16.4.

Additionally, dogma suggests that the cycle proceeds clockwise, in an oxidative direction.  Can it return counterclockwise in a reductive fashion?  And if so, under what conditions?  We'll explore that elsewhere as well.

We will pair reaction mechanism diagrams that illustrate electron flow during bond formation and bond breaking with interactive molecular models of the active site. You should rotate the models to align and identify key amino acids and ligands (substrate, substrate analogs, inhibitors, activators) shown in the static 2D mechanism diagrams. Since the active sites are often conserved across prokaryotic and eukaryotic versions, our choice of PDB structures depends on which best illustrates a conceptual point.

Note

Several ways to write abbreviated chemical equations show NAD⁺/NADH, FAD/FAD_2, and hydrogen ions in reactions. To make sense of them, consider a simplified mechanism for ethanol oxidation by alcohol dehydrogenase, as shown in Figure \(\PageIndex{3}\). Note the 2 Hs on the substrate (ethanol) that are removed, one as a hydride (H) and the other as a proton (H⁺).

Figure \(\PageIndex{3}\): Oxidation of ethanol using NAD⁺

Here is a list of different and seemingly contradictory ways to write a chemical equation to show changes in NAD⁺/NADH and hydrogen ions:

1. NAD⁺ + :H^- → NADH. This chemical equation is charge-balanced and shows only the changes in the NAD⁺/NADH pair, not the proton (H⁺) lost from the substrate.
2. NAD⁺ + 2e^- + H⁺ → NADH. This is the same as equation (1) but with the hydride separated into an electron pair and a proton.  The equation is charge-balanced.
3. NAD⁺ + H⁺ → NADH. This is balanced for Hs but not for the + charge, as it doesn't explicitly show the electron pair from the hydride added to NAD⁺.
4. NAD⁺ → NADH + H⁺. This is balanced for charge but not for Hs. The extra H⁺ is the proton from the oxidized substrate.

We will use example 4 above throughout this book. That equation is most useful for accounting for changes in the number of protons in individual reactions and entire pathways. We will also write simplified chemical equations involving FAD (in which 2Hs from the substrate are added) as: FAD → FADH₂.

Now, let's look at each step in detail.

1. Citrate Synthase (CS)

Oxaloacetate + Acetyl-CoASH + H₂O → Citrate + CoASH ΔG^o = -7.5 kcal/mol (-31 kJ/mol)

Acetyl-CoA is a thioester. Hence, it is "high energy" compared to its hydrolysis products. (Remember, there is no such thing as a "high-energy" bond.) The free energy released by its hydrolysis drives the reaction forward. This is important. Otherwise, citrate would not be formed readily. This reaction feeds the end product of glycolysis into the citric acid cycle. It is summarized in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Summary reaction - Citrate Synthase

The enzyme exists in two major conformations: open and closed. The open form binds oxaloacetate and then acetyl-CoA. The complex then shifts to the closed conformation. These changes sequester the bound substrates, exclude water, and prevent spurious hydrolysis of acetyl-CoA. Binding occurs sequentially, so the kinetics follow a sequential mechanism.

Figure \(\PageIndex{5}\) left shows an animated gif of the conformational changes between the citrate-bound version (open, green) and the citrate and CoASH-bound form (blue). The image below right shows a smoother transition between the open and closed form without bound ligands (1cts, 2cts)

Figure \(\PageIndex{5}\): Conformational changes in citrate synthase on binding substrate

For a more detailed description of the enzyme view, see the Regulation of Citrate Synthase.

The mechanism below is from https://chem.libretexts.org/Bookshel...trate_Synthase, with Contributors and Attributions from:

• Dr. Dietmar Kennepohl FCIC (Professor of Chemistry, Athabasca University)
• Prof. Steven Farmer (Sonoma State University)
• Organic Chemistry With a Biological Emphasis by Tim Soderberg (University of Minnesota, Morris)

In this reaction, a C-C bond must form between the substrates. One way to achieve this is to form a nucleophilic carbanion ion from the alpha carbon of acetyl-CoA. Remember, this is not a decarboxylation reaction, so we don't have to worry about an electron "sink" on the β carbon. Forming the carbanion is possible because the negative charge can be delocalized onto the carbonyl oxygen to form an enolate. The enolate becomes even more stable if the negative oxygen is protonated. This reaction is an aldol condensation, which involves adding an enolate to an aldehyde or ketone. The carboxylate group of aspartic acid 375 on citrate synthase removes the acidic alpha proton on acetyl-CoA. At the same time, histidine 274 donates a proton to form the neutral enol, a much more stable molecule than the enolate anion. His 274 continues to stabilize the enol during the reaction. Bound oxaloacetate is stabilized in part by Arg 329. In the next part of the mechanism, a second histidine (His 320) protonates the carbonyl oxygen of oxaloacetate, activating the carbonyl carbon for nucleophilic attack by the enol in the next step to form (S)-citryl CoA. The hydrolysis of CoASH occurs when His 320 deprotonates a water molecule, facilitating nucleophilic attack on the carbonyl carbon bonded to -SCoA, thereby forming citrate.

A plausible mechanism is shown in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Citrate synthase mechanism

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the pig citrate synthase bound to CoASH and citrate (2CTS)

Figure \(\PageIndex{7}\): pig citrate synthase bound to CoASH and citrate (2CTS). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...WXqcEsu336tcL7

The enzyme is a dimer, with its monomers depicted in different colors. Citrate and CoASH are shown as sticks and labeled. The active site residues in each subunit are shown as sticks and labeled.

2. Aconitase

Citrate ↔ Isocitrate ΔG^o = +2 (rx 2a), -0.5 (rx 2b) kcal/mol; net ΔG^o = + 1.5 kcal/mol (+6.3 kJ/mol)

Thinking like a chess player, who must anticipate future moves, the chemical rationale for this reaction is to move an OH to a beta position, which in a subsequent step is converted to a β C=O, so it can act as an electron sink to facilitate decarboxylation in a following reaction! The reaction is readily reversible (note the small ΔG^o value) because the reactant and product are simple isomers. Figure \(\PageIndex{8}\) shows the summary reaction.

Figure \(\PageIndex{8}\): Summary reaction of aconitase

The enzyme has an inorganic Fe₄S₄ cluster. Each Fe in the cluster coordinates to 4 S^2- in a cubane structure, but when either citrate or isocitrate is bound, one of the Fe ions interacts with both the Os of a substrate carboxylate. The other two carboxylates of isocitrate are stabilized through ion-ion interactions by Arg 446 and 663.

Figure \(\PageIndex{9}\) below shows a plausible partial mechanism. An active site deprotonated serine abstracts a proton at the S carbon. This is followed by the formation of the C-C double bond and the release of the resulting cis-aconitate from the FeS cluster.

Figure \(\PageIndex{9}\): Mechanism of aconitase

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the bovine S642A aconitase with bound citrate (1C97).

Figure \(\PageIndex{10}\): Bovine S642A aconitase with bound citrate (1C97). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...iJJRGaFoCDnhf7

This cis-aconitate intermediate in the interconversion of citrate and isocitrate undergoes a 180⁰ flip around the C=C double bond. It is then rehydrated to form the other isomer. The deprotonated His 101 abstracts a hydrogen from a water bound to the FeS cluster, with the hydroxide acting as a nucleophile; along with the redonation of a hydrogen ion on the protonated Ser 642, the alpha-carbon, completes the rehydration step in the formation of the other isomer.

Exercise \(\PageIndex{3}\)

Why was the S642A mutant used to produce the structure shown in the above iCn3D model?

Answer

It allows the binding of substrate/product, in this case isocitrate, to an inactive enzyme, as the active-site serine was mutated to a non-nucleophilic alanine of similar size. Hence, no bond-making/breaking occurs in the complex.

References:

https://collab.its.virginia.edu/acce...-_-/index.html

3. Isocitrate Dehydrogenase (IDH)

Isocitrate + NAD⁺ → α-ketoglutarate + NADH + H⁺ ΔG^o = -2.0 kcal/mol (-8.4 kJ/mol)

The chemical rationale should be clear. In this step, CO₂ is removed as NADH is produced via oxidative decarboxylation. NADH is reoxidized to NAD⁺ in the electron transport chain, producing ATP (see the next section). The reaction is summarized in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Summary reaction: isocitrate dehydrogenase

Exercise \(\PageIndex{4}\)

Why must the oxidation reaction precede the decarboxylation reaction?

Answer

First, a β-ketoacid intermediate must form, facilitating its easy decarboxylation.  The β-carbonyl provides an electron "sink" to facilitate the decarboxylation.

There are two forms of this enzyme (IDH), a cytoplasmic (NADP⁺) form and a mitochondrial (NAD⁺ ) form. Cytoplasmic forms in various organisms are homodimers and share a common catalytic mechanism. In contrast, yeast mitochondrial IDH has two subunits: IDH1 (regulatory, which binds the allosteric activators citrate and AMP) and IDH2 (catalytic, which binds isocitrate and NAD⁺). 

Mammalian NAD-IDHs are even more complex than yeast NAD-IDH. These enzymes have three subunit types, α, β, and γ, which share 40–52% sequence identity. The α and β form an αβ dimer, while the α and γ subunits form αγ. These then interact to form the α₂βγ heterotetramer, which effectively forms the holoenzyme. It can also form an active heterooctamer. Citrate and ADP regulate the αγ heterodimer. Citrate binding to the allosteric site induces a conformational change that enhances isocitrate binding. ADP enhances the binding of the allosteric regulator citrate. The α chain is the catalytic subunit.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model that shows the superposition of the α chains of cytoplasmic IDH (NADP, sky blue, 4L03) and mitochondrial IDH (NAD) (6KDY, salmon).

Figure \(\PageIndex{12}\): Superposition of the A chains of cytoplasmic IDH (4L03) and mitochondrial IDH (NAD) (6KDY) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...zy9yn4NaF9g1c9

Click the 3-bar menu (hamburger, =) icon (top left) in the model in the window and toggle the "a" key to move between the two forms. Although the alpha chains are not identical, they align well.

Figure \(\PageIndex{13}\) shows a probable mechanism for the reaction based on the conserved catalytic site shown in the model above.

 

Figure \(\PageIndex{13}\): Mechanism of isocitrate dehydrogenase (after https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706558/)

Figure \(\PageIndex{14}\) shows an interactive iCn3D model of the active site of the cytoplasmic human IDH1 in complex with NADP⁺ and Ca²⁺/α-ketoglutarate (4L03).

Figure \(\PageIndex{14}\): Active site of the cytoplasmic human IDH1 in complex with NADP⁺ (NAP1) and Ca²⁺/ alpha-ketoglutarate (4L03) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...P9DqLoPowEV2d9

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the active site of the αβ heterodimer of human IDH3 (6kdy) in complex with NAD⁺.

Figure \(\PageIndex{15}\): αβ heterodimer of human IDH3 (6kdy) in complex with NAD⁺(6kdy) . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...hyyJiMFEqwktB7

The α subunit is shown in gray and the β in cyan.

Gliomas, a type of brain cancer, are the most common brain tumors in adults under 50.  These contain mutations in isocitrate dehydrogenase (IDH).  IDH mutations in glial progenitor cells appear to play a key role in glial tumor development.  Jung Won Park et al.  IDH-mutant gliomas arise from glial progenitor cells harboring the initial driver mutation. Science 391,eadt0559(2026). DOI:10.1126/science.adt0559

4. α-ketoglutarate dehydrogenase

α-ketoglutarate + NAD⁺ + CoASH → succinyl CoA + CO₂ + NADH + H⁺ ΔG^o = -7.2 kcal/mol (-30 kJ/mol)

In the last reaction, α-ketoglutarate is formed. Oh no, you might say! It would have been nice to form a β-ketoacid, which could easily undergo decarboxylation. No worries, though. We spent section 16.1 explaining the biochemistry of decarboxylating another α-ketoacid, pyruvate. The same chemistry accomplishes the oxidative decarboxylation of α-ketoglutarate. Hence, we won't expand on the mechanism here. The reaction is shown in Figure \(\PageIndex{16}\).

Figure \(\PageIndex{16}\): Summary reaction for α-ketoglutarate dehydrogenase

5. Succinyl-CoA synthetase (SCS)

succinyl CoA + GDP + P_i ↔ succinate + CoASH + GTP ΔG^o = -0.8 kcal/mol (-3.3 kJ/mol)

This is the first step in which the energy change in the cycle is explicitly captured in the form of a high-energy (with respect to its hydrolysis product) phosphoanhydride bond in the form of GTP (ATP in some organisms). At the chemical level, cleavage of the thermodynamically unstable thioester is coupled to the endergonic synthesis of GTP. This can transfer its terminal phosphate to ADP to make ATP in a reaction with a ΔG^o _≈ 0 kcal/mol. The reaction is shown in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): Summary reaction for succinyl-CoA synthetase

Succinyl-CoA synthases have two subunits, α and β. The enzyme in E. coli is a tetramer (α₂β₂) with the catalysis occurring at the αβ interface. The alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the alpha-beta dimers with CoA bound in each α subunit to a nucleotide-binding loop.

Two histidines, His 246 and His 142, are involved in the reaction, with His 246 becoming phosphorylated to form an intermediate. A mutation of His 142 to an asparagine (H142N) essentially abolishes enzyme activity. Different SCSs have different specificities for purine nucleoside triphosphates. Organisms, including mammals, may have two different isoforms, one that binds ADP and one that uses GDP (as shown in most diagrams of the citric acid cycle). In E. coli, the α subunit binds CoASH and contains His246, which gets phosphorylated. The β subunit determines the specificity for either GTP or ATP. In E. coli, the ATP binding site (Site II "in the ATP-grasp fold") is quite distant from the CoASH site (Site II), so phospho-His 246 must move between the sites in the dimer interface.

The three steps in the reaction are shown below, where E is the free enzyme, a ^. indicates a noncovalent complex, and a - represents a covalent bond (after Biochemistry 2002, 41, 537-546)

1. E + succinyl-CoA + P_i ↔ E ^. succinyl-PO₃ + CoASH
2. E ^. succinyl-PO₃ ↔ E-PO₃ + succinate
3. E-PO₃ + NDP ↔ E + NTP

Figure \(\PageIndex{18}\) shows an abbreviated mechanism that shows only the involvement of His 246.

Figure \(\PageIndex{18}\): Abbreviated mechanism for succinyl-CoA synthase

Kinetic analysis suggests that the three substrates bind in a specific order, catalysis occurs, and then the three products leave. This type of reaction is called an ordered ter ter (tertiary - 3 reactants - in both the forward and reverse) and is shown in Figure \(\PageIndex{19}\).

Figure \(\PageIndex{19}\): Ordered ter ter reaction for succinyl-CoA synthase

The iCn3D model below shows key α-chain residues in the active site, including the phosphorylated His246 and the bound CoASH. Use iCn3D to visualize 1CQJ, the nonphosphorylated form.

Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the complex of ADP and Mg²⁺ with Dephosphorylated E. Coli Succinyl-CoA Synthetase (1CQI)

Figure \(\PageIndex{20}\): Complex of ADP and Mg²⁺ with Dephosphorylated E. Coli Succinyl-CoA Synthetase (1CQI). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...BssxsabV1S9cz6

Just one αβ dimer is shown. The α subunit is shown in gray and the β in cyan. Bound P_i and CoASH are labeled. The active site His246 in the α chain is shown in sticks and labeled.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the active site of pig GTP-specific succinyl-CoA synthetase in complex with succinate and CoASH (5CAE).

Figure \(\PageIndex{21}\): Active site of pig GTP-specific succinyl-CoA synthetase in complex with succinate and CoASH (5CAE). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...HjH9ER5wLqb1T7

6. Succinate Dehydrogenase

succinate + FAD ↔ fumarate + FADH₂ ΔG^o = 0 kcal/mol

The enzyme is yet another step in closing the cycle to reform oxaloacetate. It is also our first encounter with FAD as an oxidizing agent. Its reduction product, FADH₂, will be reoxidized in the electron transport chain (the mitochondrial inner membrane in eukaryotes), producing energy for ATP synthesis. Thus, it can be considered a proxy for ATP generation. The reaction is shown in Figure \(\PageIndex{22}\).

Figure \(\PageIndex{22}\): Summary reaction for succinate dehydrogenase

Succinate dehydrogenase is part of the larger Complex II of the electron transport chain. Complex II utilizes numerous cofactors in its overall function. It also utilizes an iron-sulfur cluster cofactor, similar to aconitase, to produce a C=C double-bond intermediate. We will discuss it in greater detail in the chapter on electron transport. Let's concentrate on this new cofactor and oxidizing agent, FAD. Many enzymes use FAD/FADH₂ in redox chemistry.

In contrast to NAD⁺/NADH, the FAD/FADH₂ pair stays tightly bound to the enzyme and doesn't readily dissociate. This means that after one catalytic cycle (after FAD is converted to FADH₂), the enzyme is functionally "dead". Another oxidizing agent must bind to the enzyme and reoxidize FADH₂ back to FAD. The dissociation constants for FAD/FADH₂ and its protein binder in a flavoprotein are often in the nanomolar range. In around 10% of flavoproteins, FAD/FADH₂ is covalently bonded to the enzyme.

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of the Avian respiratory complex II FAD-binding subunit with FAD and a malate-like intermediate (1YQ3).

Figure \(\PageIndex{23}\): Avian respiratory complex II FAD binding subunit with FAD and a malate-like intermediate (1YQ3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SGac3NuN4XBWk7

Note how deeply buried the FAD is within the dehydrogenase subunit. The malate-like intermediate (TEO) is shown next to the FAD in yellow spacefill.

Exercise \(\PageIndex{51}\)

Succinate dehydrogenase is irreversibly inhibited by the toxin 3-nitropropionic acid (3np) made by some plants and fungi. Eating moldy sugar cane has led to reported deaths.

1. Draw the Lewis structures of succinate and 3-nitropropionic acid. Compare them and the total number of valence electrons in each.

2. Here is a link to an iCn3D model showing the interaction of 3np with the enzyme. Explain the mode of action of the toxin.

https://structure.ncbi.nlm.nih.gov/i...cja9VKU2ZMCac9

Answer

These molecules are structurally similar and are isoelectronic (the same number of electrons in their Lewis structures.

The inhibitor 3np forms a covalent adduct through the guanidino group of Arg 297. This is a key catalytic residue that acts as a general base, accepting a proton from succinate in the reaction.

Figure \(\PageIndex{24}\) (top) shows a very general and abbreviated mechanism for the enzyme for the immediate reaction of succinate with FAD. The bottom part of the image shows the amino acids surrounding succinate in the avian (bird) version of the enzyme (pdb 1yq4)

Figure \(\PageIndex{24}\): Top - a very general and abbreviated mechanism for the enzyme's immediate reaction of succinate with FAD. Bottom - amino acids surrounding succinate in the avian (bird) version of the enzyme (1yq4)

Exercise \(\PageIndex{6}\)

One of the amino acids surrounding succinate in the figure above acts as a general base and abstracts a proton from succinate as a hydride is transferred (from a plane above) to FAD. Go to this iCn3D of the active site bound to FAD. Which amino acid is the likely general base? (Note: the figure below shows general protonated states of side chains and not necessarily those involved in the proton abstraction. Go to Analysis, Distance, and Distance between 2 atoms to find the likely general base.

Answer

Arg 297

7. Fumarase

fumarate + H₂O ↔ L-malate ΔG^o = -0.9 kcal/mol (-3.8 kJ/mol)

The chemical rationale for this reaction is clear.  It is the penultimate step in the resynthesis of oxaloacetate, one of the reactants that start the cycle. This reaction introduces oxygen via hydration, which can then be oxidized in the next step to produce NADH for electron transport and ATP production. The reaction is shown in Figure \(\PageIndex{25}\).

Figure \(\PageIndex{25}\): Summary reaction for fumarase

There are Class I (dimers containing an unstable FeS cluster, examples A and B) and Class II (tetramer, no bound iron, oxygen stable, example C) fumarases. Humans possess both cytoplasmic and mitochondrial type II fumarases, which result from the alternative transcription of the fumarase genes. We will consider type II fumarase C from E. Coli.

The tetramer consists solely of α-helices and random coils and features two distinct binding sites. Site A appears to be the active site and contains a buried water molecule. Site A, formed from three subunits, binds competitive inhibitors such as citrate and β-(trimethylsilyl)maleate, a cis substrate for fumarase, and is buried. 12 Å away is site B, which is found in only one of the subunits near a pi-helix (H129 through N135) and is more surface-exposed. Each site has a histidine, but mutation of only H188N in the A site disrupts enzyme activity. Both sites bind multi-carboxylates. The role of site B is somewhat unclear, but it is most likely an allosteric site that transfers product (malate) from the buried site to the surface for dissociation.

Figure \(\PageIndex{26}\) shows an interactive iCn3D model of the fumarase with beta-(trimethylsilyl)maleate and citrate (1fuq).

Figure \(\PageIndex{26}\): Fumarase with beta-(trimethylsilyl)maleate and citrate (1fuq). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...MewEQFebi4VL48

The monomers in the tetramer are shown in different colors. Citrate (Cit) and beta-(trimethylsilyl)maleate (SIF) are shown in spacefill.

Figure \(\PageIndex{27}\) shows an interactive iCn3D model of the binding site of citrate, a competitive inhibitor of fumarase (1FUQ).

Figure \(\PageIndex{27}\): Binding site of the competitive inhibitor citrate in fumarase (1FUQ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...yfPc5iPWbExr86

Figure \(\PageIndex{28}\) shows a plausible mechanism for the trans-addition of water to fumarate.

Figure \(\PageIndex{28}\): Mechanism for fumarase

8. Malate Dehydrogenase (MDH)

L-malate+ NAD⁺ ↔ oxaloacetate + NADH + H⁺ ΔG^o = +7.1 kcal/mol (30 kJ/mol)

We are finally there! This last reaction of the citric acid cycle produces oxaloacetate, the starting reactant, allowing the cycle to continue. It also produces NADH for mitochondrial electron transport and ATP production. Notice that the reaction is thermodynamically unfavorable (in the standard state), but the reaction is pulled to citrate formation by the next (and first step of the cycle, citrate synthase. The reaction is shown in Figure \(\PageIndex{29}\).

Figure \(\PageIndex{29}\): Summary reaction for malate dehydrogenase

Malate dehydrogenases are located in the cytoplasm and are part of the aspartate-malate shuttle, which transports cytoplasmic malate (and, indirectly through MDH, NADH) into the mitochondria.  It is also found in the mitochondria, where it is part of the citric acid cycle. NAD⁺- and NADP⁺-dependent forms also exist. Malate can undergo two oxidation reactions: one using NAD⁺ to produce oxaloacetate, and another, an oxidative decarboxylation, using NADP⁺ to produce pyruvate and CO₂. The latter is sometimes called the malic enzyme.

Humans have two forms (MDH 1 and MDH 2) that use NAD⁺. In humans, the enzyme is a homodimer, with binding sites on both subunits. Its activity is allosterically
regulated by citrate, and ATP, ADP, AMP, fumarate, and aspartate inhibit it.  High concentrations of oxaloacetate also inhibit it.
The enzyme is similar to lactate dehydrogenase, which we encountered in glycolysis. Kinetic analyses show that NAD⁺ binds first, followed by malate.

Figure \(\PageIndex{30}\) shows an interactive iCn3D model of NAD⁺ and malate bound to human malate dehydrogenase 2 (4wlu).

Figure \(\PageIndex{30}\): NAD⁺ and malate bound to human malate dehydrogenase 2 (4wlu). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...DrAXBdu64GPzx8

Just one monomer is shown. NAD⁺ and malate (LMR) are shown as sticks, labeled.

Figure \(\PageIndex{31}\) shows an abbreviated mechanism for malate dehydrogenase. The numbers refer to the E. Coli enzyme.

Figure \(\PageIndex{31}\): Abbreviated mechanism for malate dehydrogenase

Note that the hydride transferred from the malate is shown in red as a deuterium (D). Deuterium (D) was used to indicate the stereochemistry of the removed "H" and to assign that "H" to the proR position in NADH.  (Remember that the carbon with the deuterium in NADH is prochiral. D has higher priority than H in the Cahn/Ingold-Prelog system.  It is transferred to the re face of NAD⁺ to form NADH.  In the reverse reaction, the D atom, which is above the plane of the ring, occupies the proR position. The proR deuterium is transferred back in this reversible reaction.

Figure \(\PageIndex{32}\) shows an interactive iCn3D model of the active site of the E. Coli malate dehydrogenase with bound citrate and NAD⁺ (1EMD).

Figure \(\PageIndex{32}\): Active site of the E. Coli malate dehydrogenase with bound citrate and NAD⁺ (1EMD). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...mHhmeFQyPpZj18

We have shown many renderings of the enzymes involved in the cycle. Yet another one is shown below. Figure \(\PageIndex{33}\) shows an interactive iCn3D model of the electrostatic surface potential of malate dehydrogenase (4WLU).

Figure \(\PageIndex{33}\): Electrostatic surface potential of malate dehydrogenase (4WLU)with bound NAD⁺ (4WLU). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?izf2dsWXTw2bE8YD7

The displayed surface is the enzyme's electrostatic surface potential map. Red shows the more anionic surfaces with a negative electrostatic potential to which cations would be attracted. At the same time, blue represents more cationic surfaces that attract anions. Note that the bound NAD⁺, which has many oxygens that are slightly or entirely negative, is bound in a blue, positive electrostatic potential region.

Summary of the Cycle

Let's do some stoichiometry for the full cycle. Here is the net reaction (assuming that the GTP produced by succinyl-CoA synthetase is equivalent to 1 ATP).

Acetyl-CoA + 3NAD⁺ + FAD + ADP + P_i + 2H₂O → 2CO₂ + 3NADH + FADH₂ + ATP + 2H⁺ + CoASH

It must seem like a lot of work to produce just 1 ATP, especially since the partial anaerobic oxidation of glucose in glycolysis produces 2 ATPs in net fashion. The key is to realize that 3 NADHs and 1 FADH2 are produced, which, when reoxidized in mitochondrial electron transport/oxidative phosphorylation, produce many ATP.

As with glycolysis, this main energy-extracting pathway is highly regulated. We will discuss this in the next section.

Figure \(\PageIndex{34}\) highlights (colored boxes) some key features of molecules involved in the citric acid cycle.

Figure \(\PageIndex{34}\): Summary of the citric acid cycle.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter provides a mechanistically detailed account of the citric acid cycle — the central oxidative pathway of aerobic metabolism — tracing the fate of the two-carbon acetyl group from acetyl-CoA through eight enzymatic steps that regenerate oxaloacetate while extracting electrons into NADH and FADH₂ for subsequent ATP production by oxidative phosphorylation.

The chapter opens by establishing the rationale for a cyclic rather than linear pathway. Because several cycle intermediates are continuously withdrawn for biosynthesis (of amino acids, heme, nucleotides, and lipids), a linear pathway would rapidly deplete its intermediates and halt. The cyclic architecture, in which oxaloacetate is regenerated at the end of each turn, allows continuous operation as long as acetyl-CoA is supplied. When intermediates are withdrawn, anaplerotic reactions — most notably pyruvate carboxylase — replenish them. Hans Krebs's historical discovery of the cycle, using ¹⁴C-labeled intermediates and minced pigeon flight muscle, established both the pathway's cyclic nature and citrate as the first product. The overall stoichiometry per turn is: acetyl-CoA + 3NAD⁺ + FAD + ADP + Pᵢ + 2H₂O → 2CO₂ + 3NADH + FADH₂ + GTP + CoASH. Only one GTP is produced directly; the primary energetic output is three NADH and one FADH₂, which collectively drive the synthesis of approximately 10 ATP equivalents through mitochondrial electron transport.

The eight steps are described in mechanistic detail with a recurring emphasis on chemical logic. Step 1 (citrate synthase) condenses oxaloacetate and acetyl-CoA in an aldol condensation, generating a carbanion enolate from the α-carbon of acetyl-CoA through the action of Asp375 (base) and His274 (proton donor); the thermodynamic driving force is provided by hydrolysis of the thioester bond of acetyl-CoA (ΔG°′ = −7.5 kcal/mol). The enzyme undergoes an induced-fit conformational change from an open to a closed state upon sequential substrate binding, excluding water and preventing wasteful hydrolysis of acetyl-CoA. Step 2 (aconitase) isomerizes citrate to isocitrate through a cis-aconitate intermediate, catalyzed by an Fe₄S₄ cluster; the mechanistic purpose of this seemingly simple isomerization is to reposition the hydroxyl group from the tertiary carbon of citrate to the secondary carbon of isocitrate, placing it at the β-position relative to a carboxylate — the arrangement required for the oxidative decarboxylation that follows.

Step 3 (isocitrate dehydrogenase, IDH) performs this oxidative decarboxylation: oxidation of the C2 hydroxyl to a ketone produces the β-keto acid intermediate oxalosuccinate, which then decarboxylates spontaneously with the adjacent carbonyl serving as the electron sink, releasing CO₂ and forming α-ketoglutarate while reducing NAD⁺ to NADH. Both cytoplasmic (NADP⁺-dependent) and mitochondrial (NAD⁺-dependent) isoforms exist; the mitochondrial form is an α₂βγ heterotetramer, allosterically regulated by citrate and ADP. IDH mutations in glial progenitor cells are associated with glioma development. Step 4 (α-ketoglutarate dehydrogenase) addresses the problem of decarboxylating a second α-keto acid using the same TPP–lipoamide–FAD–CoASH–NAD⁺ mechanism as the pyruvate dehydrogenase complex, yielding succinyl-CoA, CO₂, and NADH. At this point, the two carbons of the original acetyl-CoA have been fully released as CO₂.

The remaining four steps regenerate oxaloacetate. Step 5 (succinyl-CoA synthetase) couples thioester hydrolysis to substrate-level GTP synthesis via a phosphohistidine intermediate (His246) and proceeds through an ordered ter-ter kinetic mechanism. Step 6 (succinate dehydrogenase) oxidizes succinate to fumarate using FAD — necessary because the reduction potential of the fumarate/succinate couple (E°′ = +0.03 V) is insufficiently negative to allow NAD⁺ to act as the oxidizing agent — and is embedded in the inner mitochondrial membrane as Complex II of the electron transport chain, with the FADH₂ produced being directly oxidized by ubiquinone. The mechanism involves an Fe-S cluster. Step 7 (fumarase) performs a stereospecific trans-hydration of fumarate across the double bond to yield L-malate, with His188 in the buried active site serving as the critical catalytic residue. Step 8 (malate dehydrogenase) oxidizes L-malate to oxaloacetate using NAD⁺, with stereospecific hydride transfer from the proR position of malate to the re face of NAD⁺. Although this step is thermodynamically unfavorable under standard conditions (ΔG°′ = +7.1 kcal/mol), the rapid consumption of oxaloacetate in step 1 (citrate synthase, ΔG°′ = −7.5 kcal/mol) drives it forward, illustrating how thermodynamically linked reactions within a pathway can drive otherwise unfavorable steps.