• Introduction
  • The basics of the citric acid cycle
  • Step 1: Citrate synthesis
  • Step 2: Isomerization of citrate
  • Step 3: Oxidative decarboxylation of isocitrate
  • Step 4: Oxidative decarboxylation of α-ketoglutarate
  • Step 5: Cleavage of succinyl coenzyme-A
  • Step 6: Oxidation of succinate
  • Step 7: Hydration of fumarate
  • Step 8: Oxidation of malate
  • What happens to NADH and FADH₂?
  • Final thoughts
  • Sources
  • Acetyl-CoA: An acetylated form of coenzyme A, an important intermediate in the oxidation of carbohydrates, fats, and protein. 
  • FADH₂: Also known as Flavin adenine dinucleotide. It is an electron carrier that transports electrons from glycolysis and the citric acid cycle to the electron transport chain.
  • Electron carrier: Small organic molecules that switch between oxidized and reduced forms and transport electrons during metabolic reactions.
  • NADH: A reduced form of Nicotinamide adenine dinucleotide that carries electrons from one reaction to another. An important cofactor in metabolism consisting of two nucleotides and their phosphate groups.
  • NAD+: An oxidized form of Nicotinamide adenine dinucleotide that accepts electrons from other reactions and becomes reduced.
  • Substrate phosphorylation: Formation of ATP from ADP by the transfer of a phosphate group.

Introduction

Cellular respiration is based on interconnected pathways that break down glucose to generate fuel in the form of adenosine triphosphate (ATP). With the presence of sufficient amounts of oxygen, this process consists of glycolysis, the citric acid cycle, and oxidative phosphorylation.

Each of these phases has a specific function allowing a glucose molecule to be oxidized. Once fully oxidized, a single glucose molecule produces 30-32 molecules of ATP. This is used during exercise as well as maintaining regular bodily functions (breathing, heartbeat, hormonal activity, etc.).

The citric acid cycle is the second phase of cellular respiration. It utilizes the end products of glycolysis and further breaks them down into molecules that can be used in oxidative phosphorylation. By itself, the citric acid cycle produces 2 ATP.

This post explains the basic processes of the citric acid cycle, and why it is such a crucial part of aerobic respiration.

The basics of the citric acid cycle

The citric acid cycle, also known as the Krebs cycle, starts with the end products of glycolysis when a 6-carbon glucose molecule is broken down to a three-carbon molecule called pyruvate. Then, pyruvate is decarboxylated (i.e.loses a carbon) by pyruvate dehydrogenase forming a two-carbon molecule called acetyl-CoA. This process also produces carbon dioxide (CO2) and NADH. Both the conversion of pyruvate to acetyl-CoA and the citric acid cycle takes place in the mitochondrial matrix. 

Acetyl-CoA then enters the citric acid cycle, where it is ultimately oxidized into CO2 after eight redox, dehydration, hydration, and decarboxylation reactions. For every added pyruvate molecule, the citric acid cycle produces:

  • 2 molecules of carbon dioxide (CO₂)
  • 3 molecules of nicotinamide adenine dinucleotide (NADH)
  • 1 molecule of flavin adenine dinucleotide (FADH₂)
  • 1 molecule of guanosine triphosphate (GTP) or adenosine triphosphate (ATP) depending on the cell type

The reason why these end products are important is that NADH and FADH₂ are electron carriers capable of transporting electrons during metabolic reactions. They also allow the oxidation of glucose to proceed to the last phase of cellular respiration, oxidative phosphorylation. But before we jump too far ahead, let’s take a closer look at each reaction. 

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The Citric Acid Cycle


The second phase of cellular respirationSplits pyruvate into Acetyl-CoAAerobic processTakes place in the mitochondriaProduces 2 CO₂ + 3 NADH + 1 FADH₂ + 1 GTP/ATP

Step 1: Citrate synthesis

The first reaction of the citric acid cycle is a condensation step catalyzed by an enzyme called citric acid synthase. In this step, the acetyl group from acetyl-CoA (C23H38N7O17P3S) is attached to a four-carbon oxaloacetate molecule, forming a six-carbon molecule called citrate. 

CoA is bound to a sulfhydryl group (-SH) and diffuses away to combine with another acetyl group. This step is exergonic (reaction that releases free energy), making the process irreversible. The rate of which this reaction occurs is determined by the amount of ATP available. The less there is, the faster the reaction is – and vice versa.

Interestingly, oxaloacetate is also the final product of the citric acid cycle, making the whole process a closed loop. 

Step 2: Isomerization of citrate

In the second step of the citric acid cycle, citrate (C6H5O7-3) loses a water molecule and gains another. This reaction is catalyzed by aconitase, which essentially converts citrate into its isomer, isocitrate. 

Because this step consists of removing and attaching a water molecule, some scientists consider that the citric acid cycle consists of nine steps instead of eight. 

Step 3: Oxidative decarboxylation of isocitrate

The third step is catalyzed by isocitrate dehydrogenase, which oxidizes isocitrate (C6H8O7) and removes a molecule of carbon dioxide, producing a five-carbon molecule α-ketoglutarate. The process also releases two electrons that reduce NAD+ to NADH and H+. This is also the first step of the cycle that produces a reduced coenzyme. 

This step is regulated by the positive feedback from ADP and negative feedback from NADH and ATP. Thus, a higher concentration of NADH and ATP inhibits this reaction and vice versa.

Step 4: Oxidative decarboxylation of α-ketoglutarate

In the fourth step of the citric acid cycle, α-ketoglutarate (C5H6O5) loses a carbon dioxide molecule and coenzyme A takes its place, forming a four-carbon molecule called succinyl-CoA. This process is catalyzed by the enzyme α-ketoglutarate dehydrogenase.

Steps three and four are oxidation and decarboxylation steps. They release electrons that reduce NAD+ into NADH and release carboxyl groups that form CO2 molecules.

The product of step three is α-ketoglutarate, whereas the products of step four is a succinyl group. CoA then binds the succinyl group to form succinyl-CoA. The enzyme that catalyzes this reaction is regulated by feedback inhibition of ATP, NADH, and succinyl CoA.

Step 5: Cleavage of succinyl coenzyme-A

The fifth step of the citric acid cycle is catalyzed by succinyl-CoA synthetase. Here, the CoA from succinyl-CoA (C25H40N7O19P3S) is removed to produce a four-carbon molecule called succinate. The energy released is then used in substrate-level phosphorylation to form guanosine triphosphate (GTP) from guanosine diphosphate (GDP) and Pi. 

These enzymes, called isoenzymes, come in two different forms depending on what type of animal tissue they are found. The first one (ATP) is found in tissues with a large amounts of ATP, such as skeletal muscle tissue, heart, etc. The other form (GTP) is found in tissues with a high number of anabolic pathways, such as the liver.

The newly formed GTP is energetically similar to ATP, but its use is more limited. One example of this is protein synthesis, which mainly uses GTP.

Step 6: Oxidation of succinate

Step six is a dehydration process, where succinate (C4H6O4) is converted by the enzyme succinate dehydrogenase, producing a four-carbon molecule called fumarate. This oxidation process also transfers two hydrogen atoms and their electrons from succinate to FAD, reducing it to FADH₂. 

Because the enzyme that catalyzes this reaction is embedded in the inner membrane of the mitochondrion, FADH₂ can immediately transfer its electrons into the electron transport chain. 

Step 7: Hydration of fumarate

In the seventh step of the citric acid cycle, water is added to fumarate (C4H4O4), producing a four-carbon molecule called malate. This reaction is catalyzed by the enzyme fumarase.

Step 8: Oxidation of malate

In the eighth and last step of the citric acid cycle, malate (C4H4O5-2) is oxidized back into oxaloacetate with the help of an enzyme called malate dehydrogenase. This step also reduces NAD+ into another molecule of NADH as well as H+. 

Because the final pathway of the citric acid cycle produces the same four-carbon compound used in the first step (oxaloacetate), the entire process is considered a closed loop. This essentially allows the cycle to run indefinitely as long as there are sufficient reactants present. 

The final phase of the citric acid cycle produces the same compound used in the first step of the process - oxaloacetate.

What happens to NADH and FADH₂?

The NADH and FADH₂ molecules formed during glycolysis and the Krebs cycle are known as electron carriers capable of transporting electrons into the inner membrane of the mitochondria. NADH especially has the capacity to produce vast amounts of ATP.

In the mitochondria, NADH and FADH₂ transfer their electrons into the electron transport chain. Through a series of reactions, these electrons provide the energy to pump protons from the mitochondrial matrix into the inner mitochondrial membrane. This gradual release of electrons creates a proton gradient that drives the synthesis of ATP.

This final phase of cellular respiration is known as oxidative phosphorylation and it produces a substantial amount of energy – 28 ATP.

Final thoughts

As you can see, the citric acid cycle is an incredibly complex process that may sometimes seem daunting to try and understand. Thus, it is no surprise that its discoverer Hans Krebs was awarded a Nobel prize in physiology or medicine in 1953. This is also why it is often referred to as the Krebs cycle. 

The citric acid cycle is an important phase of cellular respiration that sits between glycolysis and oxidative phosphorylation. It utilizes the end products from glycolysis and breaks them down further in a series of eight reactions. 

Once all eight reactions have taken place, a single pyruvate molecule is broken down into 2 CO2, 3 NADH, 1 FADH₂, and 1 GTP. While this entire process produces only 2 ATP by itself, the remaining electron carriers also drive the electron transport chain and oxidative phosphorylation. This allows the oxidation of glucose to continue, producing vast amounts of more ATP. 

Did you learn anything new about the citric acid cycle? Let us know in the comments. 

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