Ippei Kawano
Dedicated to all the medical students who never understood what was going on...
In the human body, the main job of the Krebs cycle is to take a two-carbon compound (acetyl-CoA) that you obtain from metabolizing food, and completely oxidizing it to two molecules of carbon dioxide. We live on the energy harvested by this oxidation. To say it plays a central role in life is an understatement—the Krebs cycle is the business end of life itself, there’s nothing more alive than the Krebs cycle! The 8 chemical reactions require many cofactors, and medical students are often made to memorize the intermediates, the enzymes that perform the reaction, the cofactors required, and how the TCA cycle connects to other parts of metabolism. This is tedious and dry for most, but let me give you a crash course to convince you that it is far from dry.
It is not easy to do all kinds of chemistry with just two carbons, so the cycle starts by attaching the two-carbon that we want to oxidize to a scaffold four-carbon compound. Now, this sounds simple, but attaching one carbon to another is no easy task. Organic chemistry is like Lego, but instead of a physical bump to fit into a physical notch, we have an electron density bump to fit into an electron density notch. Carbon has a problem, because on its own it has neither a bump nor a notch of electron density, so how can we stick two carbons together? This puzzled the organic chemists for a really long time until organometallic chemistry was discovered, where a metallic atom forces electron density onto the carbon to make a bump. We can also suck the electron density away from carbon using highly electronegative oxygen to make a notch. Now we can make carbon-carbon bonds. This reaction was so revolutionary that it got a Nobel prize. In biochemistry, however, organometallic compounds cannot be used, because they will react with water. So biology uses another chemistry, alpha-carbon chemistry, to create a bump on the two-carbon compound, that can be inserted into the notch-carbon of the four-carbon scaffold that has its electron density sucked away by its double bond to oxygen. It’s an elegant and ingenious reaction, all the more impressive considering this chemistry happens in neutral pH at body temperature (the wonder of enzymes!!).
Once the two carbons are attached to make a six-carbon metabolite, we must proceed with carbon oxidation to harvest energy. But alas, immediately there’s a problem. Chemically, to produce carbon dioxide from this metabolite, the electron density from the leaving carbon must go somewhere, but there is nowhere suitable on this compound. If we have a keto-group somewhere, this keto-group can serve as the electron “sink” to allow the reaction to complete. Currently, there’s only a tertiary alcohol, which cannot be further oxidized. So this alcohol group is shifted to a neighboring carbon to make secondary alcohol. Now it can be oxidized to make a keto group!
For the sake of simplicity, let’s just think that there are two kinds of keto groups, alpha-keto acid and beta-keto acid. Alpha-keto acid has its carbons organized in a way that the electron density cannot just go to the oxygen and would rather be stuck on the carbon. Beta-keto acid, on the other hand, has the perfect arrangement for electrons to go into the oxygen. Fortunately, the first compound that forms after the oxidation of secondary alcohol is indeed a beta-keto acid, so the first carbon dioxide comes off easily without extra help. But after this first carbon comes off, the remaining five-carbon compound (now known as alpha-ketoglutarate), is an alpha-keto acid! How can biology go around the forbidden reaction to take off carbon dioxide from this alpha-keto acid?
The solution is to use a different electron “sink”—a vitamin known as vitamin B1, or thiamine (this is why it is vital for life). This takes the electron density from the leaving carbon group onto its thiazole ring temporarily to allow carbon dioxide to come off from alpha-keto acid. The thiazole ring then returns this electron density back to the now four-carbon compound, leaving thiamine chemically unchanged (a catalyst!). Now we have successfully oxidized two carbons all the way to two molecules of carbon dioxide! The redox energy obtained through these reactions is stored in the form of redox cofactors to be harvested by the mitochondrial transport chain, all these have their separate chemical drama that I will not get into here.
The remaining four-carbon molecule can again serve the function of scaffold for the next two-carbon molecule from food, but now the four-carbon compound is “bare”—there are only two carboxyl carbons with two hydrocarbons, all of which terrible for doing chemistry. We want to regenerate the keto “notch” for the next two-carbon to attach. This is a relatively easy synthesis question even for a beginner student of organic chemistry, we will simply introduce an alcohol group and then oxidize it to a keto group. But this alcohol carbon must be introduced to a double bond, for which we must use the middle two hydrocarbons of the four-carbon compound. To take hydrogens off hydrocarbons is chemically difficult, but biology has evolved an elegant solution. Rather than the usual redox cofactor NAD that is used for other oxidation reactions, this step uses FAD, a stronger redox cofactor that can also do the forbidden one-electron chemistry (usually in biochemistry, two-electron chemistry is standard, to avoid unlocking the reactive nature of oxygen, which is usually kept unreactive by spin-restriction—this is also super interesting but beyond the scope here). FAD uses this one-electron chemistry to transfer electrons from the hydrocarbons to the iron-sulfur cluster directly. Essentially, this allows the enzyme to directly use the oxygen’s ability to suck electrons towards itself to achieve the difficult chemistry (since you ask—efficient quantum tunneling through the iron-sulfur clusters is ultimately driven by the highly electronegative oxygen keeping the flux going. Indeed with a lack of oxygen, the flux of electrons immediately reverses). Once this step is achieved, the rest is simple straightforward chemistry.
The beauty of the Krebs cycle lies not only in the central role it plays in metabolism but also in how it elegantly solves many fundamental chemical challenges with a very limited set of strategies. Biochemistry must operate in an aqueous environment, at neutral pH and body temperature, all while maintaining precise control to prevent unwanted reactivity that could damage the cell. Yet, within these constraints, the Krebs cycle achieves remarkable feats of molecular engineering.
Students of biochemistry and medicine are often forced to memorize its intermediates and enzymes without ever grasping the reasoning behind each step. But true understanding transforms what seems like tedious memorization into a story of elegant problem-solving. Once the "drama" of the Krebs cycle unfolds, no one would call it boring—I truly believe it is one of the most beautiful lessons science has to offer.
