Thursday, May 10, 2018

Fixing carbon by reversing the citric acid cycle

The citric acid cycle1 is usually taught as depicted in the diagram on the right.2 A four-carbon compound called oxaloaceate is joined to a two-carbon compound called acetyl-CoA to produce a six-carbon tricarboxylic acid called citrate. In subsequent reactions, two carbons are released in the form of carbon dioxide to regenerate the original oxaloacetate. The cycle then repeats. The reactions produce one ATP equivalent (ATP or GTP), three NADH molecules, and one QH2 molecule.

The GTP/ATP molecule and the reduced coenzymes (NADH and QH2) are used up in a variety of other reactions. In the case of NADH and QH2, one of the many pathways to oxidation is the membrane-associated electron transport system that creates a proton gradient across a membrane. The electron transport complexes are buried in membranes—plasma and internal membranes in bacteria and the inner mitochondrial membrane in eukaryotes. Students are often taught that this is the only fate of NADH and QH2 but that's not true.

One of the other common misconceptions is that the citric acid cycle runs exclusively in one direction; namely, the direction shown in the diagram. That's also not true. The reactions of the citric acid cycle are near-equilibrium reactions like most reactions in the cell. What this means is that the concentrations of the reactants and products are close to the equilibrium values so that a slight increase in one of them will lead to a rapid equilibration. The reactions can run in either direction.3

Furthermore, the citric acid cycle does not exist in isolation. Many of the intermediates are also intermediates in other reactions such as the synthesis and degradation of amino acids and fatty acids, to mention just two possibilities. The best biochemistry courses will make sure that students understand this and make sure they understand that all these molecules exist in a "soup" inside the cell where the fate of any one molecule depends on its concentration and the concentration of surrounding molecules. The very best courses will explain that many species of bacteria have some of the enzymes but can't make a "cycle" because one of the key enzymes is missing. This leads to an understanding of how such an irreducibly complex pathway could have evolved [The evolution of the citric acid cycle].

Sorry for the long-winded introduction. What I want to tell you about is a couple of papers that were recently published in Science along with a summary in the news section of the journal.
Ragsdale, S. W. (2018) Stealth reactions driving carbon fixation. Science 359:517-518. [doi: 10.1126/science.aar6329]

Nunoura, T., Chikaraishi, Y., Izaki, R., Suwa, T., Sato, T., Harada, T., Mori, K., Kato, Y., Miyazaki, M., Shimamura, S., Yanagawa, K., Shuto, A., Ohkouchi, N., Fujita, N., Takak, Y., Atomi, H., and Takai, K. (2018) A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science 359:559-563. [doi: 10.1126/science.aao3407]

Mall, A., Sobotta, J., Huber, C., Tschirner, C., Kowarschik, S., Bačnik, K., Mergelsberg, M., Boll, M., Hügler, M., Eisenreich, W., and Berg, I.A. (2018) Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium. Science 359:563-567. [doi: 10.1126/science.aao2410]
The papers report solid evidence that carbon dioxide can be fixed in two different species of bacteria by reversing the citric acid cycle. This shouldn't come as a big surprise given everything that I said in the first part of the post. If all of the reactions are really near-equilibrium reactions then the enzymes can catalyze reactions in either direction. This is what I taught students in introductory biochemistry and it's what's in my textbook.

So, why do these papers deserve to be published in one of the most prestigious journals? It's because most biochemists have a very different view of biochemistry than the one I described. That view is false, in my opinion, but it leads to the conclusion that reversal of the citric acid cycle is impossible. That's why the two papers seem so revolutionary. They "refute" a concept that my students already knew was false!

Most biochemists think that some reactions are irreversible because of unfavorable thermodynamics. One of these reactions is catalyzed by citrate synthase, the enzyme that interconverts oxaloacetate and citrate in the citric acid cycle [EC].
acetyl-CoA + H2O + oxaloacetate = citrate + HS-CoA + H+
The standard Gibbs free energy change for this reaction in the direction written above is about −36kJ/mol (ΔG°′=−36 kJ/mol). This is a very big number; one that's normally associated with reactions such as the hydrolysis of ATP to ADP + Pi. Scientists such as Stephen Ragsdale—the author of the news article—hold to the view that these reactions are highly thermodynamically favorable such that the reverse reaction was thought to be impossible. That's why the papers are thought to be so important. They challenge the prevailing (false) view that standard Gibbs free energy changes determine whether reactions are "exergonic" (release energy) or "endergonic" (absorb energy).

This is why many biochemists will be surprised to discover that a reaction with what was thought to be a highly favorable Gibbs free energy change can be reversible. They shouldn't be surprised because, in fact, the free energy change inside the cell is close to zero because it's at equilibrium. It's not a surprise to some that the reaction is readily reversible.

(I need to acknowledge one of the earlier co-authors on my textbook, Ray Ochs of St. John's University in Queens, New York, USA. He taught me how to understand the difference between standard Gibbs free energy changes and the real free energy changes that take place inside cells.)

1. Otherwise known as the tricarboxylic acid (TCA) cycle or the Krebs cycle,

2. Image Credit: Moran, L.A., Horton, H.R., Scrimgeour, K.G., and Perry, M.D. (2012) Principles of Biochemistry 5th ed., Pearson Education Inc. page 391 and page 409. © Pearson/Prentice Hall

3. You would think that every single biochemistry course and textbook would at least get all the reactions correct. Unfortunately, this is not the case. Most teachers and most textbook authors have copied errors that were introduced decades ago. If you search the web you will find that's it's almost impossible to find a site where all the reactions are depicted correctly [Biochemistry on the Web: The Citric Acid Cycle].


  1. So do I have it right that citrate can be converted back into oxaloacetate, if the concentration of oxaloacetate drops below a certain threshold compared to the concentrations of malate and citrate?

    1. Yes. The concentration of oxaloacetate inside the cell is extremely low because of the unfavourable equilibrium of all reactions that it’s involved with. If it drops below a certain threshold then citrate and maleate can be used to replenish it. I most species, oxaloacetate is an important source of carbon for gluconeogenesis.

      One of the papers I referenced actually measured the concentration of oxaloacetate and found that it was about 1000 times les than the concentration of citrate.

  2. This misunderstanding is a favorite creationist bugaboo as well, as in, "how could one make [insert name of compound or polymer] here] chemically when the delta G is so unfavorable?"