Science: Metabolic Poison, Rotenone

Tuba (Derris ellliptica, Fabaceae) is the plant from which rotenone was first isolated.

 

Today, we are going to examine the metabolic poison rotenone, which targets complex I of the electron transport chain. Rotenone is found in various plants of the legume family (Fabaceae), particularly the genus Derris (tuba), Lonchocarpus (cubé) and Pachyrhizus (jicama). Rotenone was first characterised in its pure crystalline form from Derris elliptica (called rōten) by a Japanese chemist in 1902. The suffix '-one' means isoflavone skeleton, which is present in rotenone. An isoflavone (Figure 1) contains two aromatic rings merged back to back with a phenyl group at carbon position 2. Rotenone contains two additional rings (rings A and D) that are incorporated into an isoflavone skeleton (rings B, C and E). Plants synthesise rotenone from the amino acid L-phenylalanine, which is converted into the upstream precursor cinnamoyl-CoA via the shikimate pathway. Cinnamoyl-CoA then undergoes chain lengthening by six carbon units, followed by intramolecular cyclisation by aldol condensation to produce a flavone intermediate (liquiritigenin). The flavone intermediate is then converted into an isoflavone (formononetin) via oxidation-reduction steps. Finally two additional rings were incorporated into formononetin to produce rotenone. I have attached the simplified biosynthesis pathway of rotenone in Figure 2, you can have fun to make sense of this chemistry challenge.

Figure 1: Chemical structure of rotenone.

Figure 2: Simplified biosynthesis pathway of rotenone.

Rotenone is a potent inhibitor of complex I of the electron transport chain (ETC). Complex I is a transmembrane enzyme called NADH-ubiquinone oxidoreductase. Examine Figure 3 carefully, NADH is a complex molecule that contains three principal components, namely, an adenine base, a dinucleotide (phosphate and sugar), and a nicotinamide group. It is aromatic nicotinamide and its nitrogen atom group that participate in electron transfer. On the other hand ubiquinone is also an a large molecule that comprises of two components, i.e., a long hydrocarbon chain, and a quinone group (Figure 4). Ubiquinone is so named because it is ubiquitous to almost all life forms, and it relies on the aromatic quinone group to give or take electrons. You may even be taking ubiquinone as a health supplement called co-enzyme Q10 (10 for the 10 polymeric units in the hydrocarbon tail). In general, complex I catalyses the oxidation of NADH to supply ETC with one proton (H⁺) and two electrons. The electrons and protons are subsequently transferred to ubiquinone, which is reduced (gained hydrogen and electrons) in the process. Find the reaction mechanism in Figure 3, and bear in mind that reduction-oxidation (redox) steps are crucial in the understanding of biochemistry. Note that the reactions are reversible,  [NADH/NAD+] and [ubiquinone/semiubiquinone/ubiquinol] pairs can be inter-converted via redox steps. That's why they are used in the cycle of ETC to supply and transfer electrons. 

Figure 3: Redox mechanisms in NADH and Ubiquinone.

 

Rotenone binds to the ubiquinol binding site of complex I, but in a slightly different manner. Thus, rotenone is expected to share structural similarities with ubiquinone, while exhibiting extra features to allow slightly different but strong binding interactions with complex I. Once rotenone is bound, complex I can no longer transfer its electrons to ubiquinone, and we basically end up with electron constipation! This causes two devastating effects. Firstly, the ETC loses its ability to make ATP because no electron transfer means no proton gradient. If the concentration of rotenone is high enough, it can cause rapid death by internal suffocation due to a lack of ATP. This is the reason why rotenone is used as a fish poison because fishes require high oxygen (energy) demand in water, making them especially sensitive to rotenone toxicity. Insects and invertebrates that rely on simple air diffusion to breathe (less efficient than lungs) are also very prone to rotenone. The fact that rotenone killed fishes are edible is because rotenone is rapidly metabolised into less toxic compounds by human liver. Nonetheless fatal instances of poisoning due to rotenone are well known to literature, some due to ingestion of rotenone-containing plants such as jicáma seeds. Symptoms of acute rotenone toxicity resembles cyanide poisoning, just without an antidote. Patients develop weakness, shortness of breathe, cyanosis (blue skin) and lactic acidosis, leading to multi-organ failure and death. Acidosis occurs because the body tries to generate energy by a back-up route called anaerobic respiration, which makes the blood acidic in the process. I'm sure we have all experienced that 'sour' feeling in muscles after intense exercise, that's due to local formation of lactic acid via anaerobic respiration. Secondly, the built-up electrons can react with oxygen atoms to make free radicals or superoxide anions, which are extremely reactive. Free radicals can damage cell membrane and DNA, causing cell death or mutation (cancer). The brain's dopaminergic neurons are particularly sensitive to free radical damage induced by rotenone. Thus, when enough neurons are damaged (neurons cannot regenerate!),  Parkinson's disease develop. There is currently no cure for Parkinson's disease, and patients permanently lose their ability to coordinate movements. Rotenone is so good at inducing Parkinson's disease, it is currently used by scientists to induce Parkinson's symptoms in animal models. This is also the reason why rotenone was banned and phased out globally as an organic pesticide. It is still used as a mass-killing agent to control invasive fishes, but even then rotenone remains a dangerous and controversial pesticide. 

Figure 4: List of complex I inhibitors.

Let's compare the structure of rotenone with ubiquinone in Figure 4. Rotenone contains ketone and aromatic methoxy groups (highlighted in red), which are similar to ubiquinone. However, instead of a long hydrocarbon chain, rotenone has additional cyclic ether (furan and pyran) rings that may enable extra binding with complex I, perhaps in a different region than ubiquinone (allosteric binding). That makes rotenone a non-competitive inhibitor and once it is bound, it cannot be displaced by more ubiquinol. In other words, once rotenone binds to complex I, the ETC is forever screwed. There are other inhibitors of complex I, which I have listed in Figure 4. The acetogenins (bullatacin), which are found in many plants of the Annonaceae family are even more potent than rotenone. The chemical structures of acetogenins are almost identical to ubiquinol, while having extra furan rings of rotenone. Toxicity wise, acetogenins may have the best of ubiquinone and rotenone. Cases of Parkinson's disease due to chronic ingestion of Annonaceae fruits containing acetogenins are well-documented. Recent research have uncovered favourable anti-cancer activity of acetogenins and rotenone, but scientists still have to figure out a ways of circumventing their profound neurotoxicity. After all, you wouldn't want to get Parkinson's disease to cure cancer!

 

That's all for today and here's your homework. Do all inhibitors of complex I come from plants? What about piericidin A, where does it come from and what purpose would it serve the organism that produce it?