Isoprenoid Chain Research Articles

Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

Location and interaction of idebenone and mitoquinone in a membrane similar to the inner mitochondrial membrane. Comparison with ubiquinone 10

Oxygen is essential for aerobic life on earth but it is also the origin of harmful reactive oxygen species (ROS). Ubiquinone is par excellence the endogenous cellular antioxidant, but a very hydrophobic one. Because of that, other molecules have been envisaged, such as idebenone (IDE) and mitoquinone (MTQ), molecules having the same redox active benzoquinone moiety but higher solubility. We have used molecular dynamics to determine the location and interaction of these molecules, both in their oxidized and reduced forms, with membrane lipids in a membrane similar to that of the mitochondria. Both IDE and reduced IDE (IDOL) are situated near the membrane interface, whereas both MTQ and reduced MTQ (MTQOL) locate in a position adjacent to the phospholipid hydrocarbon chains. The quinone moieties of both ubiquinone 10 (UQ10) and reduced UQ10 (UQOL10) in contraposition to the same moieties of IDE, IDOL, MTQ and MTQOL, located near the membrane interphase, whereas the isoprenoid chains remained at the middle of the hydrocarbon chains. These molecules do not aggregate and their functional quinone moieties are located in the membrane at different depths but near the hydrophobic phospholipid chains whereby protecting them from ROS harmful effects.

Biosynthesis of GMGT lipids by a radical SAM enzyme associated with anaerobic archaea and oxygen-deficient environments

Archaea possess characteristic membrane-spanning lipids that are thought to contribute to the adaptation to extreme environments. However, the biosynthesis of these lipids is poorly understood. Here, we identify a radical S-adenosyl-L-methionine (SAM) enzyme that synthesizes glycerol monoalkyl glycerol tetraethers (GMGTs). The enzyme, which we name GMGT synthase (Gms), catalyzes the formation of a C(sp3)–C(sp3) linkage between the two isoprenoid chains of glycerol dialkyl glycerol tetraethers (GDGTs). This conclusion is supported by heterologous expression of gene gms from a GMGT-producing species in a methanogen, as well as demonstration of in vitro activity using purified Gms enzyme. Additionally, we show that genes encoding putative Gms homologs are present in obligate anaerobic archaea and in metagenomes obtained from oxygen-deficient environments, and appear to be absent in metagenomes from oxic settings.

Chemical Dynamics of Selenium Nanoparticles in Archaeal Systems.

Methanogenic archaea, characterized by their cell membrane lipid molecules consisting of isoprenoid chains linked to glycerol-1-phosphate via ether bonds, exhibit exceptional adaptability to extreme environments. However, this distinct lipid architecture also complicates the interactions between methanogenic archaea and nanoparticles. This study addresses this challenge by exploring the interaction and transformation of selenium nanoparticles (SeNPs) within archaeal Methanosarcina acetivorans C2A. We demonstrated that the effects of SeNPs are highly concentration-dependent, with chemical stimulation of cellular processes at lower SeNPs concentrations as well as oxidative stress and metabolic disruption at higher concentrations. Notably, we observed the formation of a protein corona on SeNPs, characterized by the selective adsorption of enzymes critical for methylotrophic methanogenesis and those involved in selenium methylation, suggesting potential alterations in protein function and metabolic pathways. Furthermore, the intracellular transformation of SeNPs into both inorganic and organic selenium species highlighted their bioavailability and dynamic transformation within archaea. These findings provide vital insights into the nano-bio interface in archaeal systems, contributing to our understanding of archaeal catalysis and its broader applications.

Understanding coenzyme Q.

Coenzyme Q (CoQ), also known as ubiquinone, comprises a benzoquinone head group and a long isoprenoid side chain. It is thus extremely hydrophobic and resides in membranes. It is best known for its complex function as an electron transporter in the mitochondrial electron transport chain (ETC) but is also required for several other crucial cellular processes. In fact, CoQ appears to be central to the entire redox balance of the cell. Remarkably, its structure and therefore its properties have not changed from bacteria to vertebrates. In metazoans, it is synthesized in all cells and is found in most, and maybe all, biological membranes. CoQ is also known as a nutritional supplement, mostly because of its involvement with antioxidant defenses. However, whether there is any health benefit from oral consumption of CoQ is not well established. Here we review the function of CoQ as a redox-active molecule in the ETC and other enzymatic systems, its role as a prooxidant in reactive oxygen species generation, and its separate involvement in antioxidant mechanisms. We also review CoQ biosynthesis, which is particularly complex because of its extreme hydrophobicity, as well as the biological consequences of primary and secondary CoQ deficiency, including in human patients. Primary CoQ deficiency is a rare inborn condition due to mutation in CoQ biosynthetic genes. Secondary CoQ deficiency is much more common, as it accompanies a variety of pathological conditions, including mitochondrial disorders as well as aging. In this context, we discuss the importance, but also the great difficulty, of alleviating CoQ deficiency by CoQ supplementation.

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyses the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one (SDO) to a conjugated (22, 24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an FMN cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by SDO but cannot be oxidized even with strong oxidising agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2 (S26DH2). In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Mechanistic docking in terpene synthases using EnzyDock.

Terpene Synthases (TPS) catalyze the formation of multicyclic, complex terpenes and terpenoids from linear substrates. Molecular docking is an important research tool that can further our understanding of TPS multistep mechanisms and guide enzyme design. Standard docking programs are not well suited to tackle the unique challenges of TPS, like the many chemical steps which form multiple stereo-centers, the weak dispersion interactions between the isoprenoid chain and the hydrophobic region of the active site, description of carbocation intermediates, and finding mechanistically meaningful sets of docked poses. To address these and other unique challenges, we developed the multistate, multiscale docking program EnzyDock and used it to study many TPS and other enzymes. In this review we discuss the unique challenges of TPS, the special features of EnzyDock developed to address these challenges and demonstrate its successful use in ongoing research on the bacterial TPS CotB2.

Hyperforin modulates MAPK/CCL11 signaling to reduce the inflammatory response of nasal mucosal epithelial cells caused by allergic rhinitis by targeting BCL6.

Hyperforin is a type of bicyclic tetraketone with four isoprenoid chains extracted from Hypericum perforatum L. that has multiple biological activities such as anti-diabetes, antitumor and anti-inflammation. However, the role and potential mechanism of hyperforin in allergic rhinitis (AR) remains to be clarified. In the present study, cell viability was analyzed using Cell Counting Kit-8 assay, while inflammation was detected using ELISA and reverse transcription-quantitative PCR. Epithelial cell barrier damage was measured using western blotting and immunofluorescence staining. The expression levels of B-cell lymphoma 6 (BCL6) and the p38 MAPK/C-C motif chemokine 11 (CCL11) pathway were detected using western blotting. In addition, the association between hyperforin and BCL6 was analyzed by SWISS TargetPrediction, DisGeNET, Gene Ontology and Pathway databases. Molecular docking was performed using AutoDockTools 1.5.6 and Discovery Studio 4.5 software. The data demonstrated that there were 16 interlinking target genes of hyperforin with AR, in which BCL6 was the most relevant one with hyperforin in AR. The binding between hyperforin and BCL6 was verified, and molecular docking was modeled. The results revealed that hyperforin inhibited IL-13-induced nasal epithelial inflammatory cytokine release and repressed the damage to the cellular barrier from IL-13 stimulation. In addition, hyperforin activated BCL6 expression and significantly suppressed the expression of p38 MAPK/CCL11. Silencing of BCL6 reversed the effects of hyperforin on IL-13-induced inflammation and barrier damage. In summary, the present results revealed that hyperforin suppressed IL-13-induced nasal epithelial cell inflammation and barrier damage by targeting BCL6/p38 MAPK/CCL11, which may provide promising therapeutic targets for AR.

On the construction of some bioconjugate networks and their structural modeling via irregularity topological indices.

Bioconjugate networks refer to networks that are formed by connecting different molecules or particles (such as proteins, enzymes, or nanoparticles) through covalent or non-covalent interactions. These networks are often used in various biological and biomedical applications, such as drug delivery, biosensors, and tissue engineering. The specific properties and behavior of these networks depend on the types of molecules used and the nature of their interactions, which can be studied using various computational and experimental techniques. Farnesyl and geranyl groups are types of isoprenoid chains that are commonly found in various biological molecules such as proteins, lipids, and pigments. The addition of these groups to penicillin molecules may alter their physical and chemical properties, such as solubility, stability, and bioavailability. To gain a better understanding of the structure-property relationships of these antibiotics, this study computes various irregularity indices such as the Albertson index, irregularity index, total irregularity index, Randić irregularity index, and other degree-based indices for two types of sensitive bonds of bioconjugate networks. Numerical results and graphical representations are used to illustrate these findings. The obtained results provide valuable insights into the structure-property relationships of penicillins, which will aid in a better understanding of their behavior and developing more effective antibiotics.

Preference of Bacterial Rhamnosyltransferases for 6-Deoxysugars Reveals a Strategy To Deplete O-Antigens.

Bacteria synthesize hundreds of bacteria-specific or "rare" sugars that are absent in mammalian cells and enriched in 6-deoxy monosaccharides such as l-rhamnose (l-Rha). Across bacteria, l-Rha is incorporated into glycans by rhamnosyltransferases (RTs) that couple nucleotide sugar substrates (donors) to target biomolecules (acceptors). Since l-Rha is required for the biosynthesis of bacterial glycans involved in survival or host infection, RTs represent potential antibiotic or antivirulence targets. However, purified RTs and their unique bacterial sugar substrates have been difficult to obtain. Here, we use synthetic nucleotide rare sugar and glycolipid analogs to examine substrate recognition by three RTs that produce cell envelope components in diverse species, including a known pathogen. We find that bacterial RTs prefer pyrimidine nucleotide-linked 6-deoxysugars, not those containing a C6-hydroxyl, as donors. While glycolipid acceptors must contain a lipid, isoprenoid chain length, and stereochemistry can vary. Based on these observations, we demonstrate that a 6-deoxysugar transition state analog inhibits an RT in vitro and reduces levels of RT-dependent O-antigen polysaccharides in Gram-negative cells. As O-antigens are virulence factors, bacteria-specific sugar transferase inhibition represents a novel strategy to prevent bacterial infections.

A specific reagent to study trypanosomatids respiration through polyprenyl diphosphate synthases inhibition: 1-[(n-oct-1-ylamino) ethyl] 1,1-bisphosphonic acid.

Background: Ubiquinone or Coenzyme Q (CoQ) is a molecule performing an essential function as an electron carrier in the respiratory chain. Besides, it functions as a proton and electron acceptor and as an antioxidant. It is composed of a benzoquinone ring and an isoprenoid chain, the last one being synthesized by a polyprenil diphosphate synthase. The inhibition of the Trypanosoma cruzi enzyme by 1-[(n-oct-1-ylamino) ethyl] 1,1-bisphosphonic acid (OBA) is lethal, as well as the interference of the homologous enzyme in T. brucei brucei. We now studied the OBA sensitivities of the different forms of human pathogenic trypanosomatids.

A trick of the tail: computing the entropic contribution to the energetics of quinone-protein unbindung.

We estimate the entropic contributions to the free energy of quinone unbinding in bacterial and mitochondrial respiratory chains using molecular dynamics (MD) and Monte Carlo (MC) computer simulations. For a varying length of the isoprenoid side chain, MD simulations in lipid bilayers and in unpolar solvents are used to assess the dihedral angle distributions along the chain. These form the basis of a MC estimate of the number of molecular structures that do not exhibit steric self-overlap and that are confined to the bilayer. We obtain an entropy drive of TΔS = 1.4 kcal mol-1 for each isoprene unit, which in sum is comparable to the redox potential differences involved in respiratory chain electron transfer. We postulate an entropy-driven zipper for quinone unbinding and discuss it in the context of the bioenergetics and the structure of complex I, and we indicate possible consequences of our findings for MD-based free energy computations.

Disentangling the lipid divide: Identification of key enzymes for the biosynthesis of membrane-spanning and ether lipids in Bacteria

Bacterial membranes are composed of fatty acids (FAs) ester-linked to glycerol-3-phosphate, while archaea have membranes made of isoprenoid chains ether-linked to glycerol-1-phosphate. Many archaeal species organize their membrane as a monolayer of membrane-spanning lipids (MSLs). Exceptions to this "lipid divide" are the production by some bacterial species of (ether-bound) MSLs, formed by tail-to-tail condensation of FAs resulting in the formation of (iso) diabolic acids (DAs), which are the likely precursors of paleoclimatological relevant branched glycerol dialkyl glycerol tetraether molecules. However, the enzymes responsible for their production are unknown. Here, we report the discovery of bacterial enzymes responsible for the condensation reaction of FAs and for ether bond formation and confirm that the building blocks of iso-DA are branched iso-FAs. Phylogenomic analyses of the key biosynthetic genes reveal a much wider diversity of potential MSL (ether)-producing bacteria than previously thought, with importantt implications for our understanding of the evolution of lipid membranes.

Parent anion radical formation in coenzyme Q0: Breaking ubiquinone family rules

We report electron attachment (EA) measurements for the parent anion radical formation from coenzyme Q0 (CoQ0) at low electron energies (<2 eV) along with quantum chemical calculations. CoQ0 may be considered a prototype for the electron withdrawing properties of the larger CoQn molecules, in particular ubiquinone (CoQ10), an electron carrier in aerobic cell respiration. Herein, we show that the mechanisms for the parent anion radical formation of CoQ0 and CoQn (n = 1,2,4) are remarkably distinct. Reported EA data for CoQ1, CoQ2, CoQ4 and para-benzoquinone indicated stabilization of the parent anion radicals around 1.2–1.4 eV. In contrast, we observe for the yield of the parent anion radical of CoQ0 a sharp peak at ∼ 0 eV, a shoulder at 0.07 eV and a peak around 0.49 eV. Although the mechanisms for the latter feature remain unclear, our calculations suggest that a dipole bound state (DBS) would account for the lower energy signals. Additionally, the isoprenoid side chains in CoQn (n = 1,2,4) molecules seem to influence the DBS formation for these compounds. In contrast, the side chains enhance the parent anion radical stabilization around 1.4 eV. The absence of parent anion radical formation around 1.4 eV for CoQ0 can be attributed to the short auto-ionization lifetimes. The present results shed light on the underappreciated role played by the side chains in the stabilization of the parent anion radical. The isoprenoid tails should be viewed as co-responsible for the electron-accepting properties of ubiquinone, not mere spectators of electron transfer reactions.

The mevalonate suppressor δ-tocotrienol increases AMPA receptor-mediated neurotransmission

Synaptic dysfunction is a hallmark of aging and is found in several neurological disorders such as Alzheimer's disease. A common mechanism related to synaptic dysfunction is dysregulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which mediate excitatory neurotransmission and synaptic plasticity. Accumulating evidence suggests that tocotrienols, vitamin E molecules that contain an isoprenoid side chain, may promote cognitive improvement in hippocampal-dependent learning tasks. Tocotrienols have also been shown to reduce the secretion of β-amyloid (Aβ) and cholesterol biosynthesis in part by downregulating 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme that controls flux of the mevalonate pathway and cholesterol biosynthesis. We hypothesized that tocotrienols might promote cognitive improvement by increasing AMPA receptor-mediated synaptic transmission. Here, we found that δ-tocotrienol increased surface levels of GluA1 but not the GluA2 AMPA receptor subunit in primary hippocampal neurons. Unexpectedly, δ-tocotrienol treatment caused a decrease in the phosphorylation of GluA1 at Serine 845 with no significant changes in GluA1 at Serine 831. Moreover, δ-tocotrienol increased spontaneous excitatory postsynaptic current (sEPSC) amplitude and reduced the secretion of Aβ40 in primary hippocampal neurons. Taken together, our findings suggest that δ-tocotrienol increases AMPA receptor-mediated neurotransmission via noncanonical changes in GluA1 phosphorylation status. These findings suggest that δ-tocotrienol may be beneficial in ameliorating synaptic dysfunction found in aging and neurological disease.

Conjugate reduction of vinyl bisphosphonates

Vinyl bisphosphonates can be readily prepared by condensation of an aromatic aldehyde with the tetraester of a methylenebisphosphonate, and reduction of the resulting olefin is an attractive strategy for the preparation of monoalkyl geminal bisphosphonates. Conjugate reduction through use of variations on the Stryker approach has proven to be an efficient method for that reduction, even in the presence of aromatic substituents that also could be reduced. Furthermore, remote olefins in an isoprenoid chain survive this conjugate reduction unaffected, allowing access to isoprenoid-substituted triazole bisphosphonates of interest as potential inhibitors of terpenoid biosynthesis.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Identification of acetylated diether lipids in halophilic Archaea.

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects.

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.