An organic O donor for biological hydroxylation reactions.

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2, or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

The role of coenzyme Q10 as a preventive and therapeutic agent for the treatment of cancers.

Currently, several options are available for the prevention and treatment of cancers; however, many limitations remain with these approaches. Recently, antioxidants have become important preventive and therapeutic alternatives with few adverse events and minimum cost. Coenzyme Q10 (CoQ10) is a naturally occurring component that performs an anticancer function by reducing oxidative stress. CoQ10 supplementation as an adjuvant therapy offers more progress in the elimination and development of cancers. This review aimed to critically assess and summarize the implication of CoQ10 in cancers, highlighting possible mechanisms, and future directions of research for the standardization of the current regimen for cancer prevention and treatment.

Mutations of GEMIN5 are associated with coenzyme Q10 deficiency: long-term follow-up after treatment.

GEMIN5 exerts key biological functions regulating pre-mRNAs intron removal to generate mature mRNAs. A series of patients were reported harboring mutations in GEMIN5. No treatments are currently available for this disease. We treated two of these patients with oral Coenzyme Q10 (CoQ10), which resulted in neurological improvements, although MRI abnormalities remained. Whole Exome Sequencing demonstrated compound heterozygosity at the GEMIN5 gene in both cases: Case one: p.Lys742* and p.Arg1016Cys; Case two: p.Arg1016Cys and p.Ser411Hisfs*6. Functional studies in fibroblasts revealed a decrease in CoQ10 biosynthesis compared to controls. Supplementation with exogenous CoQ10 restored it to control intracellular CoQ10 levels. Mitochondrial function was compromised, as indicated by the decrease in oxygen consumption, restored by CoQ10 supplementation. Transcriptomic analysis of GEMIN5 patients compared with controls showed general repression of genes involved in CoQ10 biosynthesis. In the rigor mortis defective flies, CoQ10 levels were decreased, and CoQ10 supplementation led to an improvement in the adult climbing assay performance, a reduction in the number of motionless flies, and partial restoration of survival. Overall, we report the association between GEMIN5 dysfunction and CoQ10 deficiency for the first time. This association opens the possibility of oral CoQ10 therapy, which is safe and has no observed side effects after long-term therapy.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis.

Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce ubiquinone (oxidized form of Q) to ubiquinol (QH2); however, in eukaryotes, only oxidative phosphorylation (OXPHOS) complex III (CIII) oxidizes QH2 to Q. The mechanism of action of CIII is still debated. Herein, we show that the Q reductase electron-transfer flavoprotein dehydrogenase (ETFDH) is essential for CIII activity in skeletal muscle. We identify a complex (comprising ETFDH, CIII and the Q-biosynthesis regulator COQ2) that directs electrons from lipid substrates to the respiratory chain, thereby reducing electron leaks and reactive oxygen species production. This metabolon maintains total Q levels, minimizes QH2-reductive stress and improves OXPHOS efficiency. Muscle-specific Etfdh-/- mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine.

Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response to therapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy.

Endogenous chemicals guard health through inhibiting ferroptotic cell death.

Ferroptosis is a new form of regulated cell death caused by iron-dependent accumulation of lethal polyunsaturated phospholipids peroxidation. It has received considerable attention owing to its putative involvement in a wide range of pathophysiological processes such as organ injury, cardiac ischemia/reperfusion, degenerative disease and its prevalence in plants, invertebrates, yeasts, bacteria, and archaea. To counter ferroptosis, living organisms have evolved a myriad of intrinsic efficient defense systems, such as cyst(e)ine-glutathione-glutathione peroxidase 4 system (cyst(e)ine-GPX4 system), guanosine triphosphate cyclohydrolase 1/tetrahydrobiopterin (BH4) system (GCH1/BH4 system), ferroptosis suppressor protein 1/coenzyme Q10 system (FSP1/CoQ10 system), and so forth. Among these, GPX4 serves as the only enzymatic protection system through the reduction of lipid hydroperoxides, while other defense systems ultimately rely on small compounds to scavenge lipid radicals and prevent ferroptotic cell death. In this article, we systematically summarize the chemical biology of lipid radical trapping process by endogenous chemicals, such as coenzyme Q10 (CoQ10), BH4, hydropersulfides, vitamin K, vitamin E, 7-dehydrocholesterol, with the aim of guiding the discovery of novel ferroptosis inhibitors.

Mitochondrial matrix RTN4IP1/OPA10 is an oxidoreductase for coenzyme Q synthesis.

Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.

Coenzyme Q10 attenuates neurodegeneration in the cerebellum induced by chronic exposure to tramadol.

BACKGROUND: Chronic use of tramadol can cause neurotoxic effects and subsequently cause neurodegeneration in the cerebellum. The main damage mechanisms identified are oxidative stress and inflammation. Currently, we investigated the effects of coenzyme Q10 (CoQ10) in attenuates of neurodegeneration in the cerebellum induced by chronic exposure to tramadol. MATERIAL AND METHODS: Seventy-two male mature albino rats were allocated into four equal groups, including; non-treated group, CoQ10 group (which received CoQ10 at 200 mg/kg/day orally for three weeks), tramadol group (which received tramadol hydrochloride at 50 mg/kg/day orally for three weeks), and tramadol+CoQ10 group (which received tramadol and CoQ10 at the same doses as the previous groups). Tissue samples were obtained for stereological, immunohistochemical, biochemical, and molecular evaluations. Also, functional tests were performed to evaluate behavioral properties. RESULTS: We found a significant increase in stereological parameters, antioxidant factors (catalase, glutathione, and superoxide dismutase), and behavioral function scores in the tramadol+CoQ10 group compared to the tramadol group (p < 0.05). In addition, malondialdehyde levels, the density of apoptotic cells, as well as the expression of pro-inflammatory (tumor necrosis factor-alpha, interleukin 1 beta, and interleukin 6) and autophagy (lysosome-associated membrane protein 2, autophagy-related 5, beclin 1, and autophagy-related 12) genes were considerably reduced in the tramadol+CoQ10 group compared to the tramadol group (p < 0.05). CONCLUSION: We conclude that the administration of CoQ10 has neuroprotective effects in the cerebellum of rats that have chronic exposure to tramadol.

Understanding differential aspects of microdiffusion (channeling) in the Coenzyme Q and Cytochrome c regions of the mitochondrial respiratory system.

Over the past decades, models of the organization of mitochondrial respiratory system have been controversial. The goal of this perspective is to assess this "conflict of models" by focusing on specific kinetic evidence in the two distinct segments of Coenzyme Q- and Cytochrome c-mediated electron transfer. Respiratory supercomplexes provide kinetic advantage by allowing a restricted diffusion of Coenzyme Q and Cytochrome c, and short-range interaction with their partner enzymes. In particular, electron transfer from NADH is compartmentalized by channeling of Coenzyme Q within supercomplexes, whereas succinate oxidation proceeds separately using the free Coenzyme Q pool. Previous evidence favoring Coenzyme Q random diffusion in the NADH-dependent electron transfer is due to downstream flux interference and misinterpretation of results. Indeed, electron transfer by complexes III and IV via Cytochrome c is less strictly dependent on substrate channeling in mammalian mitochondria. We briefly describe these differences and their physiological implications.

Trolox aids coenzyme Q10 in neuroprotection against NMDA induced damage via upregulation of VEGF in rat model of glutamate excitotoxicity.

Glutamate induced damage to retinal ganglion cells (RGCs) requires tight physiological regulation of the N-methyl-D-aspartate (NMDA) receptors. Previously, studies have demonstrated the neuroprotective abilities of antioxidants like coenzyme Q10 (CoQ10) and vitamin E analogs like α-tocopherol against neuropathies resulting from NMDA insult, but have failed to shed light on the effect of CoQ10 and trolox, a hydrophilic analog of vitamin E, on glaucomatous neurodegeneration. In the current study, we wanted to investigate whether the combined effect of trolox with CoQ10 could alleviate NMDA-induced death of retinal cells while also trying to elucidate the underlying mechanism in relation to the yet unexplained role of vascular endothelial growth factor (VEGF) in NMDA-mediated excitotoxicity. After successful NMDA-induced degeneration, we followed it up with the treatment of combination of Trolox and CoQ10. The structural damage by NMDA was repaired significantly and retina retained structural integrity comparable to levels of control in the treatment group of Trolox and CoQ10. Detection of ROS generation after NMDA insult showed that together, Trolox and CoQ10 could significantly bring down the high levels of free radicals while also rescuing mitochondrial membrane potential (MMP). A significant increase in NMDA receptor Grin2A by CoQ10 alone as well as by CoQ10 and trolox was accompanied by a lowered Grin2B receptor expression, suggesting neuroprotective action of Trolox and CoQ10. Subsequently, lowered VEGFR1 and VEGFR2 receptor expression by NMDA treatment also recovered when subjected to combined treatment of Trolox and CoQ10. Western blot analyses also indicated the same whereby Trolox and CoQ10 could increase the diminished levels of phosphorylated VEGFR2. Immunofluorescence studies also indicated a positive correlation between recovered VEGFR2 and NMDAR2A levels and diminished levels of NMDAR2D, confirming the results obtained by RT-PCR analysis. This is the first report in our knowledge that demonstrates the efficacy of trolox in combination with CoQ10 highlighting the importance of maintaining VEGF levels that are lowered in ocular diseases due to NMDA-related toxicities.

Complex II ambiguities-FADH2 in the electron transfer system.

The prevailing notion that reduced cofactors NADH and FADH2 transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH2 in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the ß-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.

Chia seeds and coenzyme Q10 alleviate iron overload induced hepatorenal toxicity in mice via iron chelation and oxidative stress modulation.

Iron overload (IOL) can cause hepatorenal damage due to iron-mediated oxidative and mitochondrial damage. Remarkably, combining a natural iron chelator with an antioxidant can exert greater efficacy than monotherapy. Thus, the present study aimed to evaluate the efficacy of Chia and CoQ10 to chelate excess iron and prevent hepatorenal oxidative damage in IOL mice. Male Swiss albino mice (n = 49) were randomly assigned to seven groups: control, dietary Chia, CoQ10, IOL, IOL + Chia, IOL + CoQ10, and IOL + Chia + CoQ10. Computational chemistry indicates that the phytic acid found in the Chia seeds is stable, reactive, and able to bind to up to three iron ions (both Fe2+ and Fe3+). IOL induced a significant (P < 0.05) increase in serum iron, ferritin, transferrin, TIBC, TSI, RBCs, Hb, MCV, MCH, WBCs, AST, ALT, creatinine, and MDA. IOL causes a significant (P < 0.05) decrease in UIBC, platelets, and antioxidant molecules (GSH, SOD, CAT, and GR). Also, IOL elicits mitochondrial membrane change depolarization, and DNA fragmentation and suppresses mitochondrial DNA copies. Furthermore, substantial changes in hepatic and renal tissue, including hepatocellular necrosis and apoptosis, glomerular degeneration, glomerular basement membrane thickening, and tubular degeneration, were observed in the IOL group. Dietary Chia and CoQ10 induced significant (P < 0.05) amelioration in all the mentioned parameters. They can mostly repair the abnormal architecture of hepatic and renal tissues induced by IOL, as signified by normal sinusoids, normal central veins, and neither glomerular damage nor degenerated tubules. In conclusion, the combined treatment with Chia + CoQ10 exerts more pronounced efficacy than monotherapy in hepatorenal protection via chelating excess iron and improved cellular antioxidant status and hepatorenal mitochondrial function in IOL mice.

Epilepsy and Coenzyme Q10 deficiency with COQ4 variants.

Coenzyme Q10 (CoQ10) is one of the essential substances for mitochondrial energy synthesis and extra-mitochondrial vital function. Primary CoQ10 deficiency is a rare disease resulting from interruption of CoQ10 biosynthetic pathway and biallelic COQ4 variants are one of the genetic etiologies recognized in this hereditary disorder. The clinical heterogenicity is broad with wide onset age from prenatal period to adulthood. The typical manifestations include early pharmacoresistant seizure, severe cognition and/or developmental delay, dystonia, ataxia, and spasticity. Patients may also have multisystemic involvements such as cardiomyopathy, lactic acidosis or gastro-esophageal regurgitation disease. Oral CoQ10 supplement is the major therapeutic medication currently. Among those patients, c.370G > A variant is the most common pathogenic variant detected, especially in Asian population. This phenomenon also suggests that this specific allele may be the founder variants in Asia. In this article, we report two siblings with infantile onset seizures, developmental delay, cardiomyopathy, and diffuse brain atrophy. Genetic analysis of both two cases revealed homozygous COQ4 c.370G > A (p.Gly124Ser) variants. We also review the clinical manifestations of primary CoQ10 deficiency patients and possible treatment categories, which are still under survey. As oral CoQ10 supplement may improve or stabilize disease severity, early precise diagnosis of primary CoQ10 deficiency and early treatment are the most important issues. This review article helps to further understand clinical spectrum and treatment categories of primary CoQ10 deficiency with COQ4 variant.

Mutation at the entrance of the quinone cavity severely disrupts quinone binding in respiratory complex I.

In all resolved structures of complex I, there exists a tunnel-like Q-chamber for ubiquinone binding and reduction. The entrance to the Q-chamber in ND1 subunit forms a narrow bottleneck, which is rather tight and requires thermal conformational changes for ubiquinone to get in and out of the binding chamber. The substitution of alanine with threonine at the bottleneck (AlaThr MUT), associated with 3460/ND1 mtDNA mutation in human complex I, is implicated in Leber's Hereditary Optic Neuropathy (LHON). Here, we show the AlaThr MUT further narrows the Q-chamber entrance cross-section area by almost 30%, increasing the activation free energy barrier of quinone passage by approximately 5 kJ mol-1. This severely disrupts quinone binding and reduction as quinone passage through the bottleneck is slowed down almost tenfold. Our estimate of the increase in free energy barrier is entirely due to the bottleneck narrowing, leading to a reduction of the transition state entropy between WT and MUT, and thus more difficult quinone passage. Additionally, we investigate details of possible water exchange between the Q-chamber and membrane. We find water exchange is dynamic in WT but may be severely slowed in MUT. We propose that LHON symptoms caused by 3460/ND1 mtDNA mutation are due to slowed quinone binding. This leads to an increased production of reactive oxidative species due to upstream electron backup at the FMN site of complex I, thus resulting in a mt bioenergetic defect.

Uncovering Interactions between Plant Metabolism and Plant-Associated Bacteria in Huanglongbing-Affected Citrus Cultivars Using Multiomics Analysis and Machine Learning.

Huanglongbing (HLB) is a highly destructive disease that inflicts significant economic losses on the citrus industry worldwide but with no cure available. However, microbiomes formulated by citrus plants may serve as disease antagonists, increasing the level of HLB tolerance. This study established an integrated analysis of untargeted metabolomics and microbiomics data for different citrus cultivars, providing critical insights into the interactions between plant metabolism and plant-associated bacteria in the development of HLB. Machine learning models were applied to screen important metabolites and bacteria in multiple citrus materials, and the selected metabolites were then analyzed to identify essential pathways enriched in the plant and to correlate with the selected bacteria. Results demonstrated that the regulation of plant pathways, especially ABC transporters and ubiquinone and other terpene-ubiquinone biosynthesis pathways, could affect the microbial community structure, indicating potential solutions for controlling HLB by modulating bacteria in citrus plants or breeding tolerant citrus cultivars.

Diversification of Ubiquinone Biosynthesis via Gene Duplications, Transfers, Losses, and Parallel Evolution.

The availability of an ever-increasing diversity of prokaryotic genomes and metagenomes represents a major opportunity to understand and decipher the mechanisms behind the functional diversification of microbial biosynthetic pathways. However, it remains unclear to what extent a pathway producing a specific molecule from a specific precursor can diversify. In this study, we focus on the biosynthesis of ubiquinone (UQ), a crucial coenzyme that is central to the bioenergetics and to the functioning of a wide variety of enzymes in Eukarya and Pseudomonadota (a subgroup of the formerly named Proteobacteria). UQ biosynthesis involves three hydroxylation reactions on contiguous carbon atoms. We and others have previously shown that these reactions are catalyzed by different sets of UQ-hydroxylases that belong either to the iron-dependent Coq7 family or to the more widespread flavin monooxygenase (FMO) family. Here, we combine an experimental approach with comparative genomics and phylogenetics to reveal how UQ-hydroxylases evolved different selectivities within the constrained framework of the UQ pathway. It is shown that the UQ-FMOs diversified via at least three duplication events associated with two cases of neofunctionalization and one case of subfunctionalization, leading to six subfamilies with distinct hydroxylation selectivity. We also demonstrate multiple transfers of the UbiM enzyme and the convergent evolution of UQ-FMOs toward the same function, which resulted in two independent losses of the Coq7 ancestral enzyme. Diversification of this crucial biosynthetic pathway has therefore occurred via a combination of parallel evolution, gene duplications, transfers, and losses.

Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

Investigation of hydrated channels and proton pathways in a high-resolution cryo-EM structure of mammalian complex I.

Respiratory complex I, a key enzyme in mammalian metabolism, captures the energy released by reduction of ubiquinone by NADH to drive protons across the inner mitochondrial membrane, generating the proton-motive force for ATP synthesis. Despite remarkable advances in structural knowledge of this complicated membrane-bound enzyme, its mechanism of catalysis remains controversial. In particular, how ubiquinone reduction is coupled to proton pumping and the pathways and mechanisms of proton translocation are contested. We present a 2.4-Å resolution cryo-EM structure of complex I from mouse heart mitochondria in the closed, active (ready-to-go) resting state, with 2945 water molecules modeled. By analyzing the networks of charged and polar residues and water molecules present, we evaluate candidate pathways for proton transfer through the enzyme, for the chemical protons for ubiquinone reduction, and for the protons transported across the membrane. Last, we compare our data to the predictions of extant mechanistic models, and identify key questions to answer in future work to test them.