Molecular mechanism of the ischemia-induced regulatory switch in mammalian complex I.

Respiratory complex I is an efficient driver for oxidative phosphorylation in mammalian mitochondria, but its uncontrolled catalysis under challenging conditions leads to oxidative stress and cellular damage. Ischemic conditions switch complex I from rapid, reversible catalysis into a dormant state that protects upon reoxygenation, but the molecular basis for the switch is unknown. We combined precise biochemical definition of complex I catalysis with high-resolution cryo-electron microscopy structures in the phospholipid bilayer of coupled vesicles to reveal the mechanism of the transition into the dormant state, modulated by membrane interactions. By implementing a versatile membrane system to unite structure and function, attributing catalytic and regulatory properties to specific structural states, we define how a conformational switch in complex I controls its physiological roles.

Unraveling ETC complex I function in ferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

Exposure to 6-PPD quinone causes damage on mitochondrial complex I/II associated with lifespan reduction in Caenorhabditis elegans.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) is an emerging pollutant transformed from 6-PPD. However, the effect of 6-PPDQ exposure on mitochondrion and underlying mechanism remains largely unclear. Using Caenorhabditis elegans as animal model, exposed to 6-PPDQ at 0.1-10 µg/L was performed form L1 larvae to adult day-1. Exposure to 6-PPDQ (1 and 10 µg/L) could increase oxygen consumption rate and decease adenosine 5'-triphosphate (ATP) content, suggesting induction of mitochondrial dysfunction. Activities of NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) were inhibited, accompanied by a decrease in expressions of gas-1, nuo-1, and mev-1. RNAi of gas-1 and mev-1 enhanced mitochondrial dysfunction and reduced lifespan of 6-PPDQ exposed nematodes. GAS-1 and MEV-1 functioned in parallel to regulate 6-PPDQ toxicity to reduce the lifespan. Insulin peptides and the insulin signaling pathway acted downstream of GAS-1 and MEV-1 to control the 6-PPDQ toxicity on longevity. Moreover, RNAi of sod-2 and sod-3, targeted genes of daf-16, caused susceptibility to 6-PPDQ toxicity in reducing lifespan and in causing reactive oxygen species (ROS) production. Therefore, 6-PPDQ at environmentally relevant concentrations (ERCs) potentially caused mitochondrial dysfunction by affecting mitochondrial complexes I and II, which was associated with lifespan reduction by affecting insulin signaling in organisms.

Targeted degradation of NDUFS1 by agrimol B promotes mitochondrial ROS accumulation and cytotoxic autophagy arrest in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a potential anticancer drug. Our recent report showed a favorable anticancer effect of agrimol B in HCC, however, the mechanism of action remains unclear. Here, we found agrimol B inhibits the growth and proliferation of HCC cells in vitro as well as in an HCC patient-derived xenograft (PDX) model. Notably, agrimol B drives autophagy initiation and blocks autophagosome-lysosome fusion, resulting in autophagosome accumulation and autophagy arrest in HCC cells. Mechanistically, agrimol B downregulates the protein level of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) through caspase 3-mediated degradation, leading to mitochondrial reactive oxygen species (mROS) accumulation and autophagy arrest. NDUFS1 overexpression partially restores mROS overproduction, autophagosome accumulation, and growth inhibition induced by agrimol B, suggesting a cytotoxic role of agrimol B-induced autophagy arrest in HCC cells. Notably, agrimol B significantly enhances the sensitivity of HCC cells to sorafenib in vitro and in vivo. In conclusion, our study uncovers the anticancer mechanism of agrimol B in HCC involving the regulation of oxidative stress and autophagy, and suggests agrimol B as a potential therapeutic drug for HCC treatment.

The Blood-Brain Barrier Is Unaffected in the Ndufs4-/- Mouse Model of Leigh Syndrome.

Mitochondrial dysfunction plays a major role in physiological aging and in many pathological conditions. Yet, no study has explored the consequence of primary mitochondrial deficiency on the blood-brain barrier (BBB) structure and function. Addressing this question has major implications for pharmacological and genetic strategies aimed at ameliorating the neurological symptoms that are often predominant in patients suffering from these conditions. In this study, we examined the permeability of the BBB in the Ndufs4-/- mouse model of Leigh syndrome (LS). Our results indicated that the structural and functional integrity of the BBB was preserved in this severe model of mitochondrial disease. Our findings suggests that pharmacological or gene therapy strategies targeting the central nervous system in this mouse model and possibly other models of mitochondrial dysfunction require the use of specific tools to bypass the BBB. In addition, they raise the need for testing the integrity of the BBB in complementary in vivo models.

Using Gaussian accelerated molecular dynamics combined with Markov state models to explore the mechanism of action of new oral inhibitors on Complex I.

In this study, our focus was on investigating H-1,2,3-triazole derivative HP661 as a novel and highly efficient oral OXPHOS inhibitor, with its molecular-level inhibitory mechanism not yet fully understood. We selected the ND1, NDUFS2, and NDUFS7 subunits of Mitochondrial Complex I as the receptor proteins and established three systems for comparative analysis: protein-IACS-010759, protein-lead compound 10, and protein-HP661. Through extensive analysis involving 500 ns Gaussian molecular dynamics simulations, we gained insights into these systems. Additionally, we constructed a Markov State Models to examine changes in secondary structures during the motion processes. The research findings suggest that the inhibitor HP661 enhances the extensibility and hydrophilicity of the receptor protein. Furthermore, HP661 induces the unwinding of the α-helical structure in the region of residues 726-730. Notably, key roles were identified for Met37, Phe53, and Pro212 in the binding of various inhibitors. In conclusion, we delved into the potential molecular mechanisms of triazole derivative HP661 in inhibiting Complex I. These research outcomes provide crucial information for a deeper understanding of the mechanisms underlying OXPHOS inhibition, offering valuable theoretical support for drug development and disease treatment design.

CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Identification of a family of species-selective complex I inhibitors as potential anthelmintics.

Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity.

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

The alternative enzymes-bearing tunicates lack multiple widely distributed genes coding for peripheral OXPHOS subunits.

The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.

SRSF1 Is Required for Mitochondrial Homeostasis and Thermogenic Function in Brown Adipocytes Through its Control of Ndufs3 Splicing.

RNA splicing dysregulation and the involvement of specific splicing factors are emerging as common factors in both obesity and metabolic disorders. The study provides compelling evidence that the absence of the splicing factor SRSF1 in mature adipocytes results in whitening of brown adipocyte tissue (BAT) and impaired thermogenesis, along with the inhibition of white adipose tissue browning in mice. Combining single-nucleus RNA sequencing with transmission electron microscopy, it is observed that the transformation of BAT cell types is associated with dysfunctional mitochondria, and SRSF1 deficiency leads to degenerated and fragmented mitochondria within BAT. The results demonstrate that SRSF1 effectively binds to constitutive exon 6 of Ndufs3 pre-mRNA and promotes its inclusion. Conversely, the deficiency of SRSF1 results in impaired splicing of Ndufs3, leading to reduced levels of functional proteins that are essential for mitochondrial complex I assembly and activity. Consequently, this deficiency disrupts mitochondrial integrity, ultimately compromising the thermogenic capacity of BAT. These findings illuminate a novel role for SRSF1 in influencing mitochondrial function and BAT thermogenesis through its regulation of Ndufs3 splicing within BAT.

Mitochondrial complex I deficiency stratifies idiopathic Parkinson's disease.

Idiopathic Parkinson's disease (iPD) is believed to have a heterogeneous pathophysiology, but molecular disease subtypes have not been identified. Here, we show that iPD can be stratified according to the severity of neuronal respiratory complex I (CI) deficiency, and identify two emerging disease subtypes with distinct molecular and clinical profiles. The CI deficient (CI-PD) subtype accounts for approximately a fourth of all cases, and is characterized by anatomically widespread neuronal CI deficiency, a distinct cell type-specific gene expression profile, increased load of neuronal mtDNA deletions, and a predilection for non-tremor dominant motor phenotypes. In contrast, the non-CI deficient (nCI-PD) subtype exhibits no evidence of mitochondrial impairment outside the dopaminergic substantia nigra and has a predilection for a tremor dominant phenotype. These findings constitute a step towards resolving the biological heterogeneity of iPD with implications for both mechanistic understanding and treatment strategies.

CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction.

BACKGROUND: The progression of diabetic cardiomyopathy (DCM) is noticeably influenced by mitochondrial dysfunction. Variants of caveolin 3 (CAV3) play important roles in cardiovascular diseases. However, the potential roles of CAV3 in mitochondrial function in DCM and the related mechanisms have not yet been elucidated. METHODS: Cardiomyocytes were cultured under high-glucose and high-fat (HGHF) conditions in vitro, and db/db mice were employed as a diabetes model in vivo. To investigate the role of CAV3 in DCM and to elucidate the molecular mechanisms underlying its involvement in mitochondrial function, we conducted Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and functional experiments. RESULTS: Our findings demonstrated significant downregulation of CAV3 in the cardiac tissue of db/db mice, which was found to be associated with cardiomyocyte apoptosis in DCM. Importantly, cardiac-specific overexpression of CAV3 effectively inhibited the progression of DCM, as it protected against cardiac dysfunction and cardiac remodeling associated by alleviating cardiomyocyte mitochondrial dysfunction. Furthermore, mass spectrometry analysis and immunoprecipitation assays indicated that CAV3 interacted with NDUFA10, a subunit of mitochondrial complex I. CAV3 overexpression reduced the degradation of lysosomal pathway in NDUFA10, restored the activity of mitochondrial complex I and improved mitochondrial function. Finally, our study demonstrated that CAV3 overexpression restored mitochondrial function and subsequently alleviated DCM partially through NDUFA10. CONCLUSIONS: The current study provides evidence that CAV3 expression is significantly downregulated in DCM. Upregulation of CAV3 interacts with NDUFA10, inhibits the degradation of lysosomal pathway in NDUFA10, a subunit of mitochondrial complex I, restores the activity of mitochondrial complex I, ameliorates mitochondrial dysfunction, and thereby protects against DCM. These findings indicate that targeting CAV3 may be a promising approach for the treatment of DCM.

NDUFA9 and its crotonylation modification promote browning of white adipocytes by activating mitochondrial function in mice.

Protein crotonylation plays a role in regulating cellular metabolism, gene expression, and other biological processes. NDUFA9 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9) is closely associated with the activity and function of mitochondrial respiratory chain complex I. Mitochondrial function and respiratory chain are closely related to browning of white adipocytes, it's speculated that NDUFA9 and its crotonylation are associated with browning of white adipocytes. Firstly, the effect of NDUFA9 on white adipose tissue was verified in white fat browning model mice, and it was found that NDUFA9 promoted mitochondrial respiration, thermogenesis, and browning of white adipose tissue. Secondly, in cellular studies, it was discovered that NDUFA9 facilitated browning of white adipocytes by enhancing mitochondrial function, mitochondrial complex I activity, ATP synthesis, and mitochondrial respiration. Again, the level of NDUFA9 crotonylation was increased by treating cells with vorinostat (SAHA)+sodium crotonate (NaCr) and overexpressing NDUFA9, it was found that NDUFA9 crotonylation promoted browning of white adipocytes. Meanwhile, the acetylation level of NDUFA9 was increased by treating cells with SAHA+sodium acetate (NaAc) and overexpressing NDUFA9, the assay revealed that NDUFA9 acetylation inhibited white adipocytes browning. Finally, combined with the competitive relationship between acetylation and crotonylation, it was also demonstrated that NDUFA9 crotonylation promoted browning of white adipocytes. Above results indicate that NDUFA9 and its crotonylation modification promote mitochondrial function, which in turn promotes browning of white adipocytes. This study establishes a theoretical foundation for the management and intervention of obesity, which is crucial in addressing obesity and related medical conditions in the future.

Long-range electron proton coupling in respiratory complex I - insights from molecular simulations of the quinone chamber and antiporter-like subunits.

Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.

Prophylactic nicotinamide treatment protects from rotenone-induced neurodegeneration by increasing mitochondrial content and volume.

Leber's hereditary optic neuropathy (LHON) is driven by mtDNA mutations affecting Complex I presenting as progressive retinal ganglion cell dysfunction usually in the absence of extra-ophthalmic symptoms. There are no long-term neuroprotective agents for LHON. Oral nicotinamide provides a robust neuroprotective effect against mitochondrial and metabolic dysfunction in other retinal injuries. We explored the potential for nicotinamide to protect mitochondria in LHON by modelling the disease in mice through intravitreal injection of the Complex I inhibitor rotenone. Using MitoV mice expressing a mitochondrial-tagged YFP in retinal ganglion cells we assessed mitochondrial morphology through super-resolution imaging and digital reconstruction. Rotenone induced Complex I inhibition resulted in retinal ganglion cell wide mitochondrial loss and fragmentation. This was prevented by oral nicotinamide treatment. Mitochondrial ultrastructure was quantified by transition electron microscopy, demonstrating a loss of cristae density following rotenone injection, which was also prevented by nicotinamide treatment. These results demonstrate that nicotinamide protects mitochondria during Complex I dysfunction. Nicotinamide has the potential to be a useful treatment strategy for LHON to limit retinal ganglion cell degeneration.

Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

Structures of 3-acetylpyridine adenine dinucleotide and ADP-ribose bound to the electron input module of respiratory complex I.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is a major enzyme of energy metabolism that couples NADH oxidation and ubiquinone reduction with proton translocation. The NADH oxidation site features different enzymatic activities with various nucleotides. While the kinetics of these reactions are well described, only binding of NAD+ and NADH have been structurally characterized. Here, we report the structures of the electron input module of Aquifex aeolicus complex I with bound ADP-ribose and 3-acetylpyridine adenine dinucleotides at resolutions better than 2.0 Å. ADP-ribose acts as inhibitor by blocking the "ADP-handle" motif essential for nucleotide binding. The pyridine group of APADH is minimally offset from flavin, which could contribute to its poorer suitability as substrate. A comparison with other nucleotide co-structures surprisingly shows that the adenine ribose and the pyrophosphate moiety contribute most to nucleotide binding, thus all adenine dinucleotides share core binding modes to the unique Rossmann-fold in complex I.