Ubiquinone Reduction Research Articles

Mitochondrial respiratory complex I can be inhibited via bypassing the ubiquinone-accessing tunnel.

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) couples electron transfer from NADH to ubiquinone with proton translocation in its membrane part. Structural studies have identified a long (~ 30 Å), narrow, tunnel-like cavity within the enzyme, through which ubiquinone may access a deep reaction site. Although various inhibitors are considered to block the ubiquinone reduction by occupying the tunnel's interior, this view is still debatable. We synthesized a phosphatidylcholine-quinazoline hybrid compound (PC-Qz1), in which a quinazoline-type toxophore was attached to the sn-2 acyl chain to prevent it from entering the tunnel. However, PC-Qz1 inhibited complex I and suppressed photoaffinity labeling by another quinazoline derivative, [125I]AzQ. This study provides further experimental evidence that is difficult to reconcile with the canonical ubiquinone-accessing tunnel model.

Heterogeneous distribution of mitochondria and succinate dehydrogenase activity in human airway smooth muscle cells

AbstractSuccinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

Cirbp suppression compromises DHODH-mediated ferroptosis defense and attenuates hypothermic cardioprotection in an aged donor transplantation model.

Hypothermia is commonly used to protect donor hearts during transplantation. However, patients transplanted with aged donor hearts still have severe myocardial injury and decreased survival rates, but the underlying mechanism remains unknown. Because aged hearts are not considered suitable for donation, the number of patients awaiting heart transplants is increasing. In this study, we examined whether hypothermic cardioprotection was attenuated in aged donor hearts during transplantation and evaluated potential therapeutic targets. Using a rat heart transplantation model, we found that hypothermic cardioprotection was impaired in aged donor hearts but preserved in young donor hearts. RNA-Seq showed that cold-inducible RNA-binding protein (Cirbp) expression was decreased in aged donor hearts, and these hearts showed severe ferroptosis after transplantation. The young donor hearts from Cirbp-KO rats exhibited attenuated hypothermic cardioprotection, but Cirbp overexpression in aged donor hearts ameliorated hypothermic cardioprotection. Cardiac proteomes revealed that dihydroorotate dehydrogenase (DHODH) expression was significantly decreased in Cirbp-KO donor hearts during transplantation. Consequently, DHODH-mediated ubiquinone reduction was compromised, thereby exacerbating cardiac lipid peroxidation and triggering ferroptosis after transplantation. A cardioplegic solution supplemented with CIRBP agonists improved hypothermic cardioprotection in aged donor hearts, indicating that this method has the potential to broaden the indications for using aged donor hearts in transplantation.

Selenium reduction of ubiquinone via SQOR suppresses ferroptosis.

The canonical biological function of selenium is in the production of selenocysteine residues of selenoproteins, and this forms the basis for its role as an essential antioxidant and cytoprotective micronutrient. Here we demonstrate that, via its metabolic intermediate hydrogen selenide, selenium reduces ubiquinone in the mitochondria through catalysis by sulfide quinone oxidoreductase. Through this mechanism, selenium rapidly protects against lipid peroxidation and ferroptosis in a timescale that precedes selenoprotein production, doing so even when selenoprotein production has been eliminated. Our findings identify a regulatory mechanism against ferroptosis that implicates sulfide quinone oxidoreductase and expands our understanding of selenium in biology.

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

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.

Structural insights into FSP1 catalysis and ferroptosis inhibition

Ferroptosis suppressor protein 1 (FSP1, also known as AIMF2, AMID or PRG3) is a recently identified glutathione-independent ferroptosis suppressor1–3, but its underlying structural mechanism remains unknown. Here we report the crystal structures of Gallus gallus FSP1 in its substrate-free and ubiquinone-bound forms. The structures reveal a FAD-binding domain and a NAD(P)H-binding domain, both of which are shared with AIF and NADH oxidoreductases4–9, and a characteristic carboxy-terminal domain as well. We demonstrate that the carboxy-terminal domain is crucial for the catalytic activity and ferroptosis inhibition of FSP1 by mediating the functional dimerization of FSP1, and the formation of two active sites located on two sides of FAD, which are responsible for ubiquinone reduction and a unique FAD hydroxylation respectively. We also identify that FSP1 can catalyze the production of H2O2 and the conversion of FAD to 6-hydroxy-FAD in the presence of oxygen and NAD(P)H in vitro, and 6-hydroxy-FAD directly inhibits ferroptosis in cells. Together, these findings further our understanding on the catalytic and ferroptosis suppression mechanisms of FSP1 and establish 6-hydroxy-FAD as an active cofactor in FSP1 and a potent radical-trapping antioxidant in ferroptosis inhibition.

Enhanced cyanide-resistant respiration as the predominant respiratory metabolic pathway in abnormal chilling injury behavior of postharvest papaya

An increasing number of studies have demonstrated that reactive oxygen species (ROS) play a crucial role in the chilling injury (CI) of postharvest fruit and vegetables. This is not only associated with the free radical scavenging system but is also intimately connected to the electron transport chain pathway. However, it remains unclear whether ROS is associated with the electron transport chain in the abnormal chilling behavior observed in postharvest papaya. As such, this study investigated the electron transport chain of papaya fruit stored at 1 ℃ and 6 ℃ and its connection to ROS. In comparison to storage at 6 ℃, storage at 1 ℃ suppressed the expression of CpCCO, and induced the upregulation of CpAOX expression in papaya fruit. Throughout the middle and late stages of storage, there was an increase in pyruvate content and ubiquinone reduction level. The increase had two effects: it inhibited cytochrome oxidase (CCO) activity and promoted alternative oxidase (AOX) activity, leading to a blocked cytochrome pathway and an increased proportion of cyanide-resistant respiratory pathways. Consequently, the cyanide-resistant respiratory pathway supplanted the cytochrome pathway as the primary electron transport pathway. This transition effectively curbed the accumulation of ROS, thereby diminishing the damage degree of fruit cell membrane, boosting chilling tolerance, and ultimately inhibiting the CI of papaya fruit, which led to the emergence of abnormal chilling injury.

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.

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase.

The emergence of drug-resistant variants of malaria-causing Plasmodium parasites is a life-threatening problem worldwide. Investigation of the physiological function of individual parasite proteins is a prerequisite for a deeper understanding of the metabolic pathways required for parasite survival and therefore a requirement for the development of novel antimalarials. A Plasmodium membrane protein, malate-quinone oxidoreductase (MQO), is thought to contribute to the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and is an antimalarial drug target. However, there is little information on its expression and function. Here, we investigated the function of Plasmodium falciparum MQO (PfMQO) in mitochondria using a yeast heterologous expression system. Using a yeast deletion mutant of mitochondrial malate dehydrogenase (MDH1), which is expected to be functionally similar to MQO, as a background strain, we successfully constructed PfMQO-expressing yeast. We confirmed that expression of PfMQO complemented the growth defect of the MDH1 deletion, indicating that PfMQO can adopt the metabolic role of MDH1 in energy transduction for growth in the recombinant yeast. Analysis of cell fractions confirmed that PfMQO was expressed and enriched in yeast mitochondria. By measuring MQO activity, we also confirmed that PfMQO expressed in yeast mitochondria was active. Measurement of oxygen consumption rates showed that mitochondrial respiration was driven by the TCA cycle through PfMQO. In addition, we found that MQO activity was enhanced when intact mitochondria were sonicated, indicating that the malate binding site of PfMQO is located facing the mitochondrial matrix. IMPORTANCE We constructed a model organism to study the physiological role and function of P. falciparum malate-quinone oxidoreductase (PfMQO) in a yeast expression system. PfMQO is actively expressed in yeast mitochondria and functions in place of yeast mitochondrial malate dehydrogenase, which catalyzes the oxidation of malate to oxaloacetate in the TCA cycle. The catalytic site for the oxidation of malate in PfMQO, which is a membrane-bound protein, faces into the mitochondrial matrix, not the mitochondrial inner membrane space. Our findings clearly show that PfMQO is a TCA cycle enzyme and is coupled with the ETC via ubiquinone reduction.

Identification of a Ubiquinone-Ubiquinol Quinhydrone Complex in Bacterial Photosynthetic Membranes and Isolated Reaction Centers by Time-Resolved Infrared Spectroscopy.

Ubiquinone redox chemistry is of fundamental importance in biochemistry, notably in bioenergetics. The bi-electronic reduction of ubiquinone to ubiquinol has been widely studied, including by Fourier transform infrared (FTIR) difference spectroscopy, in several systems. In this paper, we have recorded static and time-resolved FTIR difference spectra reflecting light-induced ubiquinone reduction to ubiquinol in bacterial photosynthetic membranes and in detergent-isolated photosynthetic bacterial reaction centers. We found compelling evidence that in both systems under strong light illumination-and also in detergent-isolated reaction centers after two saturating flashes-a ubiquinone-ubiquinol charge-transfer quinhydrone complex, characterized by a characteristic band at ~1565 cm-1, can be formed. Quantum chemistry calculations confirmed that such a band is due to formation of a quinhydrone complex. We propose that the formation of such a complex takes place when Q and QH2 are forced, by spatial constraints, to share a common limited space as, for instance, in detergent micelles, or when an incoming quinone from the pool meets, in the channel for quinone/quinol exchange at the QB site, a quinol coming out. This latter situation can take place both in isolated and membrane bound reaction centers Possible consequences of the formation of this charge-transfer complex under physiological conditions are discussed.

A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase

Vitamin K is a vital micronutrient implicated in a variety of human diseases. Warfarin, a vitamin K antagonist, is the most commonly prescribed oral anticoagulant. Patients overdosed on warfarin can be rescued by administering high doses of vitamin K because of the existence of a warfarin-resistant vitamin K reductase. Despite the functional discovery of vitamin K reductase over eight decades ago, its identity remained elusive. Here, we report the identification of warfarin-resistant vitamin K reductase using a genome-wide CRISPR-Cas9 knockout screen with a vitamin K-dependent apoptotic reporter cell line. We find that ferroptosis suppressor protein 1 (FSP1), a ubiquinone oxidoreductase, is the enzyme responsible for vitamin K reduction in a warfarin-resistant manner, consistent with a recent discovery by Mishima et al. FSP1 inhibitor that inhibited ubiquinone reduction and thus triggered cancer cell ferroptosis, displays strong inhibition of vitamin K-dependent carboxylation. Intriguingly, dihydroorotate dehydrogenase, another ubiquinone-associated ferroptosis suppressor protein parallel to the function of FSP1, does not support vitamin K-dependent carboxylation. These findings provide new insights into selectively controlling the physiological and pathological processes involving electron transfers mediated by vitamin K and ubiquinone.

The evolution of the human mitochondrial bc1 complex- adaptation for reduced rate of superoxide production?

The mitochondrial bc1 complex is a major source of mitochondrial superoxide. While bc1-generated superoxide plays a beneficial signaling role, excess production of superoxide lead to aging and degenerative diseases. The catalytic core of bc1 comprises three peptides -cytochrome b, Fe-S protein, and cytochrome c1. All three core peptides exhibit accelerated evolution in anthropoid primates. It has been suggested that the evolution of cytochrome b in anthropoids was driven by a pressure to reduce the production of superoxide. In humans, the bc1 core peptides exhibit anthropoid-specific substitutions that are clustered near functionally critical sites that may affect the production of superoxide. Here we compare the high-resolution structures of bovine, mouse, sheep and human bc1 to identify structural changes that are associated with human-specific substitutions. Several cytochrome b substitutions in humans alter its interactions with other subunits. Most significantly, there is a cluster of seven substitutions, in cytochrome b, the Fe-S protein, and cytochrome c1 that affect the interactions between these proteins at the tether arm of the Fe-S protein and may alter the rate of ubiquinone oxidation and the rate of superoxide production. Another cluster of substitutions near heme bH and the ubiquinone reduction site, Qi, may affect the rate of ubiquinone reduction and thus alter the rate of superoxide production. These results are compatible with the hypothesis that cytochrome b in humans (and other anthropoid primates) evolve to reduce the rate of production of superoxide thus enabling the exceptional longevity and exceptional cognitive ability of humans.

Membrane-domain mutations in respiratory complex I impede catalysis but do not uncouple proton pumping from ubiquinone reduction.

Respiratory complex I [NADH:ubiquinone (UQ) oxidoreductase] captures the free energy released from NADH oxidation and UQ reduction to pump four protons across an energy-transducing membrane and power ATP synthesis. Mechanisms for long-range energy coupling in complex I have been proposed from structural data but not yet evaluated by robust biophysical and biochemical analyses. Here, we use the powerful bacterial model system Paracoccus denitrificans to investigate 14 mutations of key residues in the membrane-domain Nqo13/ND4 subunit, defining the rates and reversibility of catalysis and the number of protons pumped per NADH oxidized. We reveal new insights into the roles of highly conserved charged residues in lateral energy transduction, confirm the purely structural role of the Nqo12/ND5 transverse helix, and evaluate a proposed hydrated channel for proton uptake. Importantly, even when catalysis is compromised the enzyme remains strictly coupled (four protons are pumped per NADH oxidized), providing no evidence for escape cycles that circumvent blocked proton-pumping steps.

Binding of Natural Inhibitors to Respiratory Complex I.

NADH:ubiquinone oxidoreductase (respiratory complex I) is a redox-driven proton pump with a central role in mitochondrial oxidative phosphorylation. The ubiquinone reduction site of complex I is located in the matrix arm of this large protein complex and connected to the membrane via a tunnel. A variety of chemically diverse compounds are known to inhibit ubiquinone reduction by complex I. Rotenone, piericidin A, and annonaceous acetogenins are representatives of complex I inhibitors from biological sources. The structure of complex I is determined at high resolution, and inhibitor binding sites are described in detail. In this review, we summarize the state of knowledge of how natural inhibitors bind in the Q reduction site and the Q access pathway and how their inhibitory mechanisms compare with that of a synthetic anti-cancer agent.

A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis invivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Abstract 5658: DHODH inhibition enhances radiotherapy-induced ferroptosis by promoting mitochondrial lipid peroxidation

Abstract Background: Ferroptosis is a form of regulated cell death caused by excessive accumulation of lipid peroxides on the cellular membrane. While impaired ferroptosis has been shown to contribute to tumor development, uncontrolled ferroptosis has been associated with neurodegeneration, acute kidney injury, and cardiovascular disease. To defend against ferroptosis, cells are equipped with several antioxidant systems. Glutathione peroxidase 4 (GPX4) is a major antioxidant enzyme that localizes in both the cytosol and the mitochondria and uses glutathione (GSH) as its cofactor to convert lipid hydroperoxides into non-toxic lipid alcohols. In addition, dihydroorotate dehydrogenase (DHODH) catalyzes the reduction of ubiquinone to ubiquinol in the inner mitochondrial membrane and inhibits mitochondrial lipid peroxidation independently of the GSH-GPX4 mechanism. Radiotherapy (RT) is one of the major therapies used to treat cancers. During RT, the radiolysis of water generates reactive oxygen species (ROS), subsequently leading to lipid peroxidation and ferroptosis. In our study, we aim to determine the role of DHODH in RT-induced ferroptosis and to evaluate the therapeutic potential of DHODH inhibitors as robust radiosensitizers in cancer treatment. Methods: Lipid peroxidation and mitochondrial lipid peroxidation were evaluated by flow cytometry. Ferroptosis marker PTGS2 expression levels were evaluated by real-time PCR. CRISPR-Cas9 system used to generate knock-out cancer cell lines. Patient-derived xenografts (PDX) co-treated with RT and DHODH inhibitors were employed to access the combination effect. Lipid peroxidation marker 4-HNE levels in tumors were evaluated by immunohistochemistry. Results: RT induced lipid peroxidation and PTGS2 expression in a variety of cancer cell lines. Genetic knockout of DHODH caused cells more sensitive to RT-induced ferroptosis. Similarly, pharmacological inhibition of DHODH sensitized cancer cells to RT-induced ferroptotic cell death both in vitro and in vivo. Mechanistically, RT induces ROS production in mitochondria, resulting in mitochondrial lipid peroxidation and ferroptosis. DHODH catalyzes the reduction of ubiquinone to ubiquinol, which acts as an antioxidant to inhibit lipid peroxidation in mitochondria. In general, DHODH inhibition significantly sensitizes cancer cells to RT-induced ferroptosis by decreasing ubiquinol levels in mitochondria. Conclusion: Our study suggests a model in which DHODH inhibition enhances RT-induced ferroptosis. DHODH inhibition significantly decreases the ubiquinol/ubiquinone ratio in mitochondria, thereby sensitizing cancer cells to RT-induced ferroptosis caused by mitochondrial lipid peroxidation. Together, our results demonstrate that DHODH inhibitors can be used as radiosensitizers through inducing ferroptosis and propose a novel combination strategy for cancer therapy. Citation Format: Chao Mao, Amber Horbath, Guang Lei, Boyi Gan. DHODH inhibition enhances radiotherapy-induced ferroptosis by promoting mitochondrial lipid peroxidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5658.

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQm-1/pUQp-1 and pUQm-2/pUQp-2), with each pair having the same chemical properties except for a ∼1.0 Å difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [125I]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [125I]pUQs used, indicating that [125I]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.

Single Cell Assessment of Mitochondrial Function

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.