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Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane 1 . Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III 2 -IV 2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization., (© 2023. The Author(s).)

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)-based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures., (© 2021 Bomba-Warczak et al.)

Isolated defects in the mitochondrial respiratory chain complex II (CII; succinate-ubiquinone oxidoreductase) are extremely rare and mainly result from bi-allelic mutations in one of the nuclear encoded subunits: SDHA, SDHB and SDHD, which comprise CII and the assembly CII factor SDHAF1. We report an adolescent female who presented with global developmental delay, intellectual disability and childhood onset progressive bilateral optic atrophy. Whole exome sequencing of the patient and her unaffected parents identified the novel heterozygous de novo variant c.1984C > T [NM_004168.4] in the SDHA gene. Biochemical assessment of CII in the patient's derived fibroblasts and lymphocytes displayed considerably decreased CII residual activity compared with normal controls, when normalized to the integral mitochondrial enzyme citrate synthase. Protein modeling of the consequent p.Arg662Cys variant [NP-004159.2] suggested that this substitution will compromise the structural integrity of the FAD-binding protein at the C-terminus that will ultimately impair the FAD binding to SDHA, thus decreasing the entire CII activity. Our study emphasizes the role of certain heterozygous SDHA mutations in a distinct clinical phenotype dominated by optic atrophy and neurological impairment. This is the second mutation that has been reported to cause this phenotype. Furthermore, it adds developmental delay and cognitive disability to the expanding spectrum of the disorder. We propose to add SDHA to next generation sequencing gene panels of optic atrophy.

Mitochondrial complex II, also known as succinate dehydrogenase (SDH), is an integral-membrane heterotetramer (SDHABCD) that links two essential energy-producing processes, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. A significant amount of information is available on the structure and function of mature complex II from a range of organisms. However, there is a gap in our understanding of how the enzyme assembles into a functional complex, and disease-associated complex II insufficiency may result from incorrect function of the mature enzyme or from assembly defects. Here, we investigate the assembly of human complex II by combining a biochemical reconstructionist approach with structural studies. We report an X-ray structure of human SDHA and its dedicated assembly factor SDHAF2. Importantly, we also identify a small molecule dicarboxylate that acts as an essential cofactor in this process and works in synergy with SDHAF2 to properly orient the flavin and capping domains of SDHA. This reorganizes the active site, which is located at the interface of these domains, and adjusts the pK a of SDHA R451 so that covalent attachment of the flavin adenine dinucleotide (FAD) cofactor is supported. We analyze the impact of disease-associated SDHA mutations on assembly and identify four distinct conformational forms of the complex II flavoprotein that we assign to roles in assembly and catalysis., Competing Interests: The authors declare no competing interest.

Respiratory complexes are complicated multi-subunit cofactor-containing machines that allow cells to harvest energy from the environment. Maturation of these complexes requires protein folding, cofactor insertion, and assembly of multiple subunits into a final, functional complex. Because the intermediate states in complex maturation are transitory, these processes are poorly understood. This review gives an overview of the process of maturation in respiratory complex II with a focus on recent structural studies on intermediates formed during covalent flavinylation of the catalytic subunit, SDHA. Covalent flavinylation has an evolutionary significance because variants of complex II enzymes with the covalent ligand removed by mutagenesis cannot oxidize succinate, but can still perform the reverse reaction and reduce fumarate. Since succinate oxidation is a key step of aerobic respiration, the covalent bond of complex II appears to be important for aerobic life., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Linoleic acid is the major fatty acid moiety of cardiolipin, which is central to the assembly of components involved in mitochondrial oxidative phosphorylation (OXPHOS). Although linoleic acid is an essential nutrient, its excess intake is harmful to health. On the other hand, linoleic acid has been shown to prevent the reduction in cardiolipin content and to improve mitochondrial function in aged rats with spontaneous hypertensive heart failure (HF). In this study, we found that lower dietary intake of linoleic acid in HF patients statistically correlates with greater severity of HF, and we investigated the mechanisms therein involved., Methods: HF patients, who were classified as New York Heart Association (NYHA) functional class I (n = 45), II (n = 93), and III (n = 15), were analyzed regarding their dietary intakes of different fatty acids during the one month prior to the study. Then, using a mouse model of HF, we confirmed reduced cardiolipin levels in their cardiac myocytes, and then analyzed the mechanisms by which dietary supplementation of linoleic acid improves cardiac malfunction of mitochondria., Results: The dietary intake of linoleic acid was significantly lower in NYHA III patients, as compared to NYHA II patients. In HF model mice, both CI-based and CII-based OXPHOS activities were affected together with reduced cardiolipin levels. Silencing of CRLS1, which encodes cardiolipin synthetase, in cultured cardiomyocytes phenocopied these events. Feeding HF mice with linoleic acid improved both CI-based and CII-based respiration as well as left ventricular function, together with an increase in cardiolipin levels. However, although assembly of the respirasome (i.e., CI/CIII 2 /CIV complex), as well as assembly of CII subunits and the CIII 2 /CIV complex statistically correlated with cardiolipin levels in cultured cardiomyocytes, respirasome assembly was not notably restored by dietary linoleic acid in HF mice. Therefore, although linoleic acid may significantly improve both CI-based and CII-based respiration of cardiomyocytes, respirasomes impaired by HF were not easily repaired by the dietary intake of linoleic acid., Conclusions: Dietary supplement of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF.

Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled Escherichia coli SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in E. coli , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.

A covalently bound flavin cofactor is predominant in the succinate-ubiquinone oxidoreductase (SQR; Complex II), an essential component of aerobic electron transport, and in the menaquinol-fumarate oxidoreductase (QFR), the anaerobic counterpart, although it is only present in approximately 10 % of the known flavoenzymes. This work investigates the role of this 8α-N3-histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT calculations, classical molecular-dynamics simulations and quantum-mechanics calculations for Complex II:FAD and Complex II:FADH 2 , with and without the covalent bond, were performed. It was observed that the covalent bond is essential for the active-center arrangement of the FADH 2 /FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group, which influences interactions with the protein, flavin solvation, and possible proton-transfer pathways. Specifically, for the noncovalently bound FADH 2 cofactor, the N1 atom moves away from the His-A365 and His-A254 residues and the N5 atom moves away from the glutamine-62A residue. Both of the histidine and glutamine residues interact with a chain of water molecules that cross the enzyme, which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Patients who inherit a pathogenic loss-of-function genetic variant involving one of the four succinate dehydrogenase (SDH) subunit genes have up to an 86% chance of developing one or more cancers by the age of 50. If tumors are identified and removed early in these high-risk patients, they have a higher potential for cure. Unfortunately, many alterations identified in these genes are variants of unknown significance (VUS), confounding the identification of high-risk patients. If we could identify misclassified SDH VUS as benign or pathogenic SDH mutations, we could better select patients for cancer screening procedures and remove tumors at earlier stages. Experimental Design: In this study, we combine data from clinical observations, a functional yeast model, and a computational model to determine the pathogenicity of 22 SDHA VUS. We gathered SDHA VUS from two primary sources: The OHSU Knight Diagnostics Laboratory and the literature. We used a yeast model to identify the functional effect of a VUS on mitochondrial function with a variety of biochemical assays. The computational model was used to visualize variants' effect on protein structure. Results: We were able to draw conclusions on functional effects of variants using our three-prong approach to understanding VUS. We determined that 16 (73%) of the alterations are actually pathogenic, causing loss of SDH function, and six (27%) have no effect upon SDH function. Conclusions: We thus report the reclassification of the majority of the VUS tested as pathogenic, and highlight the need for more thorough functional assessment of inherited SDH variants. Clin Cancer Res; 23(21); 6733-43. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The respiratory megacomplex represents the highest-order assembly of respiratory chain complexes, and it allows mitochondria to respond to energy-requiring conditions. To understand its architecture, we examined the human respiratory chain megacomplex-I 2 III 2 IV 2 (MCI 2 III 2 IV 2 ) with 140 subunits and a subset of associated cofactors using cryo-electron microscopy. The MCI 2 III 2 IV 2 forms a circular structure with the dimeric CIII located in the center, where it is surrounded by two copies each of CI and CIV. Two cytochrome c (Cyt.c) molecules are positioned to accept electrons on the surface of the c 1 state CIII dimer. Analyses indicate that CII could insert into the gaps between CI and CIV to form a closed ring, which we termed the electron transport chain supercomplex. The structure not only reveals the precise assignment of individual subunits of human CI and CIII, but also enables future in-depth analysis of the electron transport chain as a whole., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mitochondrial morphology regulated by fusion and fission processes determines mitochondrial function and cell fate. Some studies showed hyperfused mitochondria could induce apoptosis in cancer cells, but the relevant molecular mechanisms remain elusive. Superoxide (O 2 •- ) and pH play vital roles in mitochondrial dysfunction and apoptosis. Therefore, it is worthwhile to explore if there is an intimate relationship between mitochondrial hyperfusion and simultaneous changes in O 2 •- and pH levels, which will be helpful to uncover relevant detailed mechanism. For this purpose, we have developed a new reversible two-photon fluorescent probe (CFT) to simultaneously monitor O 2 •- and pH in 4T1 cells and mice using dual-color imaging. With the assistance of probe, we found that inhibition of Dynamin-related protein 1 (Drp1) could transduce a signal through mitochondrial complexes I and II to enhance the O 2 •- and pH levels and eventually induced mitohyperfusion and apoptosis in breast cancer cells. Together, these data indicate that CFT provides a robust tool for unveiling the roles of O 2 •- and pH in signals associated with mitochondrial dysfunction in cells and in vivo.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: ( a ) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site II f ); ( b ) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site II f The decrease in matrix [dicarboxylate] allows O 2 access to reduced site II f , thereby making electron donation to O 2 possible, explaining the rapid increase in ROS production provided that site II f is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A series of diphenyl ether-containing pyrazole-carboxamide derivatives was designed and synthesized as new succinate ubiquinone oxidoreductase (SQR) inhibitors. This highly potent molecular scaffold was developed from a moderately activie hit 3, obtained from our previous pharmacophore-linked fragment virtual screening (PFVS) method. The results of greenhouse tests indicated that some analogues showed good SQR inhibitory activity, with promising fungicidal activity against Rhizoctonia solani and Sphaerotheca fuliginea at a dosage of 200 mg/L. Most surprisingly, compound 62 showed the highest SQR inhibitory activity with a K i value of 0.081 μM, about 4-fold more potent than penthiopyrad (K i = 0.307 μM). In addition, compounds 43 and 62 displayed excellent fungicidal activity even at a dosage as low as 6.25 mg/L, which was superior to thifluzamide. Moreover, compound 62 exhibited excellent protection effect against R. solani and provided about 81.2% protective control efficancy after 21 days with two sprayings. The present work indicated that these two compounds could be used as potential agricultural fungicides targeting SQR.

Succinate-ubiquinone oxidoreductase (SQR) is an attractive target for fungicide discovery. Herein, we report the discovery of novel SQR inhibitors using a pharmacophore-linked fragment virtual screening approach, a new drug design method developed in our laboratory. Among newly designed compounds, compound 9s was identified as the most potent inhibitor with a Ki value of 34 nM against porcine SQR, displaying approximately 10-fold higher potency than that of the commercial control penthiopyrad. Further inhibitory kinetics studies revealed that compound 9s is a noncompetitive inhibitor with respect to the substrate cytochrome c and DCIP. Interestingly, compounds 8a, 9h, 9j, and 9k exhibited good in vivo preventive effects against Rhizoctonia solani. The results obtained from molecular modeling showed that the orientation of the R(2) group had a significant effect on binding with the protein.

Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

Succinate-ubiquinone oxidoreductase (SQR, EC 1.3.5.1, complex II), an essential component of cellular respiratory chain and tricarboxylic acid (or Krebs) cycle, has been identified as one of the most significant targets for pharmaceutical and agrochemical. Herein, with the aim of discovery of new antibacterial lead structure, a series of N-benzoxazol-5-yl-pyrazole-4-carboxamides were designed, synthesized, and evaluated for their SQR inhibitory effects. Very promisingly, one candidate (Ki = 11 nM, porcine SQR) was successfully identified as the most potent synthetic SQR inhibitor so far. The further inhibitory kinetics studies revealed that the candidate is non-competitive with respect to the substrate cytochrome c and DCIP. Computational simulations revealed that the titled compounds have formed hydrogen bond with D_Y91 and B_W173 and the pyrazole ring formed cation-π interaction with C_R46. In addition, in R(1) position, -CHF2 group has increased the binding affinity and decreased the entropy contribution, while -CF3 group displayed completely opposite effect when bound with SQR. The results of the present work showed that N-benzoxazol-5-yl-pyrazole-4-carboxamide is a new scaffold for discovery of SQR inhibitors and worth further study., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.

In previous studies, 30Kc19, a lipoprotein in silkworm hemolymph, enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, we hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme. We investigated this using in vitro and cellular assessments. The activity of sialyltransferase (ST) and isolated mitochondrial complex I/III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, ST activity inside erythropoietin (EPO)-producing Chinese hamster ovary (CHO) cells increased more than 25 % and mitochondrial complex II activity in HeLa cells increased more than 50 % with 30Kc19. The increase in intracellular ST activity resulted in an increase in sialic acid content of glycoproteins produced in CHO cells supplemented with 30Kc19. Similarly, enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19 by over 50 %. Because 30Kc19 stabilized intracellular enzymes for glycosylation and enhanced protein productivity with supplementation in the culture medium, we expect that 30Kc19 can be a valuable tool for effective industrial recombinant protein production.

Defects in complex II of the mitochondrial respiratory chain are a rare cause of mitochondrial disorders. Underlying autosomal-recessive genetic defects are found in most of the 'SDHx' genes encoding complex II (SDHA, SDHB, SDHC, and SDHD) and its assembly factors. Interestingly, SDHx genes also function as tumor suppressor genes in hereditary paragangliomas, pheochromocytomas, and gastrointestinal stromal tumors. In these cases, the affected patients are carrier of a heterozygeous SDHx germline mutation. Until now, mutations in SDHx associated with mitochondrial disease have not been reported in association with hereditary tumors and vice versa. Here, we characterize four patients with isolated complex II deficiency caused by mutations in SDHA presenting with multisystem mitochondrial disease including Leigh syndrome (LS) and/or leukodystrophy. Molecular genetic analysis revealed three novel mutations in SDHA. Two mutations (c.64-2A>G and c.1065-3C>A) affect mRNA splicing and result in loss of protein expression. These are the first mutations described affecting SDHA splicing. For the third new mutation, c.565T>G, we show that it severely affects enzyme activity. Its pathogenicity was confirmed by lentiviral complementation experiments on the fibroblasts of patients carrying this mutation. It is of special interest that one of our LS patients harbored the c.91C>T (p.Arg31*) mutation that was previously only reported in association with paragangliomas and pheochromocytomas, tightening the gap between these two rare disorders. As tumor screening is recommended for SDHx mutation carriers, this should also be considered for patients with mitochondrial disorders and their family members.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation., (© FASEB.)

Succinate: quinone reductases (SQRs) are the enzymes that couple the oxidation of succinate and the reduction of quinones in the respiratory chain of prokaryotes and eukaryotes. Herein, we compare the temperature-dependent activity and structural stability of two SQRs, the first from the mesophilic bacterium Escherichia coli and the second from the thermophilic bacterium Thermus thermophilus, using a combined electrochemical and infrared spectroscopy approach. Direct electron transfer was achieved with full membrane protein complexes at single-walled carbon nanotube (SWNT)-modified electrodes. The possible structural factors that contribute to the temperature-dependent activity of the enzymes and, in particular, to the thermostability of the Thermus thermophilus SQR are discussed., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI-CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (ΔpH) and electrical charge (Δψ) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Δψ depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24h) CI inhibition in HEK293 cells induces a proton-based Δψ hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Δψ depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Δψ hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ(Nf)) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ(Ns)) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ(Nvs)) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ(Ns) signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

HDL and apolipoprotein A1 (apoA1) concentrations inversely correlate with risk of death from ischemic heart disease; however, the role of apoA1 in the myocardial response to ischemia has not been well defined. To test whether apoA1, the primary HDL apolipoprotein, has an acute anti-inflammatory role in ischemic heart disease, we induced myocardial infarction via direct left anterior descending coronary artery ligation in apoA1 null (apoA1(-/-)) and apoA1 heterozygous (apoA1(+/-)) mice. We observed that apoA1(+/-) and apoA1(-/-) mice had a 52% and 125% increase in infarct size as a percentage of area at risk, respectively, compared with wild-type (WT) C57BL/6 mice. Mitochondrial oxidation contributes to tissue damage in ischemia-reperfusion injury. A substantial defect was present at baseline in the electron transport chain of cardiac myocytes from apoA1(-/-) mice localized to the coenzyme Q (CoQ) pool with impaired electron transfer (67% decrease) from complex II to complex III. Administration of coenzyme Q10 (CoQ10) to apoA1 null mice normalized the cardiac mitochondrial CoQ pool and reduced infarct size to that observed in WT mice. CoQ10 administration did not significantly alter infarct size in WT mice. These data identify CoQ pool content leading to impaired mitochondrial function as major contributors to infarct size in the setting of low HDL/apoA1. These data suggest a previously unappreciated mechanism for myocardial stunning, cardiac dysfunction, and muscle pain associated with low HDL and low apoA1 concentrations that can be corrected by CoQ10 supplementation and suggest populations of patients that may benefit particularly from CoQ10 supplementation., (© 2014 American Society for Nutrition.)

Single electron transfers have been examined in complex II (succinate:ubiquinone oxidoreductase) by the method of pulse radiolysis. Electrons are introduced into the enzyme initially at the [3Fe-4S] and ubiquinone sites followed by intramolecular equilibration with the b heme of the enzyme. To define thermodynamic and other controlling parameters for the pathways of electron transfer in complex II, site-directed variants were constructed and analyzed. Variants at SdhB-His207 and SdhB-Ile209 exhibit significantly perturbed electron transfer between the [3Fe-4S] cluster and ubiquinone. Analysis of the data using Marcus theory shows that the electronic coupling constants for wild-type and variant enzyme are all small, indicating that electron transfer occurs by diabatic tunneling. The presence of the ubiquinone is necessary for efficient electron transfer to the heme, which only slowly equilibrates with the [3Fe-4S] cluster in the absence of the quinone.

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 - over 1000 kDa) and cultured cells (400-670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.

Parasites have developed a variety of physiological functions necessary for completing at least part of their life cycles in the specialized environments of surrounding the parasites in the host. Regarding energy metabolism, which is essential for survival, parasites adapt to the low oxygen environment in mammalian hosts by using metabolic systems that are very different from those of the hosts. In many cases, the parasite employs aerobic metabolism during the free-living stage outside the host but undergoes major changes in developmental control and environmental adaptation to switch to anaerobic energy metabolism. Parasite mitochondria play diverse roles in their energy metabolism, and in recent studies of the parasitic nematode, Ascaris suum, the mitochondrial complex II plays an important role in anaerobic energy metabolism of parasites inhabiting hosts by acting as a quinol-fumarate reductase. In Trypanosomes, parasite complex II has been found to have a novel function and structure. Complex II of Trypanosoma cruzi is an unusual supramolecular complex with a heterodimeric iron-sulfur subunit and seven additional non-catalytic subunits. The enzyme shows reduced binding affinities for both substrates and inhibitors. Interestingly, this structural organization is conserved in all trypanosomatids. Since the properties of complex II differ across a wide range of parasites, this complex is a potential target for the development of new chemotherapeutic agents. In this regard, structural information on the target enzyme is essential for the molecular design of drugs. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2013. Published by Elsevier B.V.)

Complex II (also known as Succinate dehydrogenase or Succinate-ubiquinone oxidoreductase) is an important respiratory enzyme that participates in both the tricarboxylic acid cycle and electron transport chain. Complex II consists of four subunits including a catalytic flavoprotein (SdhA), an iron-sulphur subunit (SdhB) and two hydrophobic membrane anchors (SdhC and SdhD). Complex II also contains a number of redox cofactors including haem, Fe-S clusters and FAD, which mediate electron transfer from succinate oxidation to the reduction of the mobile electron carrier ubiquinone. The flavin cofactor FAD is an important redox cofactor found in many proteins that participate in oxidation/reduction reactions. FAD is predominantly bound non-covalently to flavoproteins, with only a small percentage of flavoproteins, such as complex II, binding FAD covalently. Aside from a few examples, the mechanisms of flavin attachment have been a relatively unexplored area. This review will discuss the FAD cofactor and the mechanisms used by flavoproteins to covalently bind FAD. Particular focus is placed on the attachment of FAD to complex II with an emphasis on SdhE (a DUF339/SDH5 protein previously termed YgfY), the first protein identified as an assembly factor for FAD attachment to flavoproteins in prokaryotes. The molecular details of SdhE-dependent flavinylation of complex II are discussed and comparisons are made to known cofactor chaperones. Furthermore, an evolutionary hypothesis is proposed to explain the distribution of SdhE homologues in bacterial and eukaryotic species. Mechanisms for regulating SdhE function and how this may be linked to complex II function in different bacterial species are also discussed. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

There are two homologous membrane-bound enzymes in Escherichia coli that catalyze reversible conversion between succinate/fumarate and quinone/quinol. Succinate:ubiquinone reductase (SQR) is a component of aerobic respiratory chains, whereas quinol:fumarate reductase (QFR) utilizes menaquinol to reduce fumarate in a final step of anaerobic respiration. Although, both protein complexes are capable of supporting bacterial growth on either minimal succinate or fumarate media, the enzymes are more proficient in their physiological directions. Here we evaluate factors that may underlie this catalytic bias. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The di-heme family of succinate:quinone oxidoreductases is of particular interest, because its members support electron transfer across the biological membranes in which they are embedded. In the case of the di-heme-containing succinate:menaquinone reductase (SQR) from Gram-positive bacteria and other menaquinone-containing bacteria, this results in an electrogenic reaction. This is physiologically relevant in that it allows the transmembrane electrochemical proton potential Δp to drive the endergonic oxidation of succinate by menaquinone. In the case of the reverse reaction, menaquinol oxidation by fumarate, catalysed by the di-heme-containing quinol:fumarate reductase (QFR), evidence has been obtained that this electrogenic electron transfer reaction is compensated by proton transfer via a both novel and essential transmembrane proton transfer pathway ("E-pathway"). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory "E-pathway" appears to be required by all di-heme-containing QFR enzymes and results in the overall reaction being electroneutral. In addition to giving a brief overview of progress in the characterization of other members of this diverse family, this contribution summarizes key evidence and progress in identifying constituents of the "E-pathway" within the framework of the crystal structure of the QFR from the anaerobic epsilon-proteobacterium Wolinella succinogenes at 1.78Å resolution. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2013. Published by Elsevier B.V.)

Over a decade has passed since the elucidation of the first X-ray crystal structure of any complex II homolog. In the intervening time, the structures of five additional integral-membrane complex II enzymes and three homologs of the soluble domain have been determined. These structures have provided a framework for the analysis of enzymological studies of complex II superfamily enzymes, and have contributed to detailed proposals for reaction mechanisms at each of the two enzyme active sites, which catalyze dicarboxylate and quinone oxidoreduction, respectively. This review focuses on how structural data have augmented our understanding of catalysis by the superfamily. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Succinate Dehydrogenase (SDH) heterotetrameric complex catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and in the aerobic respiratory chains of eukaryotes and bacteria. Essential in this catalysis is the covalently-linked cofactor flavin adenine dinucleotide (FAD) in subunit1 (Sdh1) of the SDH enzyme complex. The mechanism of FAD insertion and covalent attachment to Sdh1 is unknown. Our working concept of this flavinylation process has relied mostly on foundational works from the 1990s and by applying the principles learned from other enzymes containing a similarly linked FAD. The discovery of the flavinylation factor Sdh5, however, has provided new insight into the possible mechanism associated with Sdh1 flavinylation. This review focuses on encapsulating prior and recent advances towards understanding the mechanism associated with flavinylation of Sdh1 and how this flavinylation process affects the overall assembly of SDH. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.

Increased superoxide (O2 (·-)) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occur in the post-ischemic myocardium (Zhang et al., Biochemistry 49:2529-2539, 2010; Chen et al., J Biol Chem 283:27991-28003, 2008; Chen et al., J Biol Chem 282: 32640-32654, 2007). To gain the deeper insights into ROS-mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cysteine S-glutathionylation) or OONO(-) (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.

This article reviews the structure and function of mitochondrial respiratory chain complex Ⅱ, and the clinical features, diagnosis, treatment and genetic analysis of mitochondrial respiratory chain complex Ⅱ deficiency. Mitochondrial complex Ⅱ, known as succinate dehydrogenase, is a part of the mitochondrial respiratory chain. It plays an important role in cellular oxidative phosphorylation. It is associated with oxidative stress and is a sensitive target for toxic substances and abnormal metabolin in cells. Clinical manifestations of respiratory chain complex Ⅱ deficiency are characterized by a wide variety of abnormalities. Progressive neuromuscular dysfunction is the most common syndrome. Cardiomyopathy, episodic vomit and hemolytic uremic syndrome are also encountered in a few cases. A precise diagnosis is dependent on enzyme activities assay of respiratory chain complexes and genetic analysis. Complex Ⅱ activities decreased in affected tissues. Pathogenic mutations in SDHA gene and SDHAF1 gene encoding assembly factor have been found so far. Clinical treatment aims at improving the mitochondrial function.

The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O(2), is mediated by protein-bound redox cofactors. In mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc(1) complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a(3), with Cu(B), form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc(1), whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

It is generally accepted that the mitochondria play central roles in energy production of most eukaryotes. In contrast, it has been thought that Plasmodium spp., the causative agent of malaria, rely mainly on cytosolic glycolysis but not mitochondrial oxidative phosphorylation for energy production during blood stages. However, Plasmodium spp. possesses all genes necessary for the tricarboxylic acid (TCA) cycle and most of the genes for electron transport chain (ETC) enzymes. Therefore, it remains elusive whether oxidative phosphorylation is essential for the parasite survival. To elucidate the role of TCA metabolism and ETC in malaria parasites, we deleted the gene for flavoprotein (Fp) subunit, Pbsdha, one of four components of complex II, a catalytic subunit for succinate dehydrogenase activity. The Pbsdha(-) parasite grew normally at blood stages in mouse. In contrast, ookinete formation of Pbsdha(-) parasites in the mosquito stage was severely impaired. Finally, Pbsdha(-) ookinetes failed in oocyst formation, leading to complete malaria transmission blockade. These results suggest that malaria parasite may switch the energy metabolism from glycolysis to oxidative phosphorylation to adapt to the insect vector where glucose is not readily available for ATP production.

Background: Isolated complex II deficiency is a rare form of mitochondrial disease, accounting for approximately 2% of all respiratory chain deficiency diagnoses. The succinate dehydrogenase (SDH) genes (SDHA, SDHB, SDHC and SDHD) are autosomally-encoded and transcribe the conjugated heterotetramers of complex II via the action of two known assembly factors (SDHAF1 and SDHAF2). Only a handful of reports describe inherited SDH gene defects as a cause of paediatric mitochondrial disease, involving either SDHA (Leigh syndrome, cardiomyopathy) or SDHAF1 (infantile leukoencephalopathy). However, all four SDH genes, together with SDHAF2, have known tumour suppressor functions, with numerous germline and somatic mutations reported in association with hereditary cancer syndromes, including paraganglioma and pheochromocytoma., Methods and Results: Here, we report the clinical and molecular investigations of two patients with histochemical and biochemical evidence of a severe, isolated complex II deficiency due to novel SDH gene mutations; the first patient presented with cardiomyopathy and leukodystrophy due to compound heterozygous p.Thr508Ile and p.Ser509Leu SDHA mutations, while the second patient presented with hypotonia and leukodystrophy with elevated brain succinate demonstrated by MR spectroscopy due to a novel, homozygous p.Asp48Val SDHB mutation. Western blotting and BN-PAGE studies confirmed decreased steady-state levels of the relevant SDH subunits and impairment of complex II assembly. Evidence from yeast complementation studies provided additional support for pathogenicity of the SDHB mutation., Conclusions: Our report represents the first example of SDHB mutation as a cause of inherited mitochondrial respiratory chain disease and extends the SDHA mutation spectrum in patients with isolated complex II deficiency.

Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

Mutations in cancer cells affecting subunits of the respiratory chain (RC) indicate a central role of oxidative phosphorylation for tumourigenesis. Recent studies have suggested that such mutations of RC complexes impact apoptosis induction. We review here the evidence for this hypothesis, which in particular emerged from work on how complex I and II mediate signals for apoptosis. Both protein aggregates are specifically inhibited for apoptosis induction through different means by exploiting with protease activation and pH change, two widespread but independent features of dying cells. Nevertheless, both converge on forming reactive oxygen species for the demise of the cell. Investigations into these mitochondrial processes will remain a rewarding area for unravelling the causes of tumourigenesis and for discovering interference options.

The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F₀F₁-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.

Mitochondria are both a source of ATP and a site of reactive oxygen species (ROS) production. However, there is little information on the sites of mitochondrial ROS (mROS) production or the biological role of such mROS in plants. We provide genetic proof that mitochondrial complex II (Complex II) of the electron transport chain contributes to localized mROS that regulates plant stress and defense responses. We identify an Arabidopsis mutant in the Complex II subunit, SDH1-1, through a screen for mutants lacking GSTF8 gene expression in response to salicylic acid (SA). GSTF8 is an early stress-responsive gene whose transcription is induced by biotic and abiotic stresses, and its expression is commonly used as a marker of early stress and defense responses. Transcriptional analysis of this mutant, disrupted in stress responses 1 (dsr1), showed that it had altered SA-mediated gene expression for specific downstream stress and defense genes, and it exhibited increased susceptibility to specific fungal and bacterial pathogens. The dsr1 mutant also showed significantly reduced succinate dehydrogenase activity. Using in vivo fluorescence assays, we demonstrated that root cell ROS production occurred primarily from mitochondria and was lower in the mutant in response to SA. In addition, leaf ROS production was lower in the mutant after avirulent bacterial infection. This mutation, in a conserved region of SDH1-1, is a unique plant mitochondrial mutant that exhibits phenotypes associated with lowered mROS production. It provides critical insights into Complex II function with implications for understanding Complex II's role in mitochondrial diseases across eukaryotes.

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

Mitochondria are the major source of reactive oxygen species. Both complex I and complex II mediate O2*- production in mitochondria and host reactive protein thiols. To explore the functions of the specific domains involved in the redox modifications of complexes I and II, various peptide-based antibodies were generated against these complexes, and their inhibitory effects were subsequently measured. The redox domains involved in S-glutathionylation and nitration, as well as the binding 2011. motif of the iron-sulfur cluster (N1a) of the complexes I and II were utilized to design B-cell epitopes for generating antibodies. The effect of antibody binding on enzyme-mediated O2*- generation was measured by EPR spin trapping. Binding of either antibody AbGSCA206 or AbGSCB367 against glutathione (GS)-binding domain to complex I inhibit its O2*- generation, but does not affect electron transfer efficiency. Binding of antibody (Ab24N1a) against the binding motif of N1a to complex I modestly suppresses both O2*- generation and electron transfer efficiency. Binding of either antibody Ab75 or Ab24 against nonredox domain decreases electron leakage production. In complex II, binding of antibody AbGSC90 against GS-binding domain to complex II marginally decreases both O2*- generation and electron transfer activity. Binding of antibody AbY142 to complex II against the nitrated domain modestly inhibits electron leakage, but does not affect the electron transfer activity of complex II. In conclusion, mediation of O2*- generation by complexes I and II can be regulated by specific redox and nonredox domains.

The complex II family of proteins includes succinate:quinone oxidoreductase (SQR) and quinol:fumarate oxidoreductase (QFR). In the facultative bacterium Escherichia coli both are expressed as part of the aerobic (SQR) and anaerobic (QFR) respiratory chains. SQR from E. coli is homologous to mitochondrial complex II and has proven to be an excellent model system for structure/function studies of the enzyme. Both SQR and QFR from E. coli are tetrameric membrane-bound enzymes that couple succinate/fumarate interconversion with quinone/quinol reduction/oxidation. Both enzymes are capable of binding either ubiquinone or menaquinone, however, they have adopted different quinone binding sites where catalytic reactions with quinones occur. A comparison of the structures of the quinone binding sites in SQR and QFR reveals how the enzymes have adapted in order to accommodate both benzo- and napthoquinones. A combination of structural, computational, and kinetic studies of members of the complex II family of enzymes has revealed that the catalytic quinone adopts different positions in the quinone-binding pocket. These data suggest that movement of the quinone within the quinone-binding pocket is essential for catalysis., (Published by Elsevier B.V.)

We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoA dehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G > T (exon 7) and p.P534L: c.1601 C > T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation.

Mitochondrial succinate-coenzyme Q reductase (complex II) consists of four subunits, SDHA, SDHB, SDHC and SDHD. Heterozygous germline mutations in SDHB, SDHC, SDHD and SDHAF2 [encoding for succinate dehydrogenase (SDH) complex assembly factor 2] cause hereditary paragangliomas and pheochromocytomas. Surprisingly, no genetic link between SDHA and paraganglioma/pheochromocytoma syndrome has ever been established. We identified a heterozygous germline SDHA mutation, p.Arg589Trp, in a woman suffering from catecholamine-secreting abdominal paraganglioma. The functionality of the SDHA mutant was assessed by studying SDHA, SDHB, HIF-1alpha and CD34 protein expression using immunohistochemistry and by examining the effect of the mutation in a yeast model. Microarray analyses were performed to study gene expression involved in energy metabolism and hypoxic pathways. We also investigated 202 paragangliomas or pheochromocytomas for loss of heterozygosity (LOH) at the SDHA, SDHB, SDHC and SDHD loci by BAC array comparative genomic hybridization. In vivo and in vitro functional studies demonstrated that the SDHA mutation causes a loss of SDH enzymatic activity in tumor tissue and in the yeast model. Immunohistochemistry and transcriptome analyses established that the SDHA mutation causes pseudo-hypoxia, which leads to a subsequent increase in angiogenesis, as other SDHx gene mutations. LOH was detected at the SDHA locus in the patient's tumor but was present in only 4.5% of a large series of paragangliomas and pheochromocytomas. The SDHA gene should be added to the list of genes encoding tricarboxylic acid cycle proteins that act as tumor suppressor genes and can now be considered as a new paraganglioma/pheochromocytoma susceptibility gene.