Preliminary molecular characterization and crystallization of mitochondrial respiratory complex II from porcine heart

The mitochondrial respiratory complex II, or succinate:ubiquinone oxidoreductase, is an integral membrane protein complex in both the tricarboxylic acid cycle (Krebs cycle) and aerobic respiration. The gene sequences of each complex II subunit were measured by RT-PCR. N-terminal sequencing work was performed to identify the mitochondrial targeting signal peptide of each subunit. Complex II was extracted from porcine heart and purified by the ammonium sulfate precipitation method. The sample was solubilized by 0.5% (w/v) sugar detergent n-decyl-beta-D-maltoside, stabilized by 200 mM sucrose, and crystallized with 5% (w/v) poly(ethylene glycol) 4000. Important factors for the extraction, purification and crystallization of mitochondrial respiratory complex II are discussed.     

Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

On Complex I and Other NADH:Ubiquinone Reductases of Neurospora crassa Mitochondria

The mitochondrial complex I is the first component of the respiratory chain coupling electron transfer from NADH to ubiquinone to proton translocation across the inner membrane of the organelle. The enzyme from the fungus Neurospora crassa is similar to that of other organisms in terms of protein and prosthetic group composition, structure, and function. It contains a high number of polypeptide subunits of dual genetic origin. Most of its subunits were cloned, including those binding redox groups. Extensive gene disruption experiments were conducted, revealing many aspects of the structure, function, and biogenesis of complex I. Complex I is essential for the sexual phase of the life cycle of N. crassa, but not for the asexual stage. In addition to complex I, the fungal mitochondria contain at least three nonproton-pumping alternative NAD(P)H dehydrogenases feeding electrons to the respiratory chain from either matrix or cytosolic substrates.     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme

We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 A resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg(297) as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg(297). The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg(297) to be increased by 83 Da and to have the potential of losing 44 Da, consistent with decarboxylation, during fragmentation.     

抑制线粒体复合体II诱导线粒体自噬影响细胞增殖的研究

线粒体复合体II,也被称为琥珀酸脱氢酶,参与线粒体呼吸作用及代谢重编程的调控过程。复合体II由四个亚基构成,其突变与肿瘤的发生密切相关。本论文探讨了复合体II与线粒体自噬调控及细胞增殖之间的关系。本实验采用复合体II的特异性抑制剂TTFA或敲除复合体II的B亚基SDHB使其功能缺失。结果发现,复合体II功能的缺失显著引起线粒体形态的片段化进而发生线粒体自噬,导致线粒体蛋白水平减少,抑制ATP生成,由于线粒体功能受到抑制,细胞葡萄糖消耗及乳酸产生水平增加,并显著抑制细胞的细胞的增殖。综上所述,复合体II功能缺失可能通过调控线粒体自噬而影响细胞增殖,从而在肿瘤发生中起重要作用。     

Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction

The transfer of electrons and protons between membrane-bound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH(2)). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q(1)-site and Q(2)-site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q(2)-site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.     

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.     

Issue Information

Complex II [succinate dehydrogenase (succinate‐ubiquinone oxidoreductase); EC 1.3.5.1; SDH] is the only enzyme shared by both the electron transport chain and the tricarboxylic acid (TCA) cycle in mitochondria. Complex II in plants is considered unusual because of its accessory subunits (SDH5–SDH8), in addition to the catalytic subunits of SDH found in all eukaryotes (SDH1–SDH4). Here, we review compositional and phylogenetic analysis and biochemical dissection studies to both clarify the presence and propose a role for these subunits. We also consider the wider functional and phylogenetic evidence for SDH assembly factors and the reports from plants on the control of SDH1 flavination and SDH1–SDH2 interaction. Plant complex II has been shown to influence stomatal opening, the plant defense response and reactive oxygen species‐dependent stress responses. Signaling molecules such as salicyclic acid (SA) and nitric oxide (NO) are also reported to interact with the ubiquinone (UQ) binding site of SDH, influencing signaling transduction in plants. Future directions for SDH research in plants and the specific roles of its different subunits and assembly factors are suggested, including the potential for reverse electron transport to explain the succinate‐dependent production of reactive oxygen species in plants and new avenues to explore the evolution of plant mitochondrial complex II and its utility.     

Action of respiratory inhibitors on the electron transport system of Escherichia coli]

Bacteria of two strains of Escherichia coli (Q13 and MRE 600) were disintegrated by aluminium oxide. The influence of the respiratory inhibitors RF (a protein from reticulocytes), carboxin, Dexon (fungicides), thenoylftrifluoroacetone (TTFA), rotenone, antimycin A, myristic acid and monolaurin was tested on the succinate oxidase and the NADH oxidase system, respectively, of the membrane preparation obtained in this way as well as on the NADH oxidase activity of the cytosol. Among the inhibitors listed, only TTFA (5mM) inhibited the succinate oxidase system and Dexon (10 miconr), monolaurin (100 micron) and myristic acid (100 micron) inhibited the NADH oxidase system of the membranes. KCN (10 micron) inhibited both NADH oxidase systems. The inhibitory effects by monolaurin and myristic acid were prevent by human serum albumin and were markedly weaker than those on beef heart mitochondrial particles under similar conditions. The results argue for a divergent structure of the iron-sulphur proteins in the dehydrogenase regions of the electron transport system in comparison with animal and plant mitochondria and, moreover, confirm the specificity of RF and carboxin as well as the nature of Dexon as a group reagent on pyridine nucleotide dependent flavin enzymes.     

Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.     

Exploring the inhibitor binding pocket of respiratory complex I

Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.     

One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556

Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms. The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme. The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification. In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity. This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon. The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule. The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Cytopathies involving mitochondrial complex II

Complex II (succinate-ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and contains four nuclear-encoded subunits SdhA, SdhB, SdhC, and SdhD. It functions both as a respiratory chain component and an essential enzyme of the TCA cycle. Electrons derived from succinate can thus be directly transferred to the ubiquinone pool. Major insights into the workings of complex II have been provided by crystal structures of closely related bacterial enzymes, which have also been genetically manipulated to answer questions of structure-function not approachable using the mammalian system. This information, together with that accrued over the years on bovine complex II and by recent advances in understanding in vivo synthesis of the non-heme iron co-factors of the enzyme, is allowing better recognition of improper functioning of human complex II in diseased states. The discussion in this review is thus limited to cytopathies arising because the enzyme itself is defective or depleted by lack of iron-sulfur clusters. There is a clear dichotomy of effects. Enzyme depletion and mutations in SDHA compromise TCA activity and energy production, whereas mutations in SDHB, SDHC, and SDHD induce paraganglioma. SDHC and SDHD are the first tumor suppressor genes of mitochondrial proteins.     

Human mitochondrial complex I assembly: a dynamic and versatile process

One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system, which involves the stepwise combination of >80 subunits and prosthetic groups encoded by both the mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.     

EPR Studies on 6-Benzoylaminopurine and Thenoyltrifluoroacetone Inhibition Sites of Succinate Dehydrogenase in Plant Mitochondria

6-Benzoylaminopurine (BOAP) inhibits succinate oxidation atthe level of complex II (succinate-ubiquinone reductase) inthe respiratory chain of plant mitochondria. In order to identifyits site of action, the effects of BOAP on mitochondrial membranesof potato tubers were studied using electron paramagnetic resonance(EPR), and compared to those of thenoyltrifluoroacetone (TTFA),an inhibitor of complex II. Under aerobic conditions, BOAP inducedno change in the EPR signal of the Fe-S center S-3 of succinatedehydrogenase (SDH), unlike TTFA which decreased the heightand modified the spectrum of the signal. In the presence offerricyanide (FeCN), BOAP weakly enhanced the S-3 signal whereasTTFA changed only its shape but not its height. Under anaerobicconditions, center S-1 was completely reduced in the presenceof BOAP, while in the presence of TTFA center S-1 was less reducedthan in the control, and the center S-3 signal was not differentfrom that under aerobic conditions. Reoxidation of anaerobicpreparations was obtained by O₂ or FeCN addition. Oxygen wasineffective as an oxidant in the presence of either inhibitor,but a partial reoxidation of center S-3 was obtained with FeCNin the presence of BOAP. These observations point to two differentsites for TTFA and BOAP. Both inhibitors act on the O₂ sideof center S-3, BOAP interfering with the environment of theFe-S cluster of S-3, while TTFA would act on the cluster itself. (Received March 29, 1996; Accepted July 9, 1996)     

A threonine on the active site loop controls transition state formation in Escherichia coli respiratory complex II

In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.