The exact function of some of these ligands with regard to succinate dehydrogenase remains unclear; ephrin, for example is suspected to be involved in certain cell signaling pathways in animal development [8]. The exact mechanism for the oxidation of succinate to fumarate has not yet been elucidated.
In the proposed E1cb mechanism, the deprotonation leads to the formation of an enolate intermediate; FAD then removes the hydride, as shown in Image 2 [9]. Ubiquinone is initially oriented in the active site such that the O1 carbonyl group interacts with Tyr83 of SdhD via hydrogen bonding. The electrons removed during the oxidation reaction are conveyed through the iron-sulfur clusters to 3Fe-4S; their presence on that cluster stimulates the substrate to reorient so that a second hydrogen bond between the of SdhC may form.
The electrons are transferred to the substrate individually, with the addition of the first producing a radical semiquinone and the second completing the reduction to ubiquinol.
It catalyzes succinate oxidation in the citric acid cycle and transfers the electrons to quinones in the membrane, thus constituting a part of the aerobic respiratory chain known as complex II. Complex 2 is a parallel electron transport pathway to complex 1, but unlike complex 1, no protons are transported to the intermembrane space in this pathway.
Therefore, the pathway through complex 2 contributes less energy to the overall electron transport chain process. However, patients with such SDH defect present typical Leigh syndrome and thus do not clinically differ from patients with other RC complex defects. Contrasting with the high residual activity measured in patients with mutations in the SDHA gene, SDH activity is barely detectable in tumour tissues from patients with hereditary paraganglioma.
As discussed above, the lack of SDH activity will not only cause decreased ATP production—a condition that results from severe CI or CIV defect as well—but will also deprive the RC of the only dehydrogenase activity able to maintain a high reduction status of the UQ pool.
This may in turn cause the loss of the anti-oxidant capacity of the respiratory chain and possibly an oxidative stress that is known to readily trigger tumour formation.
Observations made with the worm Caenorhabditis elegans also support the idea that the SDH plays a specific role in the handling of oxygen by mitochondria. Strangely enough, a specific SDH mutation—rather than a whole class of respiratory chain mutants—was identified in an oxygen-hypersensitivite worm. Then, being absent, complex II can be disregarded as a source of additional superoxide production. Thus, we propose that the superoxide overproduction, admittedly leading to tumour formation in human and hypersensitivity to oxygen in the mutant worm, should be ascribed to the decreased ability of the SDH to adequately reduce the UQ pool, a necessary condition to resist oxidative stress.
Studying SDH-related human diseases therefore suggests that the enzyme not only plays a central role in the Krebs cycle and the respiratory chain, but also differs from other mitochondrial dehydrogenases thanks to its unique redox properties. In partnership with ubiquinone, SDH would represent a crucial antioxidant enzyme in the mitochondria controlling superoxides scavenging activity of the RC.
It is therefore perhaps not so surprising that a wide spectrum of human diseases echoes the mutations in this multi-functional enzyme. Tzagoloff A.
Mitochondria New York, Plenum Press pp 1— Google Scholar. Article Google Scholar. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency Nature Genet 11 : — Compound heterozygous mutations in the flavoprotein gene of the respiratory chain complex II in a patient with Leigh syndrome Hum Genet : — Niemann S, Muller U.
Gene mutations in the succinate dehydrogenase subunit sdhb cause susceptibility to familial pheochromocytoma and to familial paraganglioma Am J Hum Genet 69 : 49— Gutman M. Modulation of mitochondrial succinate dehydrogenase activity, mechanism and function Mol Cell Biochem 20 : 41— Human cultured skin fibroblasts survive profound inherited ubiquinone depletion Free Rad Res 35 : 11— The consequences of a mild respiratory chain deficiency on substrate competitive oxidation in human mitochondria Biochem Biophys Res Commun : — Rustin P, Lance C.
Succinate-driven reverse electron transport in the respiratory chain of plant mitochondria. The effects of rotenone and adenylates in relation to malate and oxaloacetate metabolism Biochem J : — Redox control of beta-oxidation in rat liver mitochondria Eur J Biochem : — Among them are: blue tetrazolium chloride BT , 2,3,5-triphenyl tetrazolium chloride TTC , 3- 4,5-dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT , 5-cyano-2,3-ditolyl tetrazolium chloride CTC , 2,3-bis 2-methoxynitrosulphophenyl [ phenylamino carbonyl]-2H-tetrazolium hydroxide XTT , 4-[3- 4-idophenyl 4-nitrophenyl -2Htetrazolio]-1,3-benzene disulfonate WST1 , 2- p-iodophenyl -3 p-nitrophenyl phenyltetrazolium chloride INT or 2,2'-dibenzothiazolyl-5,5'-[4-di 2-sulfoethyl carbamoylphenyl]-3,3'- 3,3'-dimethoxy-4,4' biphenyl ditetrazolium, disodium salt WST-5 [ 19 , 22 , 23 ].
In the case of enzymatic reaction conducted in situ the plasma membrane forms a barrier with low degree of penetration. Therefore, cell permeabilization, e. According to the results obtained by Berlowska et al.
After digitonin treatment, the visible formazan crystals were observed inside the yeast cells, but not outside them Figures 8 A, B.
The formazan products are water-insoluble, but readily diffuses out of yeast cells after solubilization in DMSO. Linear correlation was observed in the concentration range of yeast cells from 7 to 8 per sample. For yeast cell concentrations below 7 per sample the formazan color intensity signals were too low to detect with good precision.
The results obtained for SDH activity were in good agreement. Yeast cells after reaction with blue tetrazolium chloride BT. A — without permeabilization; B — with permeabilization by 0. Images of light microscopy. Yeast cells after reaction with 2,3,5-triphenyl tetrazolium chloride TTC. Images of fluorescence microscopy. Yeast cell after reaction with 2,3,5-triphenyl tetrazolium chloride TTC.
Images of scanning microscopy. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of tested yeast strains [ 19 , 23 ]. Saccharomyces cerevisiae is a simple eukaryotic organism, with a complete genome sequence.
Many genetic tools that have been created during these years, including the complete collection of gene deletions and a considerable number of mechanisms and pathways existing in higher eukaryotes was first studied and described in yeast. The study of mitochondrial functions and dysfunction is of special interest in yeast because it is in this organism that mitochondrial genetics and recombination have been discovered and that nucleomitochondrial interactions have been studied in-depth.
There are also specific reasons for choosing S. This organism is petite-positive, which can successfully grow in the absence of oxygen. Therefore it can lose its mitochondrial genome provided it is supplied with a substrate for fermentation.
Consequently, all mutations of the mitochondrial genome can be studied without cell lethality. It is genetically easy to transfer mitochondria from one nuclear genetic background to another via karyogamy.
Additionally, mitochondria can be transformed making in vitro mutation analysis possible. The richness and ease of yeast molecular genetics opens big opportunities, and even the major difference existing between human and yeast mitochondrial genomes, i.
To review mitochondrial diseases may be a very difficult task because the definition might include different kinds of metabolic disorders or degenerative syndromes [ 24 ]. Moreover, some important aspects have been extensively reviewed and the reader might refer to very good recent articles by DiMauro and Garone [ 25 ] for historical aspects, by Wallace et al.
The previous review by Schwimmer et al. SDH in yeast and human are very similar. In the last ten years, deficiencies in TCA cycle enzymes have been shown to cause a wide spectrum of human diseases. For instance, mutation in the gene encoding fumarase is a rare cause of encephalomyopathy and a far more common cause of leiomyomas of the skin and uterus and of renal cancer Table 1. The TCA path dysfunction may also result from concurrent impairments in several steps of the cycle.
The ratios between TCA enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand. Consequently, in addition to the determination of residual absolute activities, estimation of ratios between enzyme activities is an effective means of detecting partial but potentially harmful deficiencies.
When used to assess respiratory chain activities, this approach enabled the identification of several gene mutations, even in patients with partial respiratory chain deficiencies. At present, TCA enzyme activities are measured using a series of independent.
Primary deficiencies in TCA cycle enzymes in humans [ 22 ]. The limited set of assays allowing both measurement of all TCA enzyme activities and detection of abnormalities in enzyme activity ratios were developed. These assays were used successfully to detect severe and partial isolated deficiencies in several TCA enzymes. The reduction of DCPIP was measured using two wavelengths nm and nm with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate.
The second assay measured -ketoglutarate dehydrogenase, aconitase, and isocitrate dehydrogenase activities. Hence, SDH 'inactivation' induces abnormal stimulation of the hypoxia-angiogenesis pathway. When complex II is absent, it can be disregarded as a source of additional superoxide production.
Thus, the superoxide overproduction would lead to tumour formation that should be ascribed to the decreased ability of the SDH to adequately reduce the Q pool, a necessary condition to resist oxidative stress [ 8 ]. Ubiquinone, beside its function in the respiratory chain as an electron carrier mediating electron transfer between the various dehydrogenases and the cytochrome path, is working as a powerful antioxidant in biological membranes [ 13 ].
It is possibly for this exact reason in much larger amounts compared to other electron carriers of the respiratory chain, including the sum of the dehydrogenases. When it is defective, the respiratory chain can produce an abnormal amount of superoxides involving additional respiratory chain components such as flavin radicals of complex I.
Delivering electrons for the full reduction of Q to QH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria. Therefore, the SDH is a key enzyme to control Q pool redox poise under these conditions, due to its unique redox properties [ 8 ]. Iron-sulfur Fe-S proteins facilitate multiple functions, including redox activity, enzymatic function, and maintenance of structural integrity.
More than 20 proteins are involved in the biosynthesis of iron-sulfur clusters in eukaryotes. Defective Fe-S cluster synthesis not only affects activities of many iron-sulfur enzymes, such as aconitase and succinate dehydrogenase, but also alters the regulation of cellular iron homeostasis, causing both mitochondrial iron overload and cytosolic iron deficiency. Fe-S cluster biogenesis takes place essentially in every tissue of humans, and products of human disease genes have important roles in the process [ 40 ].
Succinate is an oxygen sensor in the cell and can help turn on specific pathways that stimulate cells to grow in a low-oxygen environment hypoxia. In particular, succinate stabilizes a protein called hypoxia-inducible factor HIF by preventing a reaction that would allow HIF to be broken down. HIF controls several important genes involved in cell division and the formation of new blood vessels in a hypoxic environment. However, a single mutation in the SDHA gene increases the risk that an individual will develop the condition, and additional mutation that deletes the normal copy of the gene is needed to cause tumor formation.
This second mutation, called a somatic mutation, is acquired during a person's lifetime and is present only in tumor cells. As a result, there is little or no SDH enzyme activity.
Because the mutated SDH enzyme cannot convert succinate to fumarate, succinate accumulates in the cell. The excess succinate abnormally stabilizes HIF, which also builds up in cells. Excess HIF stimulates cells to divide and triggers the production of blood vessels when they are not needed. Rapid and uncontrolled cell division, along with the formation of new blood vessels, can lead to the development of tumors.
Mutations in the SDHA gene were identified in a small number of people with Leigh syndrome, a progressive brain disorder that typically appears in infancy or early childhood.
Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder. The one child died suddenly at the age of five months from a severe deterioration of neuromuscular, cardiac, and hepatic symptoms after an intermittent infection.
These genetic changes disrupt the activity of the SDH enzyme, impairing the ability of mitochondria to produce energy. This suggested a role of additional nuclear genes involved in synthesis, assembly, or maintenance of SDH.
It is not known, however, how mutations in the SDHA gene are related to the specific features of Leigh syndrome [ 41 , 42 ]. Two plausible hypotheses have been proposed to explain the peculiar linkage between disruption of electron flow through mitochondrial complex II and tumorigenesis in neuroendocrine cells. Although certain mutations in these genes result in ROS production in Saccharomyces cerevisiae and mammalian cell lines, it is not clear that ROS accumulate to levels that are mutagenic.
ROS model [ 18 ]. Succinate accumulation model [ 18 ]. Excess succinate is shuttled from the mitochondrial matrix to the cytoplasm, where it inhibits any of several aKG-dependent enzymes E that regulate levels or activities of important regulatory proteins black box.
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