Abstract
The mitochondrial succinate dehydrogenase (SDH) is a tetrameric iron-sulfur flavoprotein of the Krebs cycle and of the respiratory chain. A number of mutations in human SDH genes are responsible for the development of paragangliomas, cancers of the head and neck region. The mev-1 mutation in the Caenorhabditis elegans gene encoding the homolog of the SDHC subunit results in premature aging and hypersensitivity to oxidative stress. It also increases the production of superoxide radicals by the enzyme. In this work, we used the yeast succinate dehydrogenase to investigate the molecular and catalytic effects of paraganglioma- and mev-1-like mutations. We mutated Pro-190 of the yeast Sdh2p subunit to Gln (P190Q) and recreated the C. elegans mev-1 mutation by converting Ser-94 in the Sdh3p subunit into a glutamate residue (S94E). The P190Q and S94E mutants have reduced succinate-ubiquinone oxidoreductase activities and are hypersensitive to oxygen and paraquat. Although the mutant enzymes have lower turnover numbers for ubiquinol reduction, larger fractions of the remaining activities are diverted toward superoxide production. The P190Q and S94E mutations are located near the proximal ubiquinone-binding site, suggesting that the superoxide radicals may originate from a ubisemiquinone intermediate formed at this site during the catalytic cycle. We suggest that certain mutations in SDH can make it a significant source of superoxide production in mitochondria, which may contribute directly to disease progression. Our data also challenge the dogma that superoxide production by SDH is a flavin-mediated event rather than a quinone-mediated one.
Highlights
Succinate dehydrogenase (SDH)1 of the Krebs cycle and of the bacterial or the mitochondrial respiratory chain (MRC) is intriguing for its roles in energy generation and in mitochondria-related diseases [1,2,3]
The E. coli succinate dehydrogenase (SDH) crystal structure revealed two membrane subunits with a single heme and a single bound ubiquinone [14]; the E. coli fumarate reductases (FRD) contains a pair of membrane subunits with two menaquinone molecules but no heme [10]; the W. succinogenes FRD has a single larger membrane subunit with two hemes, but quinone molecules were absent from the crystal [11]
Mutations in the SDHB, SDHC, or SDHD genes can result in paraganglioma or pheochromocytoma [16, 17]
Summary
Plasmids, Media, and Culture Conditions—The parental strain JNY131 (ade, leu, his, trp, ura, can100, mgm101, Mata, or Mat␣) was a kind gift of Dr Jodi Nunnari [23]. An SDH2 knockout of JNY131, JG1 (SDH2⌬-1::HIS3), was constructed by replacing the entire SDH2 open reading frame with the HIS3 gene. An SDH3 knockout, JG2 (SDH3⌬-1::TRP1), was constructed by replacing the entire SDH3 open reading frame with the TRP1 gene. Isolation of Mitochondria and Enzyme Assays—YPG-grown stationary phase cells were harvested by centrifugation and lysed in a BeadBeater (BioSpec Products, Inc., Bartlesville, OK) with 0.5-mm acidwashed glass beads using five pulses of 1-min and five of 30-s duration interspersed with 3-min cooling periods. Succinate and glycerol phosphate dehydrogenase assays were performed as described in Ref. 8. Mitochondria were pre-incubated in 20 mM succinate for 15 min at 30 °C immediately prior to analysis. When measuring the effect of succinate concentration on superoxide formation, activation was achieved with 100 mM potassium phosphate [29]. Miscellaneous Methods—Measurements of FAD and protein contents, yeast and E. coli transformation, and recombinant DNA methods have been described in Ref. 30
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