Search

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the α-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four α-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2N and SDH2C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2N plus SDH2C, SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo. We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.

We characterized six novel missense mutations in mitochondrial cytochrome b (C133Y, W142R, S206L, M221K, L282F, and G340E) which impair the respiratory growth of yeast and which have differential effects on the functioning and assembly of the bc1 complex. The mutations have been mapped genetically in exons of the mitochondrial gene coding for apocytochrome b and their nucleotide sequence established. The mutants help to better define the topographical and primary sequence location of the ubiquinol oxidase (center P) and ubiquinone reductase (center N) sites on cytochrome b. Two mutants (C133Y and S206L) resulted in an active assembled complex, with selective disturbances of heme 565 and heme 562, respectively, which is consistent with the assignment of the axial ligands of these hemes; the C133Y mutation induced myxothiazol resistance, whereas the S206L did not modify the antimycin binding site, although perturbing the center N. These two amino acid replacements, along with those described elsewhere (Tron, T., and Lemesle-Meunier, D. (1990) Curr. Genet. 18, 413-419), constitute a novel class of mutants exhibiting appreciable electron transfer activity, despite their impaired ability to grow on respiratory substrates, raising the possibility that these mutants carry alleles which result in "decoupling" of proton translocation from electron transfer. Mutants W142R and M221K had an inactive but well assembled bc1 complex, whereas the G34OE and L282F mutations impaired the assembly of the bc1 complex.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

The electron acceptors in ubihydroquinone-cytochrome c reductase (Complex III) derived from beef heart mitochondria were investigated by low temperature electron paramagnetic resonance spectroscopy at ≤20 K and optical reflectance spectroscopy at 100 K. Three resonances were resolved in the region where the low field lines of low spin ferric heme compounds are usually found. These lines were assigned to cytochromes c1 (gz = 3.33), bk (gz = 3.44), and bt (gz = 3.78) on the basis of correlation with optical spectra during reduction. A broad line typical of the center line (gy) of a low spin ferric heme is seen at g ∼ 2. Other EPR-detectable electron acceptors in Complex III are the iron-sulfur center previously detected by EPR at 77° (J. S. Rieske, D. H. MacLennan, and R. Coleman (1964) Biochem. Biophys. Res. Commun. 15, 338); a species whose signal appears at g = 1.93 on reduction, presumably an iron-sulfur protein, and possibly due to contamination with succinate-ubiquinone reductase (Complex II); two acceptors of unknown structure and function, one with a signal centered at g = 2.01 (oxidized form) and the other with lines at g = 2.04 and 1.99. The signal of the latter is absent in the oxidized state, appears on partial reduction and disappears on complete reduction, indicating that three discrete oxidation states may be involved. The behavior of all of these species was studied during reductive anaerobic titration of Complex III with dithionite and of more integrated preparations, such as DPNH-cytochrome c reductase and particles derived from beef heart mitochondria by sonication, with DPNH or dithionite as reductant. An attempt is made to compare the experimentally determined capacity for electron uptake with the amounts of known electron acceptors in samples of these preparations.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.

1. A procedure is described for separating from preparations of NADH-ubiquinone reductase (Complex I), an NADH dehydrogenase which retains full activity with ferricyanide as acceptor, but which is devoid of rotenone-sensitive NADH-ubiquinone reductase activity. About 50 to 90% of the original NADH-ubiquinone reductase activity is restored by recombination with purified phospholipids in the presence of cholate, and dialysis for at least 10 hours. Reduction of ubiquinone-1 by this preparation is 80 to 90% inhibited by rotenone. 2. After reconstitution with phospholipids, the preparation reacts with ubiquinone-cytochrome c reductase (Complex III) to give rotenone- and antimycin-sensitive NADH-cytochrome c reductase activity. No activity with Complex III is observed with the preparation either before reconstitution or after dialysis without phospholipids. 3. A modification of the separation procedure leads to a preparation of NADH dehydrogenase exhibiting activity with ferricyanide as acceptor. However, after reconstitution with phospholipids, the rates of rotenone-sensitive NADH-ubiquinone reductase obtained are less than 3% of those in Complex I. The properties of the reconstitutively active and inactive NADH dehydrogenase are compared.

A dihydroorotate-oxidizing complex of high molecular weight was purified from respiratory particles of Escherichia coli, strain B. This material was found to be an active dihydroorotate-ubiquinone-30 reductase. The complex lacked cytochrome reductase and oxidase activities but catalyzed the reduction of various artificial electron acceptors, including 2,6-dichloroindophenol, simple 1,4-quinones, and ferricyanide. Molecular oxygen was also an oxidant, although a less efficient one than the artificial electron acceptors. Detergent-dispersed ubiquinone-30 was the most effective oxidizing agent that was tested. This compound was also rapidly reoxidized in a coupled reaction with 2,6-dichloroindophenol in the presence of the complex. The complex contained, in addition to the dihydroorotate-oxidizing enzymes, an active peroxidase, catalase, succinate dehydrogenase, and DPNH oxidase. In crude soluble fractions and in particulate systems of aerobic microorganisms, the oxidation of dihydroorotate is cytochrome-linked. This linkage may occur via non-heme iron proteins and ubiquinone. The oxygen-mediated oxidation of dihydroorotate leads to the quantitative conversion to orotate. Linkage of the reaction with the respiratory chain may represent an important site of respiratory control over pyrimidine biosynthesis.

The ubiquinone reductase activity of a soluble extract of beef heart submitochondrial particles can be separated into two active fractions by chromatography on a hydroxylapatite column. Fraction I was further purified by ammonium sulfate fractionation and Fraction II by chromatography on DEAE-cellulose. Both fractions catalyze the oxidation of NADH by ubiquinone-10, ubiquinone-6, ubiquinone-1, menadione, cytochrome c , and ferricyanide at nearly equal rates. Both contain FMN, nonheme iron, and acid-labile sulfide in the approximate ratio of 1:4:5, and have nearly identical spectra. Sedimentation and electrophoretic analyses indicate that the fractions are better than 95% homogeneous. The molecular weights of the two preparations are very similar (near 90,000), although their electrophoretic mobilities differ. NADH causes anaerobic bleaching of the flavin (450 mμ), but the absorption peak at 550 mμ is not altered. The reductases are inhibited by Amytal and rotenone, the latter showing an unusual biphasic curve with maximal inhibition at a rotenone to enzyme flavin ratio of nearly one. Piericidin A, dicumarol, o -phenanthrolene, or Tiron do not inhibit reductase activity.