Kinetics of ubiquinone reduction by the resolved succinate: Ubiquinone reductase

A significant lag in the thenoyltrifluoroacetone (TTFA)-sensitive succinate: ubiquinone reductase activity was observed when a ubiquinone-deficient resolved preparation of the enzyme was assayed in the presence of exogenous ubiquinone-2 (Q2) and 2,6-dichlorophenolindophenol. No such lag was seen when the free radical of N,N,N′,N′-tetramethyl-p-phenylenediamine (Wurster's Blue) was used as the terminal electron acceptor, or when the reduction of Q2 was directly measured. The apparent Km value for exogenous Q2 was determined in the Q2-mediated TTFA-sensitive succinate: Wurster's Blue reductase reaction. When the enzyme activity was measured directly by monitoring Q2 reduction without terminal acceptors, the time course of the reaction deviated from zero-order kinetics at Q2 concentrations which were much higher than those expected from the KQ2m value determined in the presence of Wurster's Blue. The time course of Q2 reduction fits a curve describing a competitive interrelationship between oxidized and reduced Q2 at the specific binding site. The data obtained are in agreement with the Q-pool behavior of ubiquinone in mitochondrial membranes and suggest that the rate of ubiquinone reduction by succinate is dependent on the ratio.     

Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b.     

Quantities of ubiquinone bound to proteins in beef heart mitochondria

Ubiquinone-binding proteins were isolated and purified from heavy beef heart mitochondria. 35% of the total ubiquinone in the mitochondria was associated with the purified proteins. About 83% of the associated ubiquinone could be released from the proteins by proteolytic treatment showing that at least 29% (0.87 nmol/mg) of the total ubiquinone (3.0 nmol ubiquinone/mg) in the mitochondria is in the bound form. The purified ubiquinone-binding proteins were resolved into 5 polypeptides with the molecular weights of 17.4, 12.9, 12.6, 9.8 and 8.6 kD on sodium dodecyl sulfate-gel electrophoresis.     

Three functionally different cytochrome b redox centres in pigeon heart mitochondria.

Three functionally different cytochrome b redox centres, apparently of high metabolic activity, were detected in intact pigeon heart mitochondria; cytochrome b(1), b(m) and b(h), with maxima of absorption at 556.6 (State 5), 560.6, and 564.5 nm, respectively (alpha-bands, 77K). 2. Cytochrome (b(l) was reduced in the presence of either antimycin or HQNO (2-heptyl-4-hydroxyquinoline N-oxide). The absorption maximum was shifted by dithionite, cyanide, NNN'N'-tetramethyl-p-phenylenediamine + ascorbate, HQNO and antimycin. The spectra obtained on simultaneous or successive addition of HQNO and antimycin favoured the assumption of a common binding site for the two inhibitors. 3. Cytochrome b(m) was reduced in the presence of HQNO, but not in the presence of antimycin. No shifts of absorption maximum was observed. 4. Cytochrome b(h) was reduced in the presence of antimycin. HQNO was unable to cause reduction of this cytochrome by endogenous substrates. The absorption maximum was shifted to lower wavelength by organic solvents. It was inseparable from that of cytochrome b(m) in the presence of 0.4% ethanol. 5. The pattern of reduction in the presence of HQNO or antimycin demonstrates the functional difference of the three redox centres and appears incompatible wih a linear respiratory chain.     

Generation of superoxide-radical by the NADH: Ubiquinone oxidoreductase of heart mitochondria

Besides major NADH-, succinate-, and other substrate oxidase reactions resulting in four-electron reduction of oxygen to water, the mitochondrial respiratory chain catalyzes one-electron reduction of oxygen to superoxide radical followed by formation of hydrogen peroxide. In this paper the superoxide generation by Complex I in tightly coupled bovine heart submitochondrial particles is quantitatively characterized.The rate of superoxide formation during -controlled respiration with succinate depends linearly on oxygen concentration and contributes approximately 0.4% of the overall oxidase activity at saturating (0.25 mM) oxygen. The major part of one-electron oxygen reduction during succinate oxidation (80%) proceeds via Complex I at the expense of its -dependent reduction (reverse electron transfer). At saturating NADH the rate of formation is substantially smaller than that with succinate as the substrate. In contrast to NADH oxidase,the rate-substrate concentration dependence for the superoxide production shows a maximum at low (50 µM)concentrations of NADH. NAD⁺ and NADH inhibit the succinate-supported superoxide generation. Deactivation of Complex I results in almost complete loss of its NADH-ubiquinone reductase activity and in increase in NADH-dependent superoxide generation. A model is proposed according to which complex I has two redox active nucleotide binding sites.One site (F) serves as an entry for the NADH oxidation and the other one (R) serves as an exit during either the succinate-supported NAD⁺ reduction or superoxide generation or NADH-ferricyanide reductase reaction.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 150–159.Original Russian Text Copyright © 2005 by Vinogradov, Grivennikova.This revised version was published online in April 2005 with corrections to the post codes.     

Possible existence of an intermediate pool of ubiquinone in rat heart mitochondria

1. The existence of an intermediate pool of ubiquinone in intact mitochondria of rat heart was investigated. 2. The incorporation of [3H-methyl]S-adenosylmethionine into ubiquinone-9 was not influenced by the co-synthesis of the intermediate, 3-nonaprenyl-4-hydroxybenzoate. 3. In the intermediate-depleted mitochondria, the synthetic rate of the intermediate, 3-nonaprenyl-4-hydroxybenzoate was similar to that of ubiquinone. 4. The possible existence of 3-nonaprenyl-4-hydroxybenzoate as a metabolic pool under physiological condition is discussed.     

Effect of ubiquinone extraction on ubiquinol-1 oxidase activity in beef heart mitochondria

Extraction of endogenous ubiquinone with different methods does not influence ubiquinol oxidase activity in lyophilized mitochondria in terms ofK _M, although a decrease ofV _max is sometimes observed. Experiments with submitochondrial particles from a UQ-deficient mutant ofS. cerevisiae confirm the results with UQ-depleted mitochondria and support the idea that endogenous ubiquinone is not required for the oxidation of exogenous ubiquinols by complex III.     

Ubiquinone biosynthesis by mitochondria, sonicated mitochondria, and mitoplasts of rat liver.

Ubiquinone was biosynthesized when rat liver mitochondria were incubated with S-adenosyl-L-methionine, solanesyl diphosphate, and [U-14C]p-hydroxybenzoate. The intermediates of ubiquinone biosynthesis but not ubiquinone were accumulated in mitochondria incubated without S-adenosyl-L-methionine and the accumulated intermediates were converted to ubiquinone by the addition of the methyl group donor and an excess of cold p-hydroxybenzoate. No solaneylated compounds except nonaprenyl p-hydroxybenzoate were found in sonicated mitochondria, while the biosynthesis of ubiquinone was observed in the sonicated preparation of mitochondria in which the intermediates accumulated. The results indicate that the initial decarboxylation reaction is completely abolished and the subsequent reactions of hydroxylation and methylation are not completely inhibited by the sonication treatment and therefore the decarboxylation reaction is the next step after nonaprenylation of p-hydroxybenzoate. Mitoplasts could biosynthesize ubiquinone with activity comparable to that of intact mitochondria, suggesting that components of the outer membrane and the intermembranous space of mitochondria are not involved in ubiquinone biosynthesis.     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Essentiality of ubiquinone for choline oxidation in rat liver mitochondria.

Rat liver mitochondria treated extensively with n-pentane are incapable of oxidizing choline. Choline oxidation is more sensitive than is succinate oxidation to serial n-pentane extraction of mitochondria. The ability to oxidize choline is restored by the addition of ubiquinone-2 or ubiquinone-10 to the oxidase assay medium.     

A role of ubiquinone in energy conservation in mitochondria

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Two distinct pools of membrane phosphatidylglycerol in Bacillus megaterium.

The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by [32P]phosphate incorporation and chase experiments. One pool (PGt) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PGs) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. [32P]phosphate- and [2-3H]glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PGt and 22 mol% PGs), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of [32P]phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35 degrees C, incorporation of [32P]phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerol expanded rapidly after cerulenin addition at 20 degrees C, but not at 35 degrees C. Moreover, incorporation of [32P]phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 35 degrees C.