Respiratory Chain Of Plant Mitochondria Research Articles

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

The effects of rotenone on the succinate-driven reduction of matrix nicotinamide nucleotides were investigated in Percoll-purified mitochondria from potato (Solanum tuberosum) tubers. Depending on the presence of ADP or ATP, rotenone caused an increase or a decrease in the level of reduction of the matrix nicotinamide nucleotides. The increase in the reduction induced by rotenone in the presence of ADP was linked to the oxidation of the malate resulting from the oxidation of succinate. Depending on the experimental conditions, malic enzyme (at pH 6.6 or in the presence of added CoA) or malate dehydrogenase (at pH 7.9) were involved in this oxidation. At pH 7.9, the oxaloacetate produced progressively inhibited the succinate dehydrogenase. In the presence of ATP the production of oxaloacetate was stopped, and succinate dehydrogenase was protected from inhibition by oxaloacetate. However, previously accumulated oxaloacetate transitorily decreased the level of the reduction of the NAD+ driven by succinate, by causing the reversal of the malate dehydrogenase reaction. Under these conditions (i.e. presence of ATP), rotenone strongly inhibited the reduction of NAD+ by succinate-driven reverse electron flow. No evidence for an active reverse electron transport through a rotenone-insensitive path could be obtained. The inhibitory effect of rotenone was masked if malate had previously accumulated, owing to the malate-oxidizing enzymes which reduced part or all of the matrix NAD+.

The Cyanide-Insensitive Oxidation of Ascorbate plus NNNN′N′-Tetramethyl-p-phenylenediamine Mixture by Mung-Bean (Phaseolus aureus) Mitochondria

The respiratory chain of plant mitochondria is branched, only the main chain being a conventional cytochrome pathway. The branch, which lacks cytochrome components, arises at the ubiquinone level and transports electrons directly to O2 via unidentified carriers. This branch is insensitive to cyanide and antimycin A, but can be inhibited by substituted hydroxyamic acids (Bendall & Bonner, 1971 ; Schonbaum eta]. , 1971). The proportion of the two oxidative pathways varies considerably, both with species and with age of tissue. Mitochondria from the spadices of Aroid plants are typically insensitive to cyanide, whereas those from plants such as the cauliflower are almost completely susceptible to cyanide inhibition. The cyanide-insensitive pathway by-passes phosphorylation sites 2 and 3 and lacks the normal tight coupling of respiration and phosphorylation. Evidence has been presented (Bonner & Bendall, 1968; Dawson & Gains, 1969) showing that energy conservation can be associated with this pathway and under certain conditions that ADP can be phosphorylated (Wilson, 1970). Only a single phosphorylation site appears to be involved in energy conservation in the cyanide-insensitive oxidase. This site differs from those of the main respiratory chain. Storey (1971) has pointed out that these observations may not apply to those species, like the mung bean and the skunk cabbage, that lack cytochrome b7. The O2 uptake that occurs when mitochondria from many plants are supplied with electrons by ascorbate + NNNN-tetramethyl-p-phenylenediamine mixture is usually totally sensitive to KCN at concentrations characteristic of the terminal oxidase. Our results demonstrate that fresh tightly coupled mung-bean mitochondria still show some O2 uptake with ascorbate+NNN’N’-tetramethyl-pphenylenediamine as substrate in the presence of 0.1 3 ~ M K C N . This concentration is 10 times that required to inhibit the terminal oxidase (lkuma & Bonner, 1967). Spectrophotometric observations and the use of electron-transport inhibitors show that the cyanide-insensitive oxidation of axorbate+ NNNN‘-tetramethyl-p-phenylenediamine involves reversed electron transport through phosphorylation site 2 and the subsequent uptake of 0 2 by the cyanide-insensitive oxidase. Energy-transfer inhibitors show that, like other energy-linked functions of plant mitochondria (Wilson & Bonner, 1970), this cyanide-insensitive oxidation is energized by ATP or by ‘high-energy’ intermediates. The latter can be generated by the passage of electrons to O2 via the cyanideinsensitive oxidase system. The oxidation is analogous to a syphon, and shows that energy conservation is associated with the cyanide-insensitive oxidase of mitochondria from mung beans, a species that lacks cytochrome b,.

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b 557, b 553, c 549 (corresponding to c 1 in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis. 2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c 549, b 557, and b 553. The intramitochondrial redox potential (IMP h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition. 3. 3. This result shows that the slow reduction of cytochrome b 557 under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b 553 and +235 mV for cytochrome c 549. Cytochrome b 557 is placed on the low potential side of coupling site II and transfers electrons to cytochrome c 549 via the coupling site. 4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP h was calculated. When replotted as IMP h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E m7.2 = + 70 mV. The minor component has a midpoint potential E m7.2 = − 12 mV; its nature is unknown.

The Respiratory Chain of Plant Mitochondria. III. Oxidation Rates of the Cytochromes c and b in Mung Bean Mitochondria Reduced With Succinate

Oxidation rates of the 2 cytochromes c and the 3 cytochromes b in mitochondria from the hypocotyls of etiolated mung beans (Phaseolus aureus) have been measured with a rapid mixing flow apparatus coupled to a dual wavelength spectrophotometer. The mitochondria were depleted with uncoupler and ADP prior to starting the experiments to minimize the complications caused by energy-linked processes on electron transport. The half-time for oxidation and absorbance change at a given wavelength for the rapidly oxidized cytochromes was plotted as a function of wavelength, with 570 mmu as reference, to yield a spectrum in which absorbance maxima and half-times were correlated. The 2 cytochrome c components have oxidation half-times of 2 milliseconds for c(549) and 3 milliseconds for c(547). (The subscripts refer to the difference absorbance maxima observed for these cytochromes in reduced-minus-oxidized difference spectra obtained at -196 degrees . The maxima observed at room temperature are 550 mmu for c(547) and 552 mmu for c(549).) Cytochrome b(557) (room temperature maximum 560 mmu) was oxidized with a half-time of 8 milliseconds, while b(553) (room temperature maximum 556 mmu) was oxidized relatively slowly with a half-time of 0.5 sec. Cytochrome b(562) (room temperature maximum 565 mmu) was oxidized with half-times in the range 15 to 35 milliseconds, depending on mitochondrial preparation. It was shown that b(562) is only partially reduced by succinate in anaerobic mitochondria previously depleted with uncoupler and ADP; this cytochrome can be fully reduced by succinate in coupled mitochondria under conditions where endogenous pyridine nucleotide is also reduced. Both b(553) and b(557) in depleted mitochondria are reduced in anaerobiosis by succinate. With ascorbate plus N,N'-tetramethylphenylenediamine as substrate, b(553) is only partially reduced in anaerobiosis, while b(557) is completely reduced in depleted mitochondria. This observed difference in ease of reducibility provides another method for differentiating the 3 cytochromes b of mitochondria isolated from plant tissues.

Effects of α-Lipoic Acid on the Respiratory Chain of Plant Mitochondria

THIS communication describes the effects of oxidized α-lipoate (6:8-dithio-n-octanoate) on the respiration and oxidative phosphorylation of mitochondria isolated from red beetroot (Beta vulgaris L.)1, Both spectrophotometric (Table 1) and manometric (Table 2) results showed that added lipoate (0.1 mM) caused substantial inhibition of the oxidation of reduced coenzyme I and of coenzyme I-linked substrates with oxygen, ferricytochrome c and 2:6-dichlorophenol indophenol as oxidants. However, the oxidation of succinate was not inhibited by lipoate at 0.1 mM concentration (Table 1). Lipoate caused a marked stimulation (about 100 per cent) of the succinate–cytochrome c reductase activity of mitochondria modified by freezing and thawing or by exposure to hypotonic conditions.