The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

The Respiratory Chain of Plant Mitochondria. I. Electron Transport Between Succinate and Oxygen in Skunk Cabbage Mitochondria

The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and the cytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobic with succinate have been measured spectrophotometrically and fluorimetrically in the absence of respiratory inhibitor and in the presence of cyanide or antimycin A. No component identifiable by these means was oxidized rapidly enough in the presence of one or the other inhibitor to qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoprotein and ubiquinone obtained by injecting 12 mum oxygen into the anaerobic mitochondrial suspension were kinetically indistinguishable in the presence of cyanide or antimycin A, implying that these 2 components are part of a respiratory pathway between succinate and oxygen which does not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. The cytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and 0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence of cyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex shows an induction period of 1 sec and a half-time of 3.5 sec. A split respiratory chain with 2 terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinone is proposed for these mitochondria.     

The Respiratory Chain of Plant Mitochondria: XVI. Interaction of Cytochrome b(562) with the Respiratory Chain of Coupled and Uncoupled Mung Bean Mitochondria: Evidence for Its Exclusion from the Main Sequence of the Chain

Cytochromes b₅₅₃, b₅₅₇, and b₅₆₂ of mung bean (Phaseolus aureus) mitochondria become partially reduced with endogenous substrate on addition of antimycin A to the aerobic mitochondrial suspension. Addition of ATP causes partial reoxidation of the three cytochromes. This partial oxidation by ATP is inhibited by oligomycin and reversed by uncoupler. Ubiquinone does not appear to act as electron acceptor for the oxidation reaction, but a nonfluorescent flavoprotein, or possibly ironsulfur protein, component does appear to act as acceptor. This is consistent with reverse electron transport driven by ATP across the first site of energy conservation of the respiratory chain. Endogenous pyridine nucleotide and the fluorescent flavoprotein with E_m7.2 = −155mv (midpoint potential at pH 7.2, referred to normal hydrogen electrode) in uncoupled mitochondria become reduced in anaerobiosis attained by oxidation of succinate in the absence of respiratory inhibitors of the cytochrome chain, provided that Pi and ATP are present. Under these same conditions, cytochrome b₅₅₇ is completely reduced but cytochrome b₅₆₂ remains nearly completely oxidized. There is no equilibration across the first site of energy conservation between the carriers on the low potential side and cytochrome b₅₆₂ with E_m7.2 = −77mv on the high potential side. It is concluded that cytochrome b₅₆₂ is not a part of the main sequence of electron transport carriers of the mitochondrial respiratory chain of plants; it can participate in redox reactions with the respiratory chain in coupled mitochondria but not in uncoupled mitochondria unless antimycin A is present.     

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp_ha and Fp_hf and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp_hf is reduced more slowly than Fp_ha by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp_m. The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp_1f. In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.     

Respiratory Chain of Plant Mitochondria: XVIII. Point of Interaction of the Alternate Oxidase with the Respiratory Chain

Oxidation of the respiratory chain carriers of anaerobic, CO-saturated skunk cabbage (Symplocarpus foetidus) mitochondria, by means of an O₂ pulse, proceeds primarily through the cyanide-insensitive alternate oxidase, since the oxidation of cytochromes a and a₃ takes place with a half-time of 3 seconds, corresponding to the rate of dissociation of CO from reduced cytochrome a₃. Ubiquinone and part of the flavoprotein are oxidized within 1 second under these conditions, and this rapid rate of oxidation is strongly inhibited by m-chlorobenzhydroxamic acid (mCLAM), a specific inhibitor of the alternate oxidase of plant mitochondria. The rate of ubiquinone oxidation under these conditions in white potato (Solanum tuberosum) mitochondria, which have no alternate oxidase, is the same as that in skunk cabbage mitochondria treated with mCLAM. Ubiquinone, thus identified as the carrier common to both the cytochrome and alternate oxidase pathways, is linked to the alternate oxidase by a flavoprotein of midpoint potential 50 millivolts more negative with which it is in equilibrium. This arrangement provides a switch for diverting electron transport primarily through the cytochrome pathway under state 3 conditions and primarily through the alternate oxidase pathway under state 4 conditions.     

The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain

The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa₃ and c₅₄₇ have unaltered oxidation half-times, designated t_{½ on}, as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t_{½ off}, decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t_{½ on}, for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t_{½ off} decreases only 30% while the extent of oxidation decreases 50%. The values of t_{½ on} and t_{½ off} and the extent of oxidation of cytochrome b₅₅₃ and flavoprotein Fp_ha were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P_i) and ATP are present. No reduction occurs in the absence of P_i, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.     

The Respiratory Chain of Chlorella protothecoides: II. Isolation and Characterization of Mitochondria

Mitochondria were isolated from glucose-bleached Chlorella protothecoides Krüger, Indiana strain 25. These mitochondria oxidized succinate, NADH, and l-malate at high rates. Oxygen uptake with these substrates was partially inhibited by 1 mmm-chlorobenzhydroxamic acid (mCLAM). Respiratory control was seen with succinate as substrate in the presence of mCLAM. The apparent Km for succinate was determined to be 0.83 mm. Chlorella mitochondria catalyzed the oxidation of reduced horse heart cytochrome c.     

The Respiratory Chain of Plant Mitochondria: V. Reaction of Reduced Cytochromes a and a3 in Mung Bean Mitochondria with Oxygen in the Presence of Cyanide

The half-times of oxidation by oxygen pulses of reduced cytochromes a and a(3) in mung bean mitochondria made anaerobic with succinate have been measured by means of a rapid mixing flow apparatus coupled to a dual wave length spectrophotometer in the presence and absence of cyanide. The absorbance changes at 438 to 455 millimicrons and 603 to 620 millimicrons are suitable for recording the time course of cytochrome a oxidation; the half-time is 2.0 milliseconds at 24 Celsius. This half-time does not change over the range 0 to 300 mum KCN, but the fraction of cytochrome a oxidized falls to a limiting value of 0.3 at the higher cyanide concentrations. The absorbance changes at 445 to 455 millimicrons record the time course of both cytochrome a and cytochrome a(3) oxidation; the former contributes 60% of the absorbance change and the latter 40%. The half-time for a(3) oxidation is calculated as 0.9 milliseconds at 24 Celsius. This half-time increases slightly to 1.3 milliseconds at 300 mum KCN. Reduced cytochrome a(3), whether uncomplexed or complexed with cyanide, becomes fully oxidized. The dissociation constant for the reduced cytochrome a(3)-cyanide complex is estimated to be 30 mum, whereas that for the oxidized a(3)-cyanide complex which inhibits electron transport is estimated to be 2 mum. This suggests two different binding sites for cyanide on the reduced and oxidized forms of cytochrome a(3). The fact that a limiting fraction of reduced cytochrome a can be oxidized at high cyanide concentrations implies that there is no interference by cyanide with electron transport from a to a(3), if cyanide remains bound to the site it occupies on reduced a(3) after this carrier becomes oxidized on reaction with molecular oxygen. Rearrangement of cyanide from this noninhibitory site to the inhibitory site occurs rapidly enough to compete with cytochrome a oxidation. The half-time for the rearrangement is calculated to be 0.9 milliseconds.     

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.     

Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 °C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2±24.7 μl h^?1g^?1) and post-thermogenic (50.5±30.4 μl h^?1g^?1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Specific Inhibition of the Cyanide-insensitive Respiratory Pathway in Plant Mitochondria by Hydroxamic Acids

Hydroxamic acids, R-CONHOH, are inhibitors specific to the respiratory pathway through the alternate, cyanide-insensitive terminal oxidase of plant mitochondria. The nature of the R group in these compounds affects the concentration at which the hydroxamic acids are effective, but it appears that all hydroxamic acids inhibit if high enough concentrations are used. The benzhydroxamic acids are effective at relatively low concentrations; of these, the most effective are m-chlorobenzhydroxamic acid and m-iodobenzhydroxamic acid. The concentrations required for half-maximal inhibition of the alternate oxidase pathway in mung bean (Phaseolus aureus) mitochondria are 0.03 mm for m-chlorobenzhydroxamic acid and 0.02 mm for m-iodobenzhydroxamic acid. With skunk cabbage (Symplocarpus foetidus) mitochondria, the required concentrations are 0.16 for m-chlorobenzhydroxamic acid and 0.05 for m-iodobenzhydroxamic acid. At concentrations which inhibit completely the alternate oxidase pathway, these two compounds have no discernible effect on either the respiratory pathway through cytochrome oxidase, or on the energy coupling reactions of these mitochondria. These inhibitors make it possible to isolate the two respiratory pathways and study their mode of action separately. These inhibitors also enhance an electron paramagnetic resonance signal near g = 2 in anaerobic, submitochondrial particles from skunk cabbage, which appears to be specific to the alternate oxidase and thus provides a means for its assay.     

The Influence of Mitochondrial Concentration and Storage on the Respiratory Control of Isolated Plant Mitochondria

The respiration of mitochondria isolated from various plant tissues was studied over a range of mitochondrial concentrations and at various times after isolation. Respiration at 25 C expressed as nanomoles of O₂ per minute per milligram of protein was constant for mitochondrial concentrations higher than some critical amount, usually 0.25 to 1.0 milligram of protein per reaction. Below this concentration the state 3 respiration rate declined and the mitochondria appeared to lose respiratory control. The respiration of isolated mitochondria stored in ice but measured at 25 C generally declined over long time periods although mitochondria from some tissues showed an initial increase. The results indicate that valid comparisons of the respiratory activity of mitochondria isolated from different tissues or from different parts of the same tissue cannot be made at least until the influence of the above factors has been evaluated.     

Oxidation of Glycine via the Respiratory Chain in Mitochondria Prepared from Different Parts of Spinach

Mitochondria were prepared from roots, stalks, leaves, and leaf veins of spinach. The mitochondrial preparations were examined for their ability to oxidize glycine via the respiratory chain. It is shown that the glycine-oxidizing capacity is restricted to photosynthetically active tissue. The activity is present in mitochondria from the green parts of the leaves, but not in mitochondria from roots, stalks, or leaf veins.