The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

The mechanism of transmembrane delta muH+ generation in mitochondria by cytochrome c oxidase.

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Electron Transport and Oxidative Phosphorylation in Arum Spadix Mitochondria

Arum spadix mitochondria exhibited a rapid cyanide-resistantoxygen uptake when oxidizing malate, NADH₂ or succinate, anda slower, cyanide-sensitive oxygen uptake when oxidizing ascorbate+tetramethylphenylenediamine(TMPD). Cytochrome oxidase does not therefore appear to functionas the terminal oxidase in the presence of cyanide, and therather low cytochrome c oxidase activity obtained using ascorbate+TMPDmay exclude it from possessing a major role even in the absenceof cyanide. ATP synthesis has been shown to accompany substrateoxidation. In the presence of antimycin A the P: O ratio accompanyingmalate oxidation was reduced by half, while phosphorylationaccompanying NADH₂ or succinate oxidation was almost completelyabolished. It is proposed that electrons from exogenous NADH₂enter the electron transport chain at a site after that whereendogenous NADH2 donates electrons and that electrons from exogenousNADH₂ are not coupled to ATP synthesis at site 1. The cyanide-resistant,non-phosphorylating electron-transport pathway may functionin the absence of cyanide and account for the low efficiencyof energy conservation observed in this tissue.     

beta-Oxidation and Glyoxylate Cycle Coupled to NADH: Cytochrome c and Ferricyanide Reductases in Glyoxysomes

Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contamination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate of 340 nanomole electrons per minute per milligram glyoxysomal protein. Palmitoyl-CoA oxidation could be coupled to ferricyanide or cytochrome c reduction. Complete oxidation of palmitoyl-CoA, yielding 14 nanomole electrons/per nanomole palmitoyl-CoA, was demonstrated with the acceptors, NAL, cytochrome c, and ferricyanide. Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by β-oxidation and the glyoxylate cycle.     

Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants

Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K⁺ + Na⁺) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

Abstract

4-Hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC₅₀ value of 8.3 ± 1.0 μM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO^? radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE–protein adducts and oxidation of membrane lipids and proteins by free radicals.     

Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols

External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues.     

Effect of cyanide on mitochondrial membrane depolarization induced by uncouplers

In this work, it was found that the ability of common uncouplers – carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP) – to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor – cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.     

Activation of the external pathway of NADH oxidation in liver mitochondria of cold-adapted rats

15 min cold exposure of rats adapted to cold results in switching on a pathway of the fast oxidation of extramitochondrial NADH in the isolated liver mitochondria. This pathway is sensitive to mersalyl and cyanide, resistant to amytal and antimycin A, and can be stimulated by dinitrophenol. A portion of the endogenous cytochrome c pool can easily be removed by washing mitochondria of the cold-exposed rats.A scheme is discussed, postulating desorption of the inner membrane-bound cytochrome c into intermembrane space of mitochondria, resulting in formation of a link between the non-phosophorylating NADH-cytochrome c reductase in the outer mitochondrial membrane and cytochrome c oxidase in the inner membrane. It is suggested that such an oxidative pathway is involved in the urgent heat production in liver in response to the cold treatment.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Evidence for a structural interacti on betwen ATP synthetase and cytochrome c oxidase in mitochondria

The reaction of cyanide with the oxidized form of cytochrome c oxidase in mitochondria is strongly inhibited by adenosine triphosphate (ATP). This inhibition is strictly dependent on the ATP concentration and is insensitive to changes in the concentrations of adenosine diphosphate (ADP) and orthophosphate. It is completely prevented by oligomycin or uncouplers of oxidative phosphorylation. The ATP is kinetically competitive with respect to cyanide and has a measured inhibitor constant of less than 2 μm The stoichiometry is one ATP/cyanide. This ATP effect is proposed to result from a structural interaction of ATP synthetase with cytochrome c oxidase, such that the formation of an ATP complex of the synthetase results in a decrease in the affinity of the oxidized form of cytochrome c oxidase for cyanide in the formation of an intermediate in the overall measured cyanide reaction.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

Cytochrome c interaction with membranes. Formylated cytochrome c.

A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 ± 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K_D of approx 0.1 μm). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K_m value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.     

Respiratory system of Fusidium coccineum and regulation of biosynthesis of fusidic acid and sporogenesis

Summary The respiratory system, sporulation, and dynamics of alkaline protease formation were studied in three strains of the fungus Fusidium coccineum, differing in their ability to make antibiotics.Oxidative phosphorylation provided most of the energy in high and low activity strains and their respiratory activity was exclusively related to mitochondria functioning.In inactive and low activity strains, the terminal oxidation of reduced equivalents proceeds mainly by the respiratory chain with cytochrome oxidase as the terminal component. In the high activity strain there is a cyanide-resistant alternative pathway which is parallel to the classical cytochrome chain. The complete transition to the use of this pathway coincides with the stage of maximum antibiotic biosynthesis. The induction of the alternative pathway in the high activity strain was not concerned with the inhibition of the cytochrome site of the respiratory chain by fusidic acid. It was shown that the quantity of the antibiotic synthesized and the character of cellular differentiation can be altered by changing the oxidation pathwats used with inhibitors such as chloramphenicol and salicyl hydroxamate.We suggest that there must be common regulation of antibiotic formation, sporulation and induction of the alternative oxidation pathway.     

Participation of plastoquinone,cytochrome c 553 and ferrodoxin-NADP+ oxido-reductase in both photosynthesis and respiration in Spirulina maxima

Dark and light oxidation of NADPH was measured in Spirulina maxima thylakoid membranes. The dark reaction was more cyanide sensitive than the light reaction. In light, 83% of the electrons from NADPH produced H₂O₂ on reducing oxygen, whereas in the dark this number was only 36%. These results are explained by assuming the presence of an electron transport segment common to the photosynthetic and the respiratory chains, so that electrons flowing through the cyanide sensitive oxidase in the dark are diverted to the photosytem (PS) I reaction center (P700). In addition, cytochrome (cyt) c ₅₅₃ was found to be an electron donor for both cyt oxidase and P700. Half maximum reduction rates were obtained with 7 M cyt c ₅₅₃. The intrathylakoidal concentration of cyt c ₅₅₃ was determined to be 83 M. About 60% of the respiratory NADPH oxidation activity was lost by extracting the membranes with pentane and was restored by adding plastoquinone (the main photosythetic quinone). NADPH oxidation activity was also inhibited upon washing the membranes with a low salt buffer. This activity was restored by adding partially purified ferredoxin-NADP⁺ oxido-reductase (FNR). A model for the electron transport in thylakoids, in which cyt c ₅₅₃, plastoquinone and FNR participate in both photosynthesis and respiration is proposed.     

Respiration Rate and Mitochondrial Activity in Iris Bulbs

The oxygen uptake of iris bulbs (Iris×hollandica‘Wedgwood’) which had been stored dry at 30 C (“retarded’ bulbs) was low (10 μmol O₂ per h and bulb), the oxygen uptake of the intact bulb, the three outer fleshy scales and the remaining central part of the bulb increased three- to fourfold, nearly twofold and fourfold, respectively. Mitochondria were isolated from the scales of retarded and activated bulbs and their oxygen consumption with succinate, l -malate (plus pyruvate). x-ketoglutarate and NADH as substrate was measured polarographically. The oxidative capacity of mitochondria isolated from the scales of activated bulbs was only slightly higher than that from retarded bulbs when calculated on a tissue basis. No difference was found between the phosphorylation efficiency, respiratory control, cytochrome c deficiency, succinate dehydrogenase, malate dehydrogenase, succinate-cytochrome c rductase, NADH-cytochrome c reductase and cytochrome oxidase activity of the retarded and activated bulbs. The increase in the in vitro oxygen uptake of the scales after transition from 30 to 13 C was not accompanied by an equal increase in the oxidative capacity of their mitochondria suggesting that they are not responsible for this rise in oxygen uptake.