Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Terminal oxidases of Crithidia oncopelti

Abstract Washed cell suspensions of Crithidia oncopelti oxidizing a variety of substrates gave complex plots for the inhibition of respiration by potassium cyanide or azide. The data indicated the presence of at least two and possibly three terminal oxidases on the basis of their differential sensitivity to these inhibitors. The oxidase most sensitive to cyanide, azide and CO accounted for approx. 65–70% of whole cell respiration and is probably cytochrome oxidase a/a₃. A second oxidase exhibiting low affinity for CO required high concentrations of KCN or azide for inhibition. This haemoprotein had the spectral characteristics of cytochrome o and accounted for 15–20% of cell respiration. Incomplete inhibition of respiration by high concentrations of KCN or azide suggested the presence of a third oxidase which was CO-unreactive.     

The Changes in the Pathways of Mitochondrial Oxidation at the Initial Period of Tuber Development

The rate of oxygen consumption and the participation of the mitochondrial oxidases cytochrome oxidase (COX) and the alternative KCN-resistant mitochondrial oxidase (AOX) were determined in nondifferentiated stolons and small newly developed tubers. High rates of about 300 l O₂/(g fr wt h) and low sensitivity to cyanide were characteristic of stolon respiration. The AOX activity comprised the major part of the latter (60%). As tubers developed, their respiration rate declined and the proportion of mitochondrial oxidases changed: COX became the major terminal oxidase, while the AOX input dropped to 15% of the total oxygen consumption. The AOX input correlated with the total monosaccharide content in stolons and tubers. These data are in line with the concept that the alternative pathway of mitochondrial oxidation serves as a mechanism of energy overflow by which to utilize excess carbohydrates that the cell can neither store nor utilize.     

Cyanide- and hydroxamate-resistant respiration in Neurospora crassa.

Strain inl-89601 of Neurospora crassa respires exclusively by means of the mitochondrial cytochrome chain. The respiration of this strain is entirely inhibited by cyanide or antimycin A, the classical inhibitors of cytochrome chain respiration. When this strain was grown in the presence of chloramphenicol, however, two additional terminal oxidases were detected. One of these oxidases is inhibited by substituted hydroxamic acids and has been described previously. The second oxidase was not inhibited by cyanide or hydroxamic acid but was inhibited by azide in the presence of both cyanide and hydroxamic acid. This azide-sensitive respiration was due to a single respiratory pathway with a Ki for azide of 200 micrometer. A small amount of azide-sensitive respiration was detected in mitochondrial fractions obtained from chloramphenicol-treated cells, and it is likely that the azide-sensitive oxidase is localized in the mitochondrion. The determinants for the azide-sensitive and hydroxamate-sensitive oxidases segregate in a Mendelian manner in crosses and are either unlinked or not closely linked to each other.     

Alternative Cyanide-sensitive Oxidase Interacting with Photosynthesis in Synechocystis PCC6803. Ancestor of the Terminal Oxidase of Chlororespiration?

Influence of respiration on photosynthesis in Synechocystis PCC6803 was studied by measuring the redox transients of cytochrome f (cyt f) upon excitation of the cells with repetitive single turnover flashes. Upon the addition of KCN the flash-induced oxidation of cyt f was increased and the rereduction of cyt f+ was accelerated. Dependence of these effects on the concentration of KCN clearly demonstrated the existence of two cyanide-sensitive oxidases interacting with photosynthesis: cyt aa3, which was sensitive to low concentrations of cyanide, and an alternative oxidase, which could be suppressed by using 1 mM KCN. The interaction between the photosynthetic and the respiratory electron transport chains was regulated mainly by the activity of the alternative cyanide-sensitive oxidase. The oxidative pathway involving the alternative cyanide-sensitive oxidase was insensitive to salicyl hydroxamic acid and azide. The close resemblance of the inhibition pattern reported here and that described for chlororespiration in algae and higher plants strongly suggest that an oxidase of the same type as the alternative cyanide-sensitive oxidase of cyanobacteria functions as a terminal oxidase in chloroplasts.     

Effects of prolonged drought stress and nitrogen deficiency on the respiratory O2 uptake of bean and pepper leaves

We analyzed the combined effects of mild drought stress and severe nitrogen (N) deprivation on respiration of acclimated mature leaves of beans (Phaseolus vulgaris L. cv. Garrofal) and peppers (Capsicum annuum L., pure line B6). Rates of oxygen uptake were measured polarographically, and inhibitors were added to the closed cuvette to compare the effects of environmental stress on the cytochrome (cyt) and alternative pathways of mitochondrial respiration. Dark oxygen uptake was decreased by the water deficit treatment in both plants, and in the case of N limitation leaf respiration rates (R_D) of peppers were also reduced. R_D of leaves of beans and peppers grown under N-limiting conditions did not follow the decrease in leaf N concentration, since R_D expressed per unit of tissue N was considerably higher in the N-stressed leaves. Values obtained with specific inhibitors of the two terminal oxidases of mitochondrial respirations suggested that the cyt pathway of respiration was affected by mild drought and severe N stress. When plants were exposed to both environmental stresses, leaf respiration response was similar to that under N limitation, in this case the most severe stress.     

The respiratory responses to cyanide of a cyanide-resistant Klebsiella oxytoca bacterial strain

The respiratory system of a cyanide-resistant Klebsiella oxytoca was analyzed by monitoring the changes in the cytochrome contents in response to various inhibitors in the presence of various concentrations of cyanide. The cells grown in the medium without cyanide (KCN) have two terminal oxidases, cytochrome d (Ki = 10(-5) M KCN) and o (Ki = 10(-3) M KCN). When cells were grown on medium with 1 mM KCN, the expression of both b-type cytochrome and cytochrome d in the plasma membranes of the cell decreased by more than 50%, while cytochrome o increased by 70%, as compared with the cells grown in the absence of KCN. Two terminal oxidases with Ki values of about 10(-3) M and 1.7 x 10(-2) M KCN were observed in the plasma membrane fractions of the cells growing on KCN enriched medium. 2-n-Heptyl-4-hydroxyquinoline-N-oxide markedly inhibited the oxidation of NADH by the plasma membranes from the cells grown in the medium without KCN, but not in those plasma membranes from KCN-grown cells. The NADH oxidases in plasma membranes of K. oxytoca grown with and without KCN were equally sensitive to UV irradiation. Adding freshly isolated quinone to the UV-damaged plasma membranes restored the NADH oxidase activity from both types of plasma membranes. From these results, we propose the presence of a non-heme type of terminal oxidase to account for the KCN resistance in K. oxytoca.     

Drought Stress Memory in Sugar Beet: Mismatch Between Biochemical and Physiological Parameters

In a greenhouse study, we aimed to determine whether a temporary water deficit induces ‘drought memory’ in sugar beet (Beta vulgaris L.), and whether this effect can be quantified by alterations in the fluorescence signature of the leaves. Plants were subjected to three consecutive water deficit phases, each followed by a recovery period, and in each cycle new, fully developed leaves were analyzed. Changes in the photosynthetic performance and pigment fluorescence were recorded with a hand-held fluorescence sensor, a laser-induced fluorescence spectrometer, and a leaf gas exchange analyzer. Parameters such as osmotic potential, proline, and chlorophyll content were used as indicators for biochemical modifications and quantification of stress intensity. In general, the evaluated cultivars showed a similar response pattern to water deficit, although the intensity of the stress-induced modification was not always on the same level in the distinct parameters. The long-term and repeated drought caused a decrease of net photosynthesis, increase of far-red fluorescence, and a decrease of both the ‘Simple Fluorescence Ratio’ and the fluorescence lifetime (LT mean) in the blue spectral region. In the second drought cycle, changes in osmotic potential and proline content were lower, but alterations in photosynthesis and fluorescence were as strong as in the first and third drought cycles. This indicates that even if a drought stress memory might occur, it was not possible to precisely identify it using gas exchange and pigment fluorescence determinations. Irrespective of that, the photosynthesis and chlorophyll fluorescence-based parameters (RF, SFR) clearly indicated with high temporal resolution the response of sugar beet plants to the stress, and their partial recovery.     

Effect of Melafen on functional efficiency of mitochondrial membranes from sugar beet taproots

Mitochondria were isolated from sugar beet (Beta vulgaris L) taproots and incubated in the presence of low concentrations of Melafen (2 × 10^?9 and 4 × 10^?12 M). This treatment of mitochondrial membranes induced an appreciable decrease in microviscosity of superficial lipids in the lipid bilayer and a parallel increase in microviscosity of the deeply immersed lipid regions adjacent to membrane proteins. Melafen had no effect on fluorescence of lipid peroxidation products in membranes of freshly prepared mitochondria but declined this fluorescence to control values in artificially aged mitochondria. Melafen raised the maximum rates for oxidation of NAD-dependent substrates, elevated the efficiency of oxidative phosphorylation, and activated electron transport in the terminal (cytochrome oxidase) step of mitochondrial respiratory chain, which implies the activation of energy metabolism within the cell. The acceleration of electron transport through the terminal step of mitochondrial respiratory chain was apparently accompanied by retardation of lipid peroxidation, which prevented impairment of mitochondrial membranes under stress conditions. A proposal is put forward that some properties of Melafen are favorable for adaptogenesis because its effects on mitochondrial energy metabolism depended on the functional state of mitochondria.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

Metabolic activity of plant mitochondria in hypertonic sucrose solutions

This study deals with effects of hypertonic sucrose solutions on respiration and oxidative phosphorylation of intact mitochondria isolated from sugar beet (Beta vulgaris L.) taproots and etiolated pea (Pisum sativum L.) seedlings. Mitochondria from plants, like those of animals, showed a trend to inhibition of oxidative phosphorylation in hypertonic sucrose solutions. The increase in sucrose concentration from 0.5 to 1.0 M suppressed malate oxidation in the presence of glutamate in state 3 by a factor of 2.5–3.5 and diminished the respiratory control ratio by a factor of 1.5–2.0. Plant mitochondria turned out remarkably resistant to osmotic stress; they retained significant respiratory control and high ADP/O ratios in a hypertonic 1 M sucrose solution. Although the origin of the observed phenomenon remains unresolved and warrants further studies, it is evident that elevated resistance of plant mitochondria to osmotic stress might be significant for energy supply under extreme environmental conditions (upon drought and salinity) when the plant organism experiences dehydration with a concomitant increase in the cytoplasmic osmolarity.     

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.     

Chlororespiration and the process of carotenoid biosynthesis

The plastoquinone pool during dark adaptation is reduced by endogenous reductants and oxidized at the expense of molecular oxygen. We report here on the redox state of plastoquinone in darkness, using as an indicator the chlorophyll fluorescence kinetics of whole cells of a Chlamydomonas reinhardtii mutant strain lacking the cytochrome b(6)f complex. When algae were equilibrated with a mixture of air and argon at 1.45% air, plastoquinol oxidation was inhibited whereas mitochondrial respiration was not. Consequently, mitochondrial oxidases cannot be responsible for the oxygen consumption linked to plastoquinol oxidation. Plastoquinol oxidation in darkness turned out to be sensitive to n-propyl gallate (PG) and insensitive to salicylhydroxamic acid (SHAM), whereas mitochondrial respiration was sensitive to SHAM and PG. Thus, both PG treatment and partial anaerobiosis allow to draw a distinction between an inhibition of plastoquinol oxidation and an inhibition of mitochondrial respiration, indicating the presence of a plastoquinol:oxygen oxidoreductase. The possible identification of this oxidase with an oxidase involved in carotenoid biosynthesis is discussed in view of various experimental data.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

The peculiarities of the structural and functional state of the cytochrome component of the liver mitochondrial respiratory chain under conditions of acetaminophen-induced hepatitis on the background of alimentary protein deprivation

The activity of a key enzyme of the cytochrome component of the respiratory chain (cytochrome oxidase), the quantitative redistribution of mitochondrial cytochromes b, c ₁, c, and aa ₃, as well as the activities of the key enzymes of cytochrome heme metabolism (δ-aminolevulinate synthase and heme oxygenase) under conditions of acetaminophen-induced liver injury were studied on the background of dietary protein deprivation. Under conditions of acetaminophen-induced hepatitis that developed on the background of alimentary protein deprivation, an inhibition of cytochrome oxidase activity and a decrease in the contents of mitochondrial cytochromes on the background of an increase in the δ-aminolevulinate synthase and heme oxygenase activity were observed. In animals with a toxic liver injury that were kept under conditions of dietary protein deprivation, the contents of mitochondrial cytochromes b, c ₁, c, and aa ₃ progressively decreased, which was accompanied by an increase in heme oxygenase activity, whereas δ-aminolevulinate synthase activity remained at the control level. It was concluded that dietary protein deprivation is a critical factor for the development of disturbances in the structural-functional integrity of the cytochrome component of the respiratory chain. The identified changes can be considered as a possible mechanism that underlies the disturbance in the function of the energy biotransformation system under conditions of dietary protein deprivation.     

Studium der terminaloxidasen in durch obligatparasiten infizierten getreideblättern

In wheat leaves infected with yellow rust (Puccinia striiformis West.) and in barley leaves infected with powdery mildew (Erysiphe graminis DC.) the effect of respiratory inhibitorsin vivo and terminal oxidases activityin vitro were studied. In the experiments the first leaves of seedlings grown in glasshouse were used. The influence of infection was shown by activation of respiration and terminal oxidases (Fe- and Cu-oxidases), first of all cytochrome oxidase and peroxidase. There might be involved also the increased role of ascorbic acid oxidase and phenolase. Peroxidase activation was found to be much higher in susceptible varieties than in resistant ones. Neither in wheat nor in barley the catecholase activity was detected; on the contrary the enzymatic oxidation of floroglucine was found to be also in barley leaves, the intensity of which being dependend on resistance or susceptibility to powdery mildew. Thus, it is not excluded, that in contact establishing between obligate parasite and the host the significant role may be played by specific phenolase and phenolic substances. It suggests itself, that due to the participating in ATP-formation the cytochrome system in terminal oxidation of cereals, infected with obligate parasites, may have centrale position.