Oxidase Of Plant Mitochondria Research Articles

Determination of ROS generation rates in plant mitochondria in vitro using fluorescent indicators: Non-specific effects of inhibitors of terminal oxidases

A possibility of quantitative analysis of the ROS production by mitochondria isolated from etiolated winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings was studied by following hydrogen peroxide accumulation in medium of organelle incubation using a new highly sensitive test-system including the fluorogenic indicator Amplex Red and horseradish peroxidase. The rates of the process were about 100 pmol H2O2/(mg protein min) under state 3 conditions; they were almost doubled after mitochondria transition into state 4, and further increased by more than 50% after the addition of alamethicin, the poreforming antibiotic inducing the organelle inner membrane permeabilization for low-molecular-weight compounds, H2O2 molecules among them. Experimental evidence is presented indicating that the classical inhibitors of the terminal oxidases of the plant mitochondrial respiratory chain, such as cyanide, salicylhydroxamic acid, and propyl gallate, can inactivate the ROS-detecting test-systems in vitro because of interactions with their functional components. These results are discussed in comparison with literature data obtained by similar test-systems and indicate that there are considerable limitations for obtaining reliable experimental evidence of the antioxidant role of alternative cyanide-resistant oxidase in plant mitochondria.

Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase

The claim of methane (CH₄) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH₄ formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non-stress conditions the plant cell cultures produced trace amounts [0.3-0.6 ng g⁻¹ dry weight (DW) h⁻¹] of CH₄ but these could be increased by one to two orders of magnitude (up to 12 ng g⁻¹ DW h⁻¹) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH₄ formation indicating that a site-specific disturbance of the ETC at complex IV causes CH₄ formation in plant cells. Our study is an important first step in providing more information on non-microbial CH₄ formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.

Cytochrome c oxidase in plant mitochondria

Rebinding of CO after flash photolysis of the carboxy mixed-valence cytochrome c oxidase in plant mitochondria has been monitored in the visible range at low temperature (160-210K). At 586/630 nm, the photodissociation of the CO-saturated suspension was followed by a largely incomplete rebinding of CO to Fea3. After a series of three to four flashes, the unbound species accumulated up to 85% of the total enzyme, the remaining 15% CO-bound form undergoing recombination repetitively. In the Soret region, the absorbance level after the first flash came back to the initial one, as if all the CO-bound form were restored. However, the amount of complex which could be photodissociated by a second flash was only part of the starting material, in agreement with observations in the alpha range. The unbound species exhibited optical features very similar to the ones of compound C. The lack of CO rebinding is interpreted as due to an electron redistribution induced by the flash photolysis. This electron transfer has been tentatively assigned to a concomitant CuB oxidation and CuA reduction on the basis of the 655 nm band as a probe for a3+3 [Beinert et al. (1976) Biochim. Biophys. Acta 423, 339-355] and of the optical features in the visible range. Results are discussed with respect to analogous investigations with beef heart mitochondria and the studies by Boelens and Wever (1979-1982) of electron transfer after photolysis of the carboxy mixed-valence state when using the isolated mammalian enzyme at room temperature.

Up-Regulation of Mitochondrial Alternative Oxidase Concomitant with Chloroplast Over-Reduction by Excess Light

Alternative oxidase (AOX), the unique terminal oxidase in plant mitochondria, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been suggested that AOX might prevent chloroplast over-reduction through the efficient dissipation of excess reducing equivalents, direct evidence for this in the physiological context has been lacking. In this study, we examined the mitochondrial respiratory properties, especially AOX, connected to the accumulation of reducing equivalents in the chloroplasts and the activities of enzymes needed to transport the reducing equivalents. We used Arabidopsis thaliana mutants defective in cyclic electron flow around PSI, in which the reducing equivalents accumulate in the chloroplast stroma due to an unbalanced ATP/NADPH production ratio. These mutants showed higher activities of the enzymes needed to transport the reducing equivalents even in low-light growth conditions. The amounts of AOX protein and CN-resistant respiration in the mutants were also higher than those in the wild type. After high-light treatment, AOX, even in the wild type, was preferentially up-regulated concomitant with the accumulation of reducing equivalents in the chloroplasts and an increase in the activities of enzymes needed to transport reducing equivalents. These results indicate that AOX can dissipate the excess reducing equivalents, which are transported from the chloroplasts, and serve in efficient photosynthesis.

Stress-induced changes in ubiquinone concentration and alternative oxidase in plant mitochondria.

We have investigated the influence of stress conditions such as incubation at 4 degrees C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4 degrees C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.

Partial male sterility in transgenic tobacco carrying an antisense gene for alternative oxidase under the control of a tapetum-specific promoter

The alternative oxidase of plant mitochondria is the terminal oxidase of the cyanide-insensitive respiratory pathway and is encoded by a nuclear gene. A 1 kb genomic fragment including exon 3 of the alternative oxidase was amplified by PCR from the genome of Arabidopsis thaliana. This fragment was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. The pollen viability in three transgenic plants ranged from 2% to 60%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. Immunolocalization of alternative oxidase protein in the immature flower bud section indicated that expression of alternative oxidase protein in tapetum of the transgenic plant was much lower than that of the non-transformant. The histological observation and protein gel-blot analysis showed that the development of pollen grains in the transgenic plant did not progress after the degradation of the tapetum, and the amount of alternative oxidase in pollen grains of the transgenic plant became lower than that of the non-transformant. These results suggested that the alternative oxidase activity in the tapetum has a significant effect on the pollen development.

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria.

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.

The alternative respiration pathway in plants: Role and regulation

In the past few years, knowledge of the nature and regulation of the alternative oxidase in plant mitochondria has increased greatly. The protein has been characterized and mechanisms that regulate its activity have been described. The consequences of these regulatory mechanisms are that in vivo the cytochrome pathway and the alternative pathway may compete for electrons. The implications for the interpretation of the ‘Bahr and Bonner’ inhibitor titrations, formerly used to estimate the partitioning of electrons over the two pathways, are discussed.It is proposed that activation and engagement of the alternative oxidase may keep Q reduction levels low in order to prevent harmful high levels of free radical production. A model is presented for the regulation of alternative oxidase protein induction, involving a signalling function of active oxygen species.

Titration of the external NADH dehydrogenase and the alternative oxidase in plant mitochondria.

Titration of the external NADH dehydrogenase and the alternative oxidase in plant mitochondria.

Random effects and variances as a synthesis of nonlinear regression analyses of mitochondrial electron transport

SUMMARY This cross-disciplinary paper develops random effects and variances methods, especially for small samples within effects, and applies them to mitochondrial enzyme kinetics. A new force flux kinetic model is introduced for the alternative oxidase of plant mitochondria, the results of 42 experiments are published and the force flux function is fitted by nonlinear regression for each experiment. The results of the regression analyses are synthesized by a random effects model specifying random nonlinear regression coefficients and random variances within experiments distributed in an inverse gamma distribution. The paper shows how random nonlinear regression coefficients analysis can summarize the results of a long series of similar biological experiments and sharpen the analysis of the results of each. A large data set often consists of a family of components each of which can be fitted with the same model. There is a within-component variation for each compo- nent and random effects variation between components. The interpretation of the data requires not only regression analysis of each component but also a subsequent synthesis of the results of such analysis into an overall summary, achieved by specifying random regression coefficients and random error variances. In the example which follows, the individual samples are so small that random variances are specified and contracted predictors of them are computed which are more accurate than the individual estimates. They are required for weights in the analysis. The residual maximum likelihood estimates of the random effects variance matrices are sometimes singular. A Lagrange multiplier method is introduced to compute them. The strength of the integrated analysis comes, however, at the price of a complex set of decisions as to how the different components of the computation are to be fitted together. The paper shows how this is done by a flow diagram for the computations, as well as describing each individual component.

Compartmentation of Alternative Oxidase in Plant Mitochondria

Compartmentation of Alternative Oxidase in Plant Mitochondria

Discrimination between duroquinol oxidase activity and the terminal oxidation step of the cyanide-resistant electron transport pathway of plant mitochondria

Comparison of the cyanide-resistant duroquinol oxidase activity of sub-mitochondrial particles from Arum maculatum L. with their ability to carry out a cyanide-resistant oxidation of NADH and succinate shows that heat-inactivation of the duroquinol oxidase activity does not proportionally affect NADH and succinate oxidation. Moreover, 1 μM antimycin inhibits duroquinol oxidase activity by 50 % while not decreasing the rates of NADH and succinate oxidation. Therefore, the cyanide-resistant electron transport does not appear to be mediated by a “duroquinol oxidase”, and a convincing proof of the existence of a specific protein acting as a cyanide-resistant oxidase in plant mitochondria is still lacking.

Dynamics of carbon monoxide recombination to fully reduced cytochrome c oxidase in plant mitochondria after low-temperature flash photolysis.

Rebinding of CO to reduced cytochrome c oxidase in plant mitochondria has been monitored optically at 590-630 nm after flash photolysis at low temperature from 160 to 200 K. (1) Under 100%-CO saturation, CO rebinding exhibits a four-step mechanism. The thermodynamic parameters of the first phase have been determined; its activation energy, Ea1, is 38.9 kJ.mol-1 and its enthalpy, delta H+/-1, and entropy, delta S+/-1, of activation are respectively 37.5 kJ.mol-1 and -75.8J.mol-1.K-1. (2) When the CO concentration is decreased to 0.2%, rebinding still occurs according to a four-step mechanism. The rate constant of the first phase is CO-concentration-independent. Under non-saturating conditions there is only one CO molecule per occupied site. The rebinding mechanism does not require additional CO molecules to be present in the haem pocket. (3) Dual-wavelength scanning experiments failed to detect optical forms correlated with the resolved phases. (4) Results are discussed with respect to previous work related to CO rebinding to mammalian cytochrome c oxidase and myoglobin.