Identification of the quinone species in cyanide-sensitive and cyanide-insensitive mitochondria of Moniliella tomentosa.
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ABSTRACT:
SUBMITTER: Vanderleyden J
PROVIDER: S-EPMC1162413 | biostudies-other | 1980 Dec
REPOSITORIES: biostudies-other
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Project description:Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.
| S-EPMC1161325 | biostudies-other
Project description:The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase.
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Project description:In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.
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Project description:1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.
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Project description:Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular energy requirements, but the mechanisms that link organelle performance to ATP levels are poorly understood. Here we confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents (that is, mitoKATP). We show that-similar to their plasma membrane counterparts-mitoKATP channels are composed of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution of MITOK together with MITOSUR recapitulates the main properties of mitoKATP. Overexpression of MITOK triggers marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our results indicate that mitoKATP channels respond to the cellular energetic status by regulating organelle volume and function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.
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Project description:Iron (Fe) is an essential micronutrient whose uptake is tightly regulated to balance both deficiency and Fe excess induced oxidative stress. The non-essential heavy metal cadmium (Cd) both induces oxidative stress and an Fe deficiency like response. It is unclear how Cd induces this response, nor how it relates to Fe status sensing. Using next generation sequencing, H2O2 quantification in both wild type and the Fe over-accumulating mutant opt3-2, we explored how Cd interferes with Fe sensing. We found a large overlap of genes consistently responsive to Fe deficiency and Cd in both leaves and roots. Two subnetworks emerged dependent on the high Fe and H2O2 concentration in opt3-2, while opt3-2 chloroplasts showed reduced photosynthetic efficiency during Cd exposure, indicating that they are one source of H2O2. We describe the hierarchical regulation of Fe deficiency responses in the context of the opt3-2 mutant, and demonstrate that the opt3 dependent induction of Fe deficiency responses can be negated, or even reversed, by H2O2 dependent signaling, while the high Fe content of opt3-2 indicates Cd induced iron deficiency is not a function of reduced Fe uptake, but rather the putative leaf Fe sensor is Cd liable.
2020-07-13 | GSE128156 | GEO
Project description:Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.
| S-EPMC1186212 | biostudies-other
Project description:The activities of the mung-bean (Phaseolus aureus L.) mitochondrial cyanide-insensitive oxidase and cytochrome pathways have been measured simultaneously. The results show that electrons can be diverted both from the alternative pathway to the cytochrome pathway and from the cytochrome to the alternative pathway. The competition of the two pathways for the available electron flux is discussed.
| S-EPMC1148698 | biostudies-other
Project description:Pseudomonas aeruginosa, an opportunistic pathogen of humans, uses quorum sensing (QS) to regulate the production of extracellular products that can benefit all members of the population. P. aeruginosa can police QS-deficient cheaters by producing hydrogen cyanide, which is also QS regulated; however, the mechanism by which cooperators selectively protect themselves from the toxicity of cyanide remained unresolved. Here, we show that a cyanide-insensitive terminal oxidase encoded by cioAB provides resistance to cyanide, but only in QS-proficient strains. QS-deficient cheaters do not activate cioAB transcription. QS-mediated regulation of cioAB expression depends on production of both cyanide by cooperators (which is QS regulated) and reactive oxygen species (ROS) from cheaters (which is not QS regulated). This type of regulatory system allows cooperating populations to respond, via ROS, to the presence of cheaters, and might allow them to defer the substantial metabolic cost of policing until cheaters are present in the population.
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Project description:Several measures of energy conservation, namely ADP/O ratio, P/O ratio, ATP/O ratio and phosphorylation detected by continuous assay with purified firefly luciferase and luciferin, all show phosphorylation can occur with mung-bean mitochondria at cyanide concentrations sufficient to inhibit the cytochrome oxidase system. Phosphorylation in the presence of cyanide is uncoupler- oligomycin- and salicylhydroxamate-sensitive. The participation of phosphorylation site 1 is excluded, phosphorylation being attributable to a single phosphorylation site associated with the cyanide-insensitive oxidase. The cyanide-insensitive oxidase has also been shown to support a variety of other energy-linked functions, namely, Ca2+ uptake, reversed electron transport and the maintenance of a membrane potential detected by the dye probes 8-anilinonaphthalene-1-sulphonate and safranine. High concentrations of cyanide have uncoupler-like activity, decreasing the ADP/O ratio and the t 1/2 for the decay of a pH pulse through the the mitochondrial membrane. This uncoupler-like effect is most marked with aged mitochondria. The observations of energy conservation attributable to the cyanide-insensitive oxidase are compared with other reports where it is concluded that the alternative oxidase is uncoupled.
| S-EPMC1162100 | biostudies-other