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.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

The Content of Alternative Oxidase of Euglena Mitochondria is Increased by Growth in the Presence of Cyanide and is not Cytochrome o.

ABSTRACT A study of the effect of respiratory inhibitors on O₂ uptake of Euglena gracilis mitochondria, isolated from cells grown in the presence of cyanide or with ethanol as carbon source, was undertaken. The contents of cytochrome c oxidase and alternative oxidase were also determined. Inhibition of respiration by antimycin and cyanide was only partial and it was dependent on the oxidizable substrate used. Succinate oxidation was the most sensitive to cyanide whereas lactate oxidation was the most resistant. Cell growth in the presence of cyanide or with ethanol as carbon source brought about an enhanced content of alternative oxidase without a concomitant increase in cytochrome aa₃ content. However, a correlation between cyanide-resistant respiration and alternative oxidase content was not found. Analysis of heme types in mitochondrial membranes revealed the absence of heme O. The data suggest the presence of an inducible alternative oxidase in Euglena mitochondria which has high resistance to cyanide and contains heme B. A close relationship between Euglena alternative oxidase and bacterial quinol oxidases containing B-type heme is proposed.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7

Abstract The role of cytochrome b ₅₆₂, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b ₅₆₂ did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form ( b ₅₆₂) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b ₅₆₂ was upstream of both the ' g _y= 1.89' Rieske FeS cluster and the a -type cytochromes. These results show that the b -type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Hexose-phosphate as an Intermediate in the Assimilation of Methanol by Candida sp

Sporobolomyces ruberrimus is insensitive to antimycin A which is a respiratory inhibitor of the cytochrome system, as cyanide is. When this red yeast was cultured in the presence of antimycin A, the growth curve showed the same pattern as that of the normal culture in the absence of it, but the growth mass was only about 70% of that of the normal culture. The antimycin A-insensitive and cyanide-insensitive respiration of Sp. ruberrimus was inhibited by pyrocatechol and salicylhydroxamic acid. Sporobolomyces red yeasts have two characteristic terminal oxidase systems; one is a cytochrome oxidase system and the other is a cyanide- and antimycin A-insensitive oxidase system. The proportions of the two respiratory systems differed among the species and strains of Sporobolomyces red yeasts examined.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

EXTRACELLULAR PEROXIDASE-MEDIATED OXYGEN CONSUMPTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

The unicellular green alga Chlamydomonas reinhardtii Dang. displays a high capacity for salicylhydroxamic acid (SHAM)—stimulated O₂ consumption, mediated by extracellular peroxidaie. Addition of exogenous NADH also resulted in stimulation of O₂ consumption. The SHAM-and NADH-stimulated peroxidase activity was partially sensitive to inhibition by exogenous superoxide dismutase, ascorbate, and gentisic acid. These compounds did not inhibit O₂ consumption in the absence of effectors. SHAM-and NADH-stimulated peroxidase activity also was sensitive to inhibition by cyanide, and cyanide titration curves indicated that O₂ consumption by peroxidase was more cyanide-sensitive than O₂ consumption by cytochrome oxidase. The differential sensitivity to cyanide was used to estimate partitioning of O₂ consumption between mitochondrial respiration and extracellular peroxidase. We suggest that, despite a large capacity for peroxidase-me-diated O₂ consumption, peroxidase did not consume O₂ at detectable rates in the absence of effectors. Therefore, in the absence of effectors, measured rates of O₂ consumption represented the rate of mitochondrial respiration .     

Effects of mitochondrial complex III disruption in the respiratory chain of Neurospora crassa

In mitochondria from most organisms, including Neurospora crassa , dimeric complex III was found associated with complex I. Additional association of complex IV with this core structure leads to the formation of a respirasome. It was recently described for bacteria and mammals that complex III is needed for the assembly/stability of complex I. To elucidate the role of complex III in the organization of the respiratory chain of N. crassa , we analysed strains devoid of either the Rieske iron-sulphur or the COREII polypeptide subunits. The mutants display reduced growth, are female sterile and lack active complex III. The supramolecular organization of the oxidative phosphorylation system was characterized by electrophoretic analyses and the efficiency of the respiratory chain analysed by oxygen consumption measurements. The results obtained indicate that absence of complex III activity is not associated with the absence of complex I or complex IV, and leads to the induction of alternative oxidase. Complex III mutant mitochondria are devoid of respirasomes but contain significant amounts of dimeric complex I (I₂) and of the supercomplex I₁IV₁. Moreso, for the first time the alternative oxidase was found associated with dimeric complex IV and with supercomplex I₁IV₁.     

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM.

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a3, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a3, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N',N'-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.     

Purification and characterization of membrane-bound CO-reactive hemoprotein from Tetrahymena pyriformis mitochondria

Abstract A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis . It showed absorption peaks at 615 and 455 nm in the reduced form and an α peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a ₆₂₀' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis , it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a ₆₂₀' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis .     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Methanol metabolism in mutants of the methylotrophic yeast Hansenula polymorpha

Abstract Three types of Hansenula polymorpha 356 (leu⁻) mutants unable to grow on methanol were isolated and characterized. The first type of mutants, M8, M14, and M41, were deficient in the alcohol oxidase activity (MOX⁻). The dihydroxyacetone synthase activity appeared after incubation of the strains in the medium with glycerol and methylamine but not with methanol. One of the mutants (W218) with the reduced activity of alcohol oxidase lacked the formate dehydrogenase activity (FDH⁻). All these mutants produced a low level of extracellular formaldehyde from methanol.
The second and third types of mutants were deficient in dihydroxyacetone synthase (DAS⁻; 349, 409, 450), and dihydroxyacetone kinase (DAK⁻; 4D1, 4D3, 4D16) activities, respectively. DAK⁻ mutants showed both the high activities of alcohol oxidase and NADH-dependent reduction of CH₂O catalyzed by alcohol dehydrogenase. This indicated the possibility that NADH, generated in the oxidation of formaldehyde to CO₂, may be oxidized by molecular oxygen via a futile cycle composed of the alcohol oxidase and alcohol dehydrogenase.     

The branched respiratory system of photosynthetically grown Rhodopseudomonas capsulata.

Various respiratory electron transport activities of Rhodopseudomonas capsulata were studied in membrane fragments prepared from photosynthetically grown cells of a parental strain and two terminal oxidase-defective mutant strains. The NADH and succinate oxidase activities of the mutant having a functional N,N,N1,N1-tetramethyl-p-phenylenediamine oxidase, M6, were consideraly more sensitive to inhibition by either antimycin A or cyanide than the corresponding activities of the mutant lacking a functional N,N,N1,N1-tetramethyl-p-phenylenediamine oxidase, M7. The parental strain, Z-1, but not the mutants, showed biphasic inhibitory responses of NADH and succinate oxidase activities with either antimycin A or cyanide. In certain reactions no differences in inhibitor susceptibility were found among the strains tested, implying that the pathways involved were unaffected in the mutants. In this category were the actions of rotenone on NADH oxidase, antimycin A on cytochrome c reductase and, in M6 and Z-1, cyanide on N,N,N'N'-tetramethyl-p-phenylenediamine oxidase. These results suggest that the respiratory chain of the parental strain branches at the ubiquinone-cytochrome b region into two pathways, each branch goes to a distinct terminal oxidase, and either may be blocked independently by genetic mutation.     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

Carbon monoxide-insensitive respiratory chain of Pseudomonas carboxydovorans.

Experiments employing electron transport inhibitors, room- and low-temperature spectroscopy, and photochemical action spectra have led to a model for the respiratory chain of Pseudomonas carboxydovorans. The chain is branched at the level of b-type cytochromes or ubiquinone. One branch (heterotrophic branch) contained cytochromes b558, c, and a1; the second branch (autotrophic branch) allowed growth in the presence of CO and contained cytochromes b561 and o (b563). Electrons from the oxidation of organic substrates were predominantly channelled into the heterotrophic branch, whereas electrons derived from the oxidation of CO or H2 could use both branches. Tetramethyl-p-phenylenediamine was oxidized via cytochromes c and a exclusively. The heterotrophic branch was sensitive to antimycin A, CO, and micromolar concentrations of cyanide. The autotrophic branch was sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide, insensitive to CO, and inhibited only by millimolar concentrations of cyanide. The functioning of cytochrome a1 as a terminal oxidase was established by photochemical action spectra. Reoxidation experiments established the functioning of cytochrome o as an alternative CO-insensitive terminal oxidase of the autotrophic branch.     

Respiration of Colpoda cucullus During Active Life and Encystment

SYNOPSIS. During encystment of Colpoda cucullus (O. F. Müller) the respiratory rate decreases from about 11.3 × 10^-5μl. O₂/hr. (mean value for active form) to about 1.3 × 10^-5μl./hr. (mean for unstable cyst); the process takes about 24 hrs. At the same time the activity of the contractile vacuole is stopped, but there is no correlation in time between the two processes: when respiration is inhibited by 1/2–2/3 the vacuolar activity is hardly changed. The respiration of unstable cysts remains approximately constant at least for 4 weeks. Experiments with methylene blue suggest that inhibition of respiration at encystment may be due to inhibition of terminal oxidase.     

Petite mutation in aged and oxidatively stressed ale and lager brewing yeast

Aims:  To determine the role of oxidative stress and chronological ageing on the propensity of brewing yeast strains to form respiratory deficient 'petites'.
Methods and Results:  Four industrial yeast strains (two ale and two lager strains) were exposed to oxidative stress in the form of H₂O₂ (5 mmol l⁻¹) for two hours. Cell viability and occurrence of petites were determined by the slide culture and TTC-overlay techniques, respectively. Increases in petite frequency were observed but only in those strains sensitive to oxidative stress. Chronological ageing under aerobic conditions led to an increase in petites in strains sensitive to oxidative stress. No such increase was observed under anaerobic conditions.
Conclusion:  Ageing may contribute to mitochondrial DNA damage and increase the propensity of brewing yeast cells to become respiratory deficient. Tolerant strains may be less likely to generate petites as a result of serial re-pitching.
Significance and Impact of the Study:  Continuous re-use of brewing yeast is associated with an increase in the frequency of petites within brewery yeast slurries, a phenomenon resulting in reduced fermentative capacity. The cause of petite generation during brewery handling is unknown. We show that endogenous oxidative stress has the potential to generate petites within brewing yeast populations.