Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate

Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide-sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.

The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate-insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns.

     

Confounding of alternate respiration by lipoxygenase activity

The initial burst of respiratory activity (Q_o2) of imbibing soybean (Glycine max [L.] Merr. var. Wayne) seed tissue is cyanide-insensitive, and sensitive to salicylhydroxamate: presumptive evidence for the presence of alternate respiration. The initial O₂ consumption is also highly sensitive to propyl gallate. Soybean lipoxygenase exhibits similar characteristics of insensitivity to cyanide and sensitivity to salicylhydroxamate and to propyl gallate. The initial burst of respiration is enhanced by the addition of linoleic acid, a lipoxygenase substrate. These results indicate that the conventional tests for alternate respiration in plant tissues can be confounded by lipoxygenase; they also suggest that propyl gallate can be used to assess the possible participation of lipoxygenase in the O₂ uptake by plant tissues.     

Respiration of Wild Type and Extrachromosomal Mutants of Neurospora crassa

The specific rates of respiration of cells of wild type and four extrachromosomal mutants of Neurospora crassa were measured throughout the vegetative growth cycle. Two forms of respiration were observed: (i) cyanide sensitive; and (ii) cyanide resistant, salicyl hydroxamate sensitive. These two forms are called terminal and alternate, respectively. The former proceeds by the mitochondrial electron transfer chain and involves the cytochromes; the latter apparently proceeds by the initial portion of the electron transfer chain and does not involve cytochromes. Large and rapid changes of both the terminal and alternate respiratory activities occurred during the vegetative growth cycle. The kinetics of these changes in wild type were compared under some conditions which inhibit protein synthesis and others in which the nitrogen source was varied. The kinetics of the changes of the two forms of respiration of mutants differed from those normally exhibited by wild type, but with varied experimental conditions wild type could be made to resemble the mutants. The results of these studies are discussed in terms of a dynamic model of regulation of mitochondrial biogenesis in the coordination of the synthesis of mitochondrial proteins encoded by nuclear and mitochondrial genomes.     

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.     

Decay of cytochromes and appearance of a cyanide-insensitive electron transfer pathway in Euglena gracilis grown in anoxia

The effect of anoxia over a 3-week period on the respiratory ability of Euglena gracilis (strains Z and ZC) was studied. Low temperature absorption spectra indicated that comparable alterations of the cytochromes occurred in both cell types studies, leading to the disappearance of cytochrome oxidase and cytochrome c558 in the aplastic strain ZC and in the wild type strain Z. Both types of cells maintained their ability to consume O₂ when they were transferred from the nitrogen atmosphere to air. Resistance of respiration to cyanide and azide, and sensitivity to propyl gallate increased during anoxia, indicating the decreasing role of cytochrome oxidase in this O₂ consumption. Quantitative changes in the O₂ consumption capacity were followed during the course of anoxia; this capacity decreased during the 5 first days of anoxia and then increased to recover its initial value during the second week. This recovery of a high O₂, consumption capacity was linked to the appearance of a cyanide- and azide-resistant, propyl gallate-sensitive O₂ consumption pathway. This paper raises the question of the physiological significance of this electron transfer pathway induced by anoxia.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.     

Alternative Respiratory Pathway: ITS ROLE IN SEED RESPIRATION AND ITS INHIBITION BY PROPYL GALLATE

Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O₂ uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O₂ uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: I. Biochemical Analyses of Pigment Mutations

Wild type Gracilaria tikvahiae, a macrophytic red alga, and fourteen genetically characterized pigment mutants were analyzed for their biliprotein and chlorophyll contents. The same three biliproteins, phycoerythrin, phycocyanin, and allophycocyanin, which are found in the wild type are found in all the Mendelian and non-Mendelian mutants examined. Some mutants overproduce R-phycoerythrin while others possess only traces of phycobiliprotein; however, no phycoerythrin minus mutants were found. Two of the mutants are unique; one overproduces phycocyanin relative to allophycocyanin while the nuclear mutant obr synthesizes a phycoerythrin which is spectroscopically distinct from the R-phycoerythrin of the wild type. The phycoerythrin of obr lacks the typical absorption peak at 545 nanometers characteristic of R-phycoerythrin and possesses a phycoerythrobilin to phycourobilin chromophore ratio of 2.6 in contrast to a ratio of 4.2 found in the wild type. Such a lesion provides evidence for the role of nuclear genes in phycoerythrin synthesis. In addition, comparisons are made of the pigment compositions of the Gracilaria strains with those of Neoagardhiella bailyei, a macrophytic red alga which has a high phycoerythrin content, and Anacystis nidulans, a cyanobacterium which lacks phycoerythrin. The mutants described here should prove useful in the study of the genetic control of phycobiliprotein synthesis and phycobilisome structure and assembly.     

Cyanide- and carbon monoxide-resistant mutants of Vitreoscilla: altered cytochromes and respiratory properties

Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide-resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type.     

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: II. Phycobilisome Characteristics of Pigment Mutants

Phycobilisomes were isolated from wild type Gracilaria tikvahiae and a number of its genetically characterized Mendelian and non-Mendelian pigment mutants in which the principal lesions result in an increase or decrease in the accumulation of phycoerythrin. Both the size and phycoerythrin content of the phycobilisomes are proportional to the phycoerythrin content of the crude algal extracts. In most of the strains examined, the structure and function of the phycocyanin-allophycocyanin phycobilisome cores are the same as in wild type. The phycobilisome architecture is derived from wild type by the addition or removal of phycoerythrin. The same pattern is observed for the phycobilisome of mos² which contains a large excess of phycocyanin that is not bound to the phycobilisome. The single exception is a yellow, non-Mendelian mutant, NMY-1, which makes functional phycobilisomes composed of phycoerythrin and allophycocyanin with almost no phycocyanin. Characterization of the `linker' polypeptides of the phycobilisome indicates that a 29 kilodalton protein is required for the stable incorporation of phycocyanin into the phycobilisome. Evidence is provided for the requirement of nuclear and cytoplasmic genes in phycobilisome synthesis and assembly. The symmetry properties of the phycobilisome are considered and a structural model for the reaction center II-phycobilisome organization is presented.     

Cell wall organisation in Chlamydomonas reinhardi

Summary Among a series of mutants regulating wall formation, two exhibit both Mendelian and non-Mendelian segregation patterns on crossing with wild type. In addition each, on crossing with an identical mutant, frequently gives wild type and pseudo-wild type forms. Diploid wild types can be generated by fusing two identical haploid mutants. The results of genetic analyses indicated that extra-nuclear information is involved in the regulation of wall formation; this is usually stable in vegetative cells but can be renewed at the diploid spore stage. The degree of autonomy of the extra-nuclear system is considered, and the possibility of its being ultimately based on nuclear information discussed.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT-PURIFIED MITOCHONDRIA

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O₂ uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A-insensitive O₂ uptake in either gradient band. Antimycin A-insensitive O₂ consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O₂ uptake. This O₂ consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O₂ consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development.     

Cyanide-Insensitive Respiration inSchizophyllum commune

Two mutants of the hymenomyceteSchizophyllum commune, unable to use acetate as the sole carbon source for growth, were isolated. Growth of the mutants on a glucose minimal medium was only slightly inhibited by sodium azide. Genetic analysis revealed mutations in different chromosomal genes in the respective mutants. Both these mutants exhibited a high cyanide-insensitive endogenous respiration. The inhibition of the respiration by 8-hydroxyquinoline showed the mutants to respire predominantly by an alternative respiratory pathway observed in many fungal species, but not in the hymenomycetes so far. An enhanced cyanide-insensitive respiration was also found in a wild-type strain ofSchizophyllum commune grown in the presence of sodium azide.     

Cyanide-insensitive respiration of Candida lipolytica

Whole cells of the yeast Candida lipolytica exhibited a high, cyanide-sensitive endogenous respiration which became completely cyanide-insensitive under certain physiological circumstances namely (1) in the stationary phase of growth and (2) upon aeration in the resting state. This cannot be due to a change in permeability of the cell wall as the respiration of protoplasts showed the same (in)sensitivity to cyanide as the cells from which they were obtained.The cyanide-insensitive respiration of C. lipolytica was located in the mitochondria and coexisted with the normal respiratory chain, as the mitochondria isolated from cyanide-insensitive cells exhibited at the same time a cyanidesensitive respiration of ascorbate and N,N,N,N-tetramethyl-p-phenylenediamine and a cyanide-insensitive respiration of succinate.The alternate respiratory pathway was sensitive to benzyl- and salicylhydroxamic acids. In this respect it resembles the alternate mitochondrial pathway described in the literature for various plants.The cyanide-insensitive respiration did not appear in the resting state when the cells were aerated in the presence of cycloheximide nor at 0 C instead of at room temperature. These facts suggest some form of induction involving new protein synthesis. The induction process depends on the presence of molecular oxygen as the cyanide-insensitive endogenous respiration did not appear during agitation of yeast cells in the resting state if the gaseous atmosphere lacked oxygen.     

Respiratory System of Rhodotorula glutinis I. Inhibitor Tolerance and Cytochrome Components

Oxygen uptake by the carotenoid-containing yeast, Rhodotorula glutinis was not affected by concentrations of cyanide and antimycin A which completely inhibit the respiration of Saccharomyces cerevisiae. The tolerance of R. glutinis to these inhibitors was somewhat dependent on the age of the cultures. Reduced minus aerated difference spectra of cells revealed spectral changes presumably due to cytochromes and carotenoids. The kinetics of these spectral changes induced by oxygen were followed. Carotenoid deficient cells were prepared by growth in the presence of diphenylamine. Difference spectra of these cells revealed the presence of flavoprotein, and a, b, and c type cytochromes. Growth of R. glutinis was completely inhibited by concentrations of cyanide which did not affect respiration. Oxidation of reduced nicotinamide adenine dinucleotide by sub-cellular fractions was sensitive to cyanide and antimycin A. Although respiration of intact cells is tolerant to these inhibitors, studies with cell-free extracts suggest the presence of a cyanide and antimycin A-sensitive, cytochrome-linked, respiratory chain.     

Effects of Respiratory Inhibitors on Respiration, ATP Contents, and the Circadian Conidiation Rhythm of Neurospora crassa

Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. All inhibitors, potassium cyanide, sodium azide, antimycin A, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), shifted the phase of the conidiation rhythm. All the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. Phase shifting by azide and CCCP was proportional to the lowering of respiratory activity and ATP content, but such a correlation was not observed for cyanide and antimycin A. In particular, cyanide at a concentration of 0.5 millimolar completely depleted ATP of the cultures but did not significantly shift their phase. Their results suggest that large shifts caused by these inhibitors are not due to a decrease in energy from respiratory activity.