ACTION OF INHIBITORS ON HYDROGENASE IN AZOTOBACTER

The inhibitors usually associated with the activity of the cytochrome oxidase system—cyanide and carbon monoxide—are also effective in reducing the oxidation of H₂ by intact cells of Azotobacter vinelandii. The hydrogenase system is more sensitive to CO than is the respiratory system. Oxidation of a carbon source and of hydrogen by Azotobacter cells is inhibited in a quantitatively different manner by the following compounds: sodium azide, hydroxylamine, sodium iodoacetate, and sodium fluoride. In every case, a concentration range which is definitely inhibitory for respiration has little or no effect on the hydrogenase activity. The differential inhibition by hydroxylamine explains certain observations in the literature which have been erroneously interpreted as demonstrating a specific inhibition by NH₂OH of biological nitrogen fixation. This supposed demonstration has been offered as support for the hypothesis that NH₂OH is an intermediate in the fixation reaction. The differential inhibitors can be used for detection of hydrogenase in cultures possessing a high endogenous respiration. The method is illustrated by an experiment with root nodule bacteria from pea and cowpea nodules. No hydrogenase was found in either.     

THE EFFECT OF SPECIFIC POISONS UPON THE PHOTO-REDUCTION WITH HYDROGEN IN GREEN ALGAE

1. The effect of poisons upon the photoreduction with hydrogen in Scenedesmus and similar algae has been studied. The poisons used were cyanide, hydroxylamine, dinitrophenol, and carbon monoxide, substances known to inhibit more or less specifically certain enzymatic reactions. 2. It was found that quite generally one has to distinguish between the action of poisons upon the photoreduction in the stationary state, once this type of metabolism has been well established in the cells, and their effects on transition phenomena, on the "adaptation" and its reversal, the "turnback" from photoreduction to photosynthesis. 3. Cyanide inhibits photoreduction more strongly than it inhibits photosynthesis in the same algae. It is concluded that the mechanism of oxygen liberation, which is idle in photoreduction, is not very sensitive to cyanide. 4. Hydroxylamine in low concentrations is a powerful inhibitor of photosynthesis but has practically no influence on the rate of photoreduction. Consequently, it is assumed that it acts in photosynthesis mainly by inhibiting the evolution of oxygen. Greater concentrations of hydroxylamine clearly inhibit photoreduction, but diminish the rate to about one-half only. A greater degree of inhibition is obtained only by prolonged incubation. 5. Dinitrophenol was found to inhibit strongly the reduction of carbon dioxide, under aerobic as well as under anaerobic conditions. A stimulating effect of dinitrophenol can be demonstrated only with respiration or fermentation, not with photosynthesis. 6. Carbon monoxide interferes with all phases of the hydrogen metabolism in algae. It is supposed therefore to be a specific inhibitor for the hydrogenase system. 7. The "adaptation" to the hydrogen metabolism, which takes place if the algae are incubated anaerobically in hydrogen for several hours, is inhibited completely by very small amounts of cyanide. The adaptation reaction is more sensitive to cyanide than most of the other metabolic processes in the same cell. Correspondingly cyanide enhances the return to aerobic conditions, the "turnback," which occurs under the influence of light of high intensities. 8. Hydroxylamine, applied aerobically, inhibits the adaptation reaction to about the same degree as it inhibits photosynthesis. Photoreduction proceeds after the adaptation in presence of hydroxylamine only at a fraction of the rate that it would have if the poison were added later. 9. Hydroxylamine in concentrations of 10^–3 M protects the anaerobic metabolism against the return to aerobic photosynthesis which normally occurs under the influence of light of too high intensity. The protection is only relative and the higher the light intensity the more hydroxylamine is needed to keep photoreduction going. Once a "turnback" occurs in presence of much hydroxylamine all photochemical gas exchange comes to an end.     

Action of Respiratory Inhibitors on Seed Germination and Oxygen Uptake in Avena fatua L.

The effects of sodium azide, potassium cyanide (cytochrome oxidaseinhibitors), and salicylhydroxamic acid (SHAM; an alternativerespiration inhibitor) on germination and respiration of Avenafatua L. seeds were studied. Azide and cyanide released seeddormancy at similar concentrations and treatment durations.Cyanide, however, stimulated germination of seeds with littleafter-ripening, whereas azide had no effect under similar conditionsunless the seeds were after-ripened for several months; theduration of after-ripening required for seeds to respond toazide varied with seed batch. There was also a greater lag priorto germination in the case of azide, compared to cyanide treatedseeds. SHAM inhibited the stimulation of germination and respirationby azide, but not by cyanide. Furthermore, respiration induced by azide or cyanide could notbe inhibited by the subsequent application of SHAM. These findingssuggest that the respiration stimulated by azide and cyanideis not alternative (SHAM-sensitive) and, therefore, this respiratorypathway cannot be involved in the stimulation of germinationby cytochrome oxidase inhibitors. While embryos excised fromcontrol, azide or cyanide pretreated seeds had the capacityto perform alternative respiration, the actual contributionof this pathway was negligible. A large proportion of respirationof embryos excised from azide or cyanide pretreated seeds wasresidual, i.e. insensitive to both SHAM and cyanide. Alternative respiration, azide, cyanide, dormancy, salicylhydroxamic acid, wild oats     

Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans

Photoinhibition of photosynthesis and its reactivation was studied in the cyanobaterium A. nidulans in the presence of the respiratory inhibitor sodium azide, the uncouplers carbonyl cyanide p -(trifluoromethoxy)-phenylhydrazone (FCCP) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) and the photosystem I elicitor phenazine methosulphate (PMS). Inhibition of dark respiration by azide increased the susceptibility of the cyanobacterium to photoinhibition. Both FCCP and CCCP also remarkably affected the process of photoinhibition in A. nidulans. The PMS at lower photoinhibitory light intensity partially protected A. nidulans from photoinhibition. The recovery from photoinhibition in the presence of azide or FCCP was slow and normal photosynthesis could not be resumed even after a longer period of incubation under suitable reactivating condition. Thus dark respiration has a key function in the process of photoinhibition of photosynthesis and its reactivation in the cyanobacterium A. nidulans.     

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum)

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.     

Sodium exclusion and potassium retention by the red marine alga,Porphyra perforata

Cells of the red marine alga, Porphyra perforata, accumulate potassium and exclude sodium, chloride, and calcium. Various metabolic inhibitors including dinitrophenol, anoxia, and p-chloromercuribenzoate partially abolish the cells' ability to retain potassium and exclude sodium. Iodoacetate induces potassium loss only in the dark; reduced sulfur compounds offer protection against the effects of p-chloromercuribenzoate; dinitrophenol stimulates respiration at concentrations which cause potassium loss and sodium gain. Following exposure to anoxia potassium accumulation and sodium extrusion take place against concentration gradients. These movements are retarded by sodium cyanide, but are stimulated by light. Sodium entry, following long exposure to 0.6 M sucrose, occurs rapidly with the concentration gradient, while potassium entry against the concentration gradient takes place slowly, and is prevented by cyanide.     

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.     

Dormancy in Cereal Seeds: I. THE EFFECTS OF OXYGEN AND RESPIRATORY INHIBITORS

A wide spectrum of respiratory inhibitors has been found tostimulate the breaking of dormancy in barley. These includecarbon monoxide, cyanide, azide, hydrogen sulphide, sodium sulphide,hydroxylamine, diethyldithiocarbamate (DIECA), fluoride, iodoacetate,malonate, monofluoroacetate, and 2,4-dinitrophenol (DNP). Inrice, only the first six of these have been shown to be effective.Apart from CO, all the above inhibitors were tested on winteroats, but in this material only cyanide, azide, and hydroxylaminewere found to increase the germination of dormant seeds. Allthe terminal-oxidase inhibitors except CO were tested on perennialryegrass, but in this case only cyanide was found to break dormancy. As compared with air, an atmosphere of 96 per cent oxygen appliedto barley during the first 24 h after the seeds have been setto germinate stimulates the breaking of dormancy. When appliedat later stages, this high oxygen tension inhibits the germinationof dormant seeds although it has no effect on nondormant seeds.Paradoxically, the stimulatory effects of respiratory inhibitorsapplied during the initial stages of germination are relatedto their ability to inhibit oxygen uptake. Thus cyanide, azide,malonate, and monofluoroacetate, while stimulating the breakingof dormancy in barley, also inhibit oxygen uptake. In rice,cyanide and azide had similar effects, but fluoride, which hadno effect on dormancy, also had no effect on the oxygen uptakeof dormant seeds. These results are compatible with the hypothesis that some oxidationreaction is necessary for germination. This oxidation is notpart of the normal respiratory pathway, and does not proceedsatisfactorily in dormant seeds. It may be stimulated, however,by increasing the oxygen tension or by reducing normal respiratorycompetition with respiratory inhibitors.     

Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O ₂ ^? ) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN₃). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.     

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.     

The development of structure and function in chloroplasts of greening mutants of Scenedesmus I. Formation of chlorophyll

Several mutant strains of Scenedesmus obliquus have been isolatedwhich show only traces of chlorophyll when grown heterotrophicallybut, when transferred to light, develop chlorophyll in amountsequivalent to the wild-type strain. The action spectrum forgreening of one of these mutants, C-2A', while in a stationarygrowth phase, revealed a predominant influence of blue lightwith a maximum effectiveness around 460 nm; possible involvementof either carotenoids or flavoproteins in the greening responseis suggested. The greening process is absolutely dependent uponlight and oxygen and the kinetics demonstrate two distinctivephases. The initial phase is blue-light dependent and apparentlylinked energeticalty to the "respiratory" capacity of the cell,whereas the second one depends upon the increased photosyntheticcapacity resulting from the initial stage of chlorophyll production.Chlorophyll synthesis ceased completely when cultures were transferredto the dark but recommensed at the same rate when once againplaced in the light. The onset of greening is accompanied byan enhancement of respiration and of polysaccharide utilization.The rate of greening of C-2A' is markedly influenced by temperaturein the range of 20 to 30°C. Greening is prevented by anaerobicconditions, potassium cyanide, dinitrophenol, sodium arsenite,sodium fluoroacetate and sodium azide. Inhibition of the secondphase also results when photosynthesis is prevented, i.e., byDCMU inhibition or carbon dioxide exclusion. Possible mechanismsfor greening in this mutant are discussed. ¹ A portion of the results presented here was reported at the"International Seminar on Physiology of Differentiation in Plants",Simla, India, May 1971. (Received February 22, 1972; )     

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.     

Differential Effect of Respiratory Inhibitors on Ergosterol Synthesis by Saccharomyces cerevisiae During Adaptation to Oxygen

The effect of different respiratory inhibitors on the ergosterol content of microaerobically grown non-proliferating yeast cultures was monitored during adaptation to oxygen. It was found that dinitrophenol, azide, and cyanide, which act on the mechanism of the respiratory chain, cause a marked stimulation of sterol production. Acriflavine and chloramphenicol, which affect the synthesis of the respiratory apparatus, caused a delay in the onset of ergosterol synthesis or a marked decrease in sterol content. The data obtained provide presumptive evidence that a component of sterol formation is synthesized on the 70S ribosomal system of the mitochondrion and induced in the presence of oxygen.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : V. CYTOCHROME OXIDASE ACTIVITY IN THE EGGS OF ARBACIA PUNCTULATA

1. An enzyme capable of oxidizing reduced cytochrome c (i.e. a cytochrome oxidase) has been obtained from Arbacia eggs. In 0.02 M hydroquinone, the cytochrome oxidase was half activated at a cytochrome c concentration of approximately 4 x 10^–6 M. The concentration of the cytochrome oxidase was found to be nearly the same in unfertilized and fertilized eggs, the amount of the enzyme—as measured by means of its activity toward cytochrome c as a representative substrate—being more than sufficient to account for the highest rate of oxygen utilization yet observed in the intact, living, fertilized eggs, and of the same order as that in certain rat tissues. 2. The Arbacia cytochrome oxidase was strongly inhibited by carbon monoxide in the dark, the inhibition being almost completely reversed by light. The inhibition constant was not greatly altered by variation in the concentration of cytochrome c or the concentration of hydroquinone used as reductant for the cytochrome c, having a value of 3 to 5 under the conditions used. The inhibition constant was about 2 with p-phenylenediamine as reductant for the cytochrome c, but apparently had the surprisingly low value of about 0.5 with 0.02 M cysteine as reductant. 3. The cytochrome oxidase was completely inhibited by sufficiently high concentrations of sodium cyanide, sodium azide, and sodium sulfide. It was also completely inhibited in 0.6 M sodium chloride. It was not inhibited by two inhibitors of copper containing enzymes, 8-hydroxyquinoline and sodium diethyldithiocarbamate. It was also not significantly inhibited by 2,4-dinitrothymol, 2,4-dinitro-o-cyclohexylphenol, phenylurethane, 5-isoamyl-5-ethylbarbituric acid, or iodoacetic acid. 4. Quantitative examination of the fertilized eggs showed that cytochrome c, if present at all, occurred in a concentration of less than 2 micrograms per gram of wet fertilized Arbacia eggs. On the basis of these data and those of Fig. 2, above, it seems safe to conclude that cytochrome c cannot carry a significant fraction of the oxygen consumption of fertilized Arbacia eggs. It was also found that, in contrast to similar preparations from certain other animal tissues, the Arbacia cytochrome oxidase preparation displayed no succinic dehydrogenase activity when tested manometrically in the presence of excess cytochrome c. 5. Extending previously reported (3) experiments with other inhibitors, the effects of sodium azide and sodium sulfide on the respiration and cell division of fertilized Arbacia eggs were determined, the eggs being initially exposed to the reagents 30 minutes after fertilization at 20°C. With either reagent cleavage was completely blocked by a concentration of reagent which reduced the respiration to approximately 50 per cent of the normal level. 6. On the basis of certain theoretical considerations regarding the possible mechanism of action of cyanide and other respiratory inhibitors it is suggested that a fraction of the respiration apparently concerned with supplying energy for division processes in the fertilized Arbacia egg may be keyed into the respiratory cycle through a carrier having a somewhat higher potential than those which carry the larger portion of the egg respiration. The theory is also employed in an effort to resolve a number of hitherto apparently paradoxical observations regarding the effects of cyanide, azide, and carbon monoxide on cell respiration.     

Rates of male pronuclear enlargement in sea urchin zygotes

Rates of male pronuclear enlargement were determined using synchronous populations of fertilized sea urchin eggs (Arbacia punctulata and Lytechinus variegatus), fixed at regular intervals following insemination and stained with Hoechst 33342. The rate of male pronuclear expansion in both Arbacia and Lytechinus zygotes was linear and significantly affected by polyspermy, temperature, metabolic inhibitors (dinitrophenol, potassium cyanide, sodium azide, antimycin A, and oligomycin), and blockage of cytoplasmic alkalinization. Inhibitors of protein synthesis (emetine and puromycin) and disrupters of cytoskeletal elements (colchicine, nocodazole and cytochalasin B) had no effect on the rate of male pronuclear enlargement.     

Electrical properties of Neurospora crassa. Respiration and the intracellular potential

The internal potential of Neurospora appears to have two components, one (a) which is reduced by anoxia or abolished by respiratory inhibitors such as azide and 2,4-dinitrophenol, and (b) a fraction that remains in the presence of respiratory inhibitors and is sensitive to the external potassium concentration. Under standard conditions 1 mM azide or dinitrophenol diminishes internal potentials from near -200 mv to about -30 mv within 1 minute and at a maximal rate of 20 mv/second. The internal potential usually recovers within 10 minutes after the inhibitor has been removed. The effect of carbon monoxide on the internal potential is similar to that of azide or dinitrophenol, but can be reversed by visible light, specifically of the wavelengths (430 mµ and 590 mµ) known to decompose cytochrome-CO complexes in yeast. Respiration and internal potentials vary proportionally with azide concentration, but dinitrophenol at low (3 x 10^-6 M) concentrations enhances oxygen consumption without affecting the internal potential. In the presence of 0.1 mM calcium, the fraction of the internal potential which persists during respiratory inhibition increases (becomes more negative) about 30 mv for each tenfold decrease of external potassium over the range 10 to 0.1 mM. The surface resistivity of Neurospora, normally about 5000 ohm.cm², is unchanged by respiratory inhibitors during the period of rapid potential shift.     

The Effect of Respiratory Inhibitors and Chelating Agents on the Frequencies of Chromosomal Aberrations Produced by X-Rays in Vicia

Nitrosophenylhydroxylamine-ammonium (cupferron), potassium cyanide, sodium azide, ethylenediaminetetraacetate (EDTA), α,α'-dipyridyl, and o-phenanthroline were tested (1) for their ability to enhance the frequencies of chromosomal aberrations produced by x-rays in the root tip cells of the broad bean, Vicia faba, and (2) for their ability to inhibit oxygen consumption of excised roots of the same plant. In all cases a close correlation was found between the inhibitory effect on respiration and the enhancement of the sensitivity to x-rays at low oxygen pressures. EDTA, dipyridyl, and o-phenanthroline did not affect respiration to any greater extent, and they were without influence on the radiosensitivity. Cyanide, azide, and cupferron, which strongly inhibited respiration, also increased the frequencies of chromosome aberrations produced by x-rays at low oxygen pressures. The relation between oxygen concentration and radiosensitivity was determined both in the presence and the absence of the respiratory inhibitor cupferron. When cupferron was present, the radiosensitivity was influenced by oxygen concentrations 30 times lower than those effective in the absence of the inhibitor. In an atmosphere of pure oxygen, an increase of radiosensitivity of about 20 per cent was obtained with cupferron, EDTA, and potassium cyanide.     

Interactions between Mitochondria and Chloroplasts in Cells: I. Action of Cyanide and of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea on the Spore of Funaria hygrometrica

The effects of cyanide and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of intact chlorophyllic moss (Funaria hygrometrica) spore was investigated. Thirty micromolar cyanide strongly inhibited dark respiration, was without effect on photosynthesis at high light intensities (above the saturation plateau values), and stimulated photosynthesis at low light intensities (below the saturation plateau values). Three hundred nanomolar DCMU inhibited the photosynthesis and was without effect, even under light conditions, on the dark respiration. It seems likely, therefore, that in the chlorophyllic moss spore the cytochrome oxidase pathway is not functioning under high light intensities unless the photosynthesis is inhibited by DCMU.     

Cyanide and sulfide interact with nitrogenous compounds to influence the relaxation of various smooth muscles

Sodium nitroprusside relaxed guinea pig ileum after the segment had been submaximally contracted by either histamine or acetylcholine, intact isolated rabbit gall bladder after submaximal contraction by either acetylcholine or cholecystokinin octapeptide, and rat pulmonary artery helical strips after submaximal contraction with norepinephrine. In each of these cases the relaxation produced by nitroprusside was at least partially reversed by the subsequent addition of excess sodium cyanide. Cyanide, however, in nontoxic concentrations did not reverse the spasmolytic effects of hydroxylamine hydrochloride, sodium azide, nitroglycerin, sodium nitrite, or nitric oxide hemoglobin on guinea pig ileum, nor did cyanide alone in the same concentrations have any effect. The similar interaction between nitroprusside and cyanide on rabbit aortic strips is not dependent on the presence of an intact endothelial cell layer. Also, on rabbit aortic strips and like cyanide, sodium sulfide reversed the spasmolytic effects of azide and hydroxylamine, but it had little or no effect on the relaxation induced by papaverine. Unlike cyanide, however, sulfide augmented the relaxation induced by nitroprusside, and it reversed the effects of nitric oxide hemoglobin, nitroglycerin, and nitrite. A direct chemical reaction between sulfide and nitroprusside may account for the difference between it and cyanide. Although evidence was obtained also for a direct chemical reaction between sulfide and norepinephrine, that reaction does not seem to have played a role in these results. These observations suggest the existence of at least three distinct subclasses of so-called nitric oxide vasodilators. At least in some cases cyanide and sulfide cannot be acting by the same mechanism in their modifications of the responses to the agonists.