Low Concentrations Of Cyanide Research Articles

Determination of manganese(II) in cyanide medium by volumetric and coulometric titration with hexacyanoferrate(III)

A volumetric titration of manganese(II) may be carried out with hexacyanoferrate(III) in cyanide medium under an inert atmosphere with a potentiometric, amperometric or biamperometric end-point. In very strong NaCN solution or at high temperature, positive errors are produced by the Fe(CN)63–-CN– reaction. At lower cyanide concentrations errors become negative owing to manganese(II) oxidation with dissolved oxygen. In 2.0 M NaCN solution, 1–5 mmol of manganese(II) may be titrated with a 0.2–0.3% relative error. A relative standard deviation for a 2.5-mmol determination is 0.14%. Foreign ion effects were studied and methods for avoiding interferences from Fe(II), Fe(III) and V(IV) are described. The proposed procedure was applied to slag analysis.Coulometric titration of 2.5–100 µmol of manganese(II) with electrogenerated hexacyanoferrate(III) may be carried out in 1.0 M NaCN solution with relative errors lower than 0.4%. The relative standard deviation for the determination of about 120 µmol of manganese(II) is 0.01%.

KINETICS AND MECHANISM OF THE REACTION BETWEEN MONO ((ETHYLENEDIOXY) DIETHYLENEDINITRILO)-TETRAACETATODINICKELATE(II) AND CYANIDE ION

Abstract The kinetics of ligand substitution at the binuclear nickel(II) chelate Ni2L, where L is EGTA ((Ethylenedioxy)diethylenedinitrilo)tetraacetic acid with cyanide ion has been investigated spectro-photometrically. The reaction conditions were pH = 11.0 ± 0.1, μ = 0.1 M (NaClO4) and T = 25 ± 0.1°. The formation of Ni(CN)4 2- is first order in Ni2L and first-order in cyanide over a large range of cyanide concentration, but below 10−3 M the reaction becomes zero order in cyanide. These results indicate the presence of a slow step in which Ni2 L dissociates to give NiL2-n and Ni(aq) 2+ at low cyanide concentrations and a cyanide assisted rapid dissociation of Ni2L to produce NiL(CN)x 2-n-x and Ni(aq) 2+ at higher cyanide concentrations. These species react with excess cyanide finally producing Ni(CN)4 2-. On the other hand, reaction of Ni(CN)4 2- with EGTA is first-order in both reactants and inverse-order in cyanide. These results lead to a five step mechanism in which the fourth step has been identifi...

Kinetic aspects of the reaction of aquaethylenediaminetetraacetatoferrate(III) with cyanide ion

The kinetics and mechanism of reaction between ethylenediaminetetraacetatoferrate(III) and cyanide ion at pH= 10.5, 25 °C and I=0.25 M have been investigated spectrophotometrically at 395 nm. The reaction consists of three observable stages. The first involves the formation of [Fe(C-N)5(OH)]3−, the second is the conversion of [Fe(CN)5(OH)]3− into [Fe(CN)6]3− and the third is its reduction to [Fe(CN)6]4− by oxidation of EDTA. The first reaction shows a variable order dependence on the concentration of cyanide, ranging from one at high cyanide concentration to three at low cyanide concentration. The second reaction exhibits first order dependence on the concentration of [Fe(CN)5(OH)]3− as well as in cyanide; while the third reaction follows over-all second order kinetics, first order each in [Fe(CN)6]3− and EDTA released in the reaction. This reaction is highly dependent on hydroxide ion concentration. The reverse reaction between [Fe(CN)5(OH)]3− and EDTA shows an inverse first-order dependence on cyanide concentration along with first-order dependence each on [Fe(C-N)5(OH)]3− and EDTA concentrations. A five-step mechanism is proposed for the first reaction.

Mixed liganded species in Native β-helix p-hemocyanin

It is known that addition of low concentrations of cyanide to oxy hemocyanin causes a rapid bleaching of the absorption band, which has been related to the displacement of oxygen [1–3]. Removal of copper is known to require higher concentrations of cyanide and occurs with different kinetics in the presence or absence of other ligands at the site [4,5]. In this communication, preliminary results of a study concerning the competition between O 2, CO and CN − for the active site of Helix p-hemocyanin are reported. The replacement of these ligands occurs in different ways and may be related to the different modes of binding at the active site. In the case of oxygenated hemocyanin a hyperbolic displacement of oxygen by cyanide is observed, the apparent partition coefficient being independent of hemocyanin concentration in the range 10–50 μ M. In the case of CO displacement by cyanide, the shape and position of the curve depend on protein concentration and at higher hemocyanin concentration a ‘lag-phase’ followed by a hyperbolic displacement is observed. This behaviour has been tentatively interpreted assuming that at low concentration of cyanide binding of the ligand to CO-saturated hemocyanin may occur to one of the two metals in a site, while CO remains bound to the other. The results of experiments in which the addition of small amounts of KNC to hemocyanin partially saturated with O 2 and CO causes an increase of CO-emission intensity and a concomitant decrease of O 2copper absorption band are fully consistent with this hypothesis. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin [6] and the formation of the complex is reversible on dialysis or oxygenation. Since mixed-liganded intermediates have also been observed in the case of half-met hemocyanin, in which one of the metals is cupric [7], it is suggested that the geometries of the active site of such species and of native hemocyanin may be quite similar.

The effects of cyanide on the growth and respiration of Enterobacter aerogenes in continuous culture.

The effect of cyanide on the physiology of lactate- and oxygen-limited Enterobacter aerogenes NCTC 10336 was studied in chemostat culture (D = 0.1 h-1). In the absence of cyanide, the molar growth yield from oxygen (YO2) under oxygen limitation was 60% of the carbon-limited value. A similar decrease in yield was observed in a lactate-limited culture (excess oxygen) which was continuously fed low concentrations of potassium cyanide. The cultures with the lower growth yields possessed respiratory systems less sensitive to inhibition by cyanide. This was particularly marked in cultures grown in the presence of cyanide. Increased cyanide resistance was associated with an increase in the concentration of a cytochrome oxidase tentatively identified as a d-type and the appearance of additional cytochromes tentatively identified as b-type.

Proton gradient linkage to active uptake of [3H]acetylcholine by Torpedo electric organ synaptic vesicles.

It has been confirmed that cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit adenosine 5'-triphosphate (ATP) dependent active uptake of [3H]acetylcholine. Active uptake can be completely inhibited by low concentrations of the mitochondrial uncouplers carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, nigericin, gramicidin, valinomycin, and A 23187. Under similar conditions uncouplers stimulate the vesicle adenosinetriphosphatase (ATPase) by from 40 to 80%. ATP-supported uptake of [3H]acetylcholine increases greatly as the external pH is increased from 6.6 to 7.6 and remains approximately constant from pH 7.8 to pH 8.6. The uptake also becomes more selective for [3H]acetylcholine compared to [14C]choline as the pH is increased from 6.6 to 7.6, achieving 12-fold selectively, in a manner similar to the increase in the amount of [3H]acetylcholine taken up. Bicarbonate stimulates both the amount and selectivity of [3H]acetylcholine uptake over the lower pH range, but it has no effect over the higher pH range. Exogenous ammonium ion completely inhibits active [3H]acetylcholine uptake, with lower concentrations of ammonium ion required at higher pH values in a manner consistent with ammonia being the active species. Adenosine 5'-diphosphate and a nonhydrolyzable ATP analogue do not support active [3H]acetylcholine uptake. It is concluded that an ATPase pumps protons into the cholinergic synaptic vesicle to produce an internally acidic and positively charged proton gradient that is linked to [3H]acetylcholine uptake.

Preliminary study of nickel, ferric, lead, and copper oxinates in reactions of reextraction through the exchange of ligands: The use of chloroform solutions of the copper oxinate in the spectrophotometric determination of cyanide

In this paper a systematic study of four metallic oxinates (ferric, nickel, lead, and copper complexes) is carried out, determining some characteristics of the organic solutions of these complexes in reactions of reextraction by the exchange of ligands. These reactions are based on the decoloration produced in organic solutions of colored complexes when they are mixed with aqueous solutions of anions. The study of some characteristics and the qualitative tests shows that only the copper oxinate is interesting for quantitative purposes because its solutions in chloroform are decolored at very low concentrations of oxalate ethylenediamine tetraacetate, sulfide, and cyanide. The application of this study to the indirect spectrophotometric determination of cyanide is carried out. The decoloration varies linearly with the cyanide concentration between 10 and 50 ppm for a phase volume ratio 1:1. Finally, the error, reproducibility of the method, nature of the exchange reaction, and interferences of foreign ions are also established.

Studies on cyclopropane fatty acid synthesis: Correlation between the state of reduction of respiratory components and the accumulation of methylene hexadecanoic acid by Pseudomonas denitrificans

A delay in the onset of accumulation of methylene hexadecanoic acid could be engendered in Pseudomonas denitrificans growing under limited oxygen conditions when the concentration of citrate but not the concentration of succinate in the medium was increased from 0.1 to 0.5%. Ascorbate, which specifically reduced a cytochrome component possessing a maximum absorbance at 551 nm, partially inhibited the accumulation of methylene hexadecanoic acid under conditions which otherwise led to maximal production. Limiting terminal cytochrome oxidase activity by controlling the oxygen supply, or by the use of low concentrations of the oxidase inhibitors cyanide or azide also prevented the accumulation of the fatty acid regardless of the nature or concentration of carbon source in the medium. Measurement of the levels of ATP, NAD and NADH as well as the steady state of reduction of respiratory components in vivo showed that the onset of accumulation of methylene hexadecanoic acid could be specifically correlated with the state of reduction of respiratory components. The uniqueness of succinate respiration in promoting the synthesis of cyclopropane synthetase (unsaturated-phospholipid methyltransferase (EC 2.1.1.16)) under limited oxygen conditions could therefore be assigned to the high degree of oxidation of respiratory components observed under this condition.

Cyanide degradation by immobilised fungi

Cyanide hydratase, which converts cyanide to formamide, was induced in mycelia of Stemphylium loti by growth in the presence of low concentrations of cyanide. Mycelia were immobilised by several methods. The most useful system was found to be treatment with flocculating agents. This technique is applicable to a wide range of easily isolated fungi that contain cyanide hydratase.

ECE reaction pathways in the electrochemical reduction of dicyanocobalamin Kinetics of ligand substitution in vitamin B12r cyanocob(II)alamin

The reduction of dicyanocob(III)alamin leads in a first stage to monocyanocob(II)alamin which can be partially converted into the base-off and base-on Co(II) complexes (B12r). The latter species are easier to reduce than the starting Co(III) complex leading to a single two-electron wave at low cyanide concentrations and/or low diffusion rates. Upon raising one of these two parameters two successive one-electron waves tend to be obtained corresponding to the Co(III)/Co(II) and Co(II)/Co(I) conversion respectively. The kinetics of the reduction process is investigated using potential-dependent potentiostatic chronoamperometry which allows a simpler analysis than cyclic voltammetry for systems involving a slow initial charge-transfer step. It is seen that the second electron, at the level of the first wave, comes from the electrode and not from the cyano-Co(II) complex in the solution. The reduction thus follows an ECE rather than a DISP-type mechanism in conditions where they can be distinguished by the usual electrochemical kinetic techniques. This contrasts with that which occurs in organic electrochemistry where the electron transfers are generally fast, while in the present case they are slow. The analysis of the reduction kinetics as a function of cyanide concentration gives some insight into the mechanism of the ligand substitution reaction at the Co(II). The kinetic data are discussed in terms of SN1-, SN2- and SNAr-like mechanisms.

ECE reaction pathways in the electrochemical reduction of dicyanocobalamin: Kinetics of ligand substitution in vitamin B12r cyanocob(II)alamin

The reduction of dicyanocob(III)alamin leads in a first stage to monocyanocob(II)alamin which can be partially converted into the base-off and base-on Co(II) complexes (B12r). The latter species are easier to reduce than the starting Co(III) complex leading to a single two-electron wave at low cyanide concentrations and/or low diffusion rates. Upon raising one of these two parameters two successive one-electron waves tend to be obtained corresponding to the Co(III)/Co(II) and Co(II)/Co(I) conversion respectively. The kinetics of the reduction process is investigated using potential-dependent potentiostatic chronoamperometry which allows a simpler analysis than cyclic voltammetry for systems involving a slow initial charge-transfer step. It is seen that the second electron, at the level of the first wave, comes from the electrode and not from the cyano-Co(II) complex in the solution. The reduction thus follows an ECE rather than a DISP-type mechanism in conditions where they can be distinguished by the usual electrochemical kinetic techniques. This contrasts with that which occurs in organic electrochemistry where the electron transfers are generally fast, while in the present case they are slow. The analysis of the reduction kinetics as a function of cyanide concentration gives some insight into the mechanism of the ligand substitution reaction at the Co(II). The kinetic data are discussed in terms of SN1-, SN2- and SNAr-like mechanisms.

Patterns of 14C-Leucine uptake in red spruce and larch seedlings

Summary Incorporation of 14 C-L-leucine into young spruce seedlings ( Picea abies ), a process innibited by NaF, FCCP and azide, is less sensitive to low cyanide concentrations than for larch ( Laris decidua ) seedlings, in keeping with previous observations on biosynthetic capacity.

Carbon monoxide oxidation by methanogenic bacteria.

Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.

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 mV ) and two cytochromes of c type ( E′ 0 = +342, +94 mV ). A clear indication of the involvement in respiration of cytochrome b 413 ), cytochrome c 342 and cytochrome b 47 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 b 47 . 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.

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na + and K + by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions. 2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin. 3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value. 4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis. 5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.

Oxidation of organic C1 compounds by Hyphomicrobium spp.

Washed cell suspensions of Hyphomicrobium spp. were able to oxidize methanol, formaldehyde and formate. This suggested that enzymes for the oxidation of these compounds were present. The pathway of the oxidation of methanol to carbon dioxide and water has been investigated using cell-free extracts. An ammonium-ion-activated, phenazine methosulphate-linked methanol dehydrogenase was detected. This enzyme has a dual substrate specificity for normal primary alcohols and formaldehyde. It has a high pH optimum for activity of 9.5. The pathway is completed by an NAD-linked formate dehydrogenase. This enzyme is inhibited by low concentrations of potassium cyanide, copper sulphate and hypophosphite.

Synergistic uncoupling of spinach chloroplasts by combinations of photophosphorylation inhibitors and carbonyl cyanide p-trifluoromethoxyphenylhydrazone

Combinations of low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhy-drazone (FCCP) with suboptimal concentrations of Dio-9, phloridzin, ajmaline, and dihydrodiscarine B synergistically inhibited cyclic and noncyclic photophosphorylation in spinach chloroplasts but their effects on the light-triggered ATPase were additive rather than synergistic. The effect was reversed by washing and prevented by dithioerythritol and by cistein. Carbonyl cyanide m-chlorophenylhydrazone (CCP) could replace FCCP but uncouplers of other types like atebrin did not substitute for FCCP. Combinations of FCCP with the four inhibitors synergistically uncoupled ferricyanide reduction in the presence of ADP and P i but not in their absence. The synergistic uncoupling was not observed on the light-dependent pH rise of chloroplast suspensions. Association of FCCP with any of the inhibitors completely abolished the stimulation of proton uptake or the inhibition of electron transport induced by low concentrations of ATP. This synergistic and peculiar uncoupling can not be ascribed to a modification of membrane permeability. One possible explanation is that the effect requires a conformational state of the membrane-bound coupling factor 1 (CF 1) induced by phosphorylating conditions which would facilitate the interaction of inhibitors and FCCP with the membrane.

The effects of halothane on hepatic microsomal electron transfer

1. The effects of halothane (CF3CHBrCl), a volatile anaesthetic agent, on electron transfer in isolated rat liver microsomal preparations were examined. 2. At halothane concentrations achieved in tissues during clinical anaesthesia (1-2mM), halothane shifts the redox equilibrium of microsomal cytochrome b5 in the presence of NADPH towards the oxidized form. Halothane accelerates stoicheiometric consumption of NADPH and O2, increases the rate of reoxidation of NADH-reduced microsomal ferrocytochrom b5, but does not affect NADPH- or NADH-cytochrome c reductase activity. The enhanced microsomal electron flow seen in the presence of halothane is not diminished by CO nor is it increased by pretreatment of the animals with phenobarbital. 3. The effects of halothane are maximum in microsomal preparations isolated from animals fed on a high-carbohydrate diet to induce stearate desaturase activity. Changes in microsomal electron transfer caused by halothane are in all cases abolished by low concentrations (1-2mM) of cyanide. Microsomal stearate desaturase activity is unaffected by halothane. 4. The first-order rate constant for oxidation of membrane-bound ferrocytochrome b5 in the absence of added substrate (k1 equals 1.5 times 10(-3)A-1) is similar to that for autoxidation of purified ferrocytochrome b5(k1 equals 7 times 10(-3)S-1) the rate of autoxidation of soluble ferrocytochrome b5 is unaffected by halothane. 5. It is concluded that the effects of halothane on microsomal electron transfer are not related to cytochrome P-450 linked metabolism but rather arise from the interaction of halothane with the cyanide-sensitive factor of the stearate desaturase pathway.

Reduced Ferredoxin:CO2Oxidoreductase FromClostridium pasteurianum.Effect of Ligands to Transition Metals on the Activity and the Stability of the Enzyme

Reduced ferredoxin: CO2 oxidoreductase (CO2-reductase) from Clostridium pasteurianum catalyzes the reduction of CO2 to formate at the expense of reduced ferredoxin, an isotopic exchange between CO2 and formate in the absence of ferredoxin, and the oxidation of formate to CO2 with oxidized ferredoxin. The three activities were found to be equally affected by monovalent anions known to be ligands to transition metals: The enzyme was reversibly inhibited by azide (Ki = 0.004mM), cyanate (Ki = 0.3 mM), thiocyanate (Ki = 1mM), nitrite (Ki = 0.4mM), nitrate (Ki = 6mM), chlorate (Ki = 3mM), fluoride (Ki = 5mM), and by chloride, bromide, iodide (Ki greater than 5mM). There was no observable effect of pH on the inhibition constants. The enzyme was not inhibited by carbon monoxide. The enzyme was irreversibly inactivated by low concentrations (10muM) of cyanide. The rate of inactivation increased with increasing pH with an inflection point near pH 9.5. Reduced ferredoxin and formate rather than oxidized ferredoxin or CO2 protected the enzyme from inactivation by cyanide. The enzyme was protected by azide and cyanate from inactivation. In the presence of high concentrations of the monovalent anions the rate of inactivation by heat (55 degrees C), by molecular oxygen, and by cyanide was decreased by a factor of more than 100. Half maximal protection was observed at the Ki concentrations of the two reversible inhibitors. The data are interpreted to indicate that a transition metal of weak "a class" character and a disulfide are catalytically significant groups of CO2-reductase from C. pasteurianum.

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g. 2. NADH, succinate, d(−)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(−)- and l(+)-lactate dehydrogenases were NAD + and NADP + independent. Ascorbate- N, N, N′, N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases. 3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(−)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(−)-lactate and l(+)-lactate oxidases. 4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation. 5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(−)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide. 6. The apparent K m for O 2 for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O 2.