Acetate oxidation by bloodstream forms of Trypanosoma cruzi.

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was a sensitive to antimycin A inhibition as was CO2 production. There was a 6--7% residual O2 uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.     

Acetate Oxidation by Bloodstream Forms of Trypanosoma cruzi*

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was as sensitive to antimycin A inhibition as was CO₂ production. There was a 6–7% residual O₂ uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity

The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K_i of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path.     

Gene mutation eliminating antimycin A-tolerant electron transport in Ustilago maydis

A class of mutants of Ustilago maydis selected on a fungitoxic oxathiin lack of antimycin A-tolerant respiratory system which is present in wild-type cells. This system provides, directly or indirectly, for considerable resistance to antimycin A because growth of mutant cells lacking the system is much more sensitive to the antibiotic than that of the wild type. Antimycin A-sensitive O(2) uptake and growth is found in half of the progeny from crosses of mutant to wild type. All antimycin A-sensitive segregants are somewhat more resistant to oxathiins than the antimycin A-resistant segregants. The respiration of the mutant is strongly inhibited by cyanide and azide at concentrations which stimulate respiration of the wild type. Respiration of both mutant and wild type is about equally inhibited by rotenone. It appears that the mutation alters some component of the respiratory system located between the rotenone inhibition site and the antimycin A inhibition site that permits shift of electron transport to an alternate terminal oxidase when the normal electron transport pathway is blocked.     

成熟和褐变荔枝果实呼吸作用和脂氧合酶活性

荔枝果实完全成熟和果皮变鮮红时,呼吸速率降低,仅相当于果皮带绿时的39.4％。此时果皮和果肉的脂氧合酶活性亦明显降低,分别相当于后者的60.2％和49.1％。成熟荔枝果实果皮呼吸作用对KCN抑制敏感。2mM KCN抑制果皮总呼吸的91.8％,而仅抑制果肉的56.9％。荔枝果皮呼吸的电了传递主要是通过细胞色素氧化酶途径,而果肉則可能一半是通过其它氧化酶途径。2mKCN和1.5mM SHAM抑制成熟果皮总呼吸97.9％,为SHAM抑制的交替途径呼吸占总呼吸5.28％。相同浓度KCN和SHAM抑制褐变果皮总呼吸79.7％,则SHAM抑制的交替途径呼吸占27.1％。果实褐变时,果成交替途径呼吸比例增高。这一变化可能促进H_2O_2积累、乙烯产生和果皮褐变深化。     

Contribution of cyanide-insensitive respiratory pathway, catalyzed by the alternative oxidase, to citric acid production in Aspergillus niger

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 microM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.     

Characterization of alternative respiratory pathways in the yeast Schwanniomyces castellii by the study of mutants deficient in cytochromes a+a3 and/or b.

After a general review of the proposed mechanisms and physiological roles of the alternative respiratory pathways found in various organisms, the studies are focussed on the amylolytic yeast Schwaniomyces castellii. In addition to the cytochrome chain, the wild type presents two alternative pathways insensitive to antimycin A. One is salicylhydroxamic acid (SHAM)-sensitive and azide-insensitive; the other is SHAM-insensitive and sensitive to high azide concentration. Conditions for mutagenesis and screening are described, which allow isolation of mutants deficient in cytochromes a+a3 and/or b in this yeast previously classified as petite negative. The relative proportions of the alternative respiratory pathways are compared in the wild type and mutant strains following inhibition by SHAM and azide at optimal concentration as determined by iso-inhibition curves. The growth of the cytochrome deficient mutants on citrate, a non-fermentable carbon source, and the ability of the wild type to grow on citrate+antimycin A, after a lag of about 10 h, indicate an involvement of the alternative pathway(s) in energy production. Rotenone sensitivity of respiration and ATP level confirm the presence of a functional phosphorylation site 1. The role of each alternative respiratory pathway in energy production is discussed.     

Influence of the alternative respiratory pathway on the adenylate pools in heterotrophic Euglena gracilis

Etiolated Euglena gracilis Pringsheim, strain Z, were cultured in a lactate medium either in the presence of 2 μ M antimycin A for cells adapted to this inhibitor, or in the absence of antimycin A for controls. The adenylates (ATP, ADP and AMP) and the energy charge (EC) were followed during the growth of both types of cells. The effects of KCN, salicylhydroxamic acid (SHAM) and rotenone on the respiration and the adenylate pool, were investigated during the exponental and stationary phases. EC values of controls and antimycin-adapted cells were not significantly different during culture. In the logarithmic phase, EC of controls was unaffected by 3 m M SHAM, an inhibitor of the alternative pathway, but markedly decreased by 0.3 m M KCN, which inhibits the cytochrome pathway. In contrast, in antimycin-adapted Euglena , in which the cytochrome pathway was blocked, ATP content and EC were markedly lowered in the presence of SHAM but slightly increased by 0.3 m M KCN. The combination of the preceeding treatments, as well as 15 m M KCN alone, were deleterious for both types of cells, in the logarithmic and the late stationary phases. The data indicate that the energy level in Euglena was dependent on the alternative pathway when the cytochrome pathway was blocked. Such dependence could be explained by the engagement of the first rotenone-sensitive site of phosphorylation. Indeed, 50 μ M rotenone caused a similar relative decrease of oxygen consumption and ATP content in controls and in antimycin-adapted Euglena . In the absence of cytochrome respiration, the alternative pathway allowed electrons to flow through this first segment of the respiratory chain, and ATP production by the first site of phosphorylation.     

The operation of the alternative electron-leak pathways mediated by cytochrome c in mitochondria

Low (1 x 10(-9)M) concentrations of cytochrome c inhibit H2O2 production in cytochrome c-depleted mitochondria, purified succinate-cytochrome c reductase (SCR) and antimycin A inhibited cytochrome c-depleted HMP. At higher concentration (2 x 10(-6)M), cytochrome c eliminates pre-existed H2O2 if feeding electrons to it by succinate. Cytochrome c also decreases the OH* produced by succinate-cytochrome c reductase oxidizing succinate. We conclude that the alternative electron-leak pathway mediated by cytochrome c operates very well. In the presence of antimycin A, ferrocytochrome c can suppress the generation of H2O2 in SCR system, but ferricytochrome c cannot. Similar results are obtained on the elimination of pre-existed H2O2 by cytochrome c. For hydroxyl radical, antimycin A abolishes the suppression caused by both ferrocytochrome c and ferricytochrome c. These results indicate that the reductive state of cytochrome c caused by electron-flow is necessary and sufficient for the operation of cytochrome c-mediated electron-leakage pathway.     

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.     

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

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.     

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth,respiration and protein biosynthesis

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O₂ consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.Abbreviations DNP dinitrophenol - PG propylgallate - SHAM salicylhydroxamic acid     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

A study of cyanide-insensitive respiration in the genus Dekkera and Brettanomyces

Dekkera intermedia and Brettanomyces custersii were shown to have a respiratory pathway resistant to cyanide, antimycin A, and azide. This respiration remained sensitive to salicylhydroxamic acid (SHAM). The "cyanide-resistant" respiration was induced mainly at the end of the growth phase and could reach 50% of total respiratory capacity. The mitochondrial "petite colony" mutation had no effect on this oxidation pathway. The presence of this respiration pathway in these strains constitutes a compensation mechanism for the reducing activity of acetaldehyde dehydrogenase. This alternate pathway would thus be a fundamental element of the Custer effect, a characteristic feature of these strains.     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H2O2-producing side-reaction of respiratory complex I with O2

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation kinetics for O2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O2-affinity system (Km app=1.5 microM) was abolished by low cyanide concentration; it corresponded to cytochrome aa3. The low-O2-affinity system (Km app=120 microM) was resistant to cyanide. Similar O2 saturation kinetics, using succinate or ascorbate-TMPD as electron donor, showed only the high-O2-affinity cyanide-sensitive component. Horse cytochrome c increased 2-3 times the rate of electron flow across the cyanide-sensitive pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant amounts of H2O2 (at least 10% of the total O2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H2O2. Production of H2O2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O2, producing H2O2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.