Cyanide Resistance in Achromobacter I. Induced Formation of Cytochrome a(2) and Its Role in Cyanide-Resistant Respiration.

Arima, Kei (University of Tokyo, Tokyo, Japan), and Tetuo Oka. Cyanide resistance in Achromobacter. I. Induced formation of cytochrome a(2) and its role in cyanide-resistant respiration. J. Bacteriol. 90:734-743. 1965.-By following the cytochrome concentrations during the growth cycle and under various conditions (aerobic, aerobic plus KCN, reduced aeration, anaerobic plus NaNO(3)) in Achromobacter strain D, a close relationship between the formation of cytochrome a(2) (and a(1)) and the difficulty of oxygen utilization was demonstrated. Cytochrome o, which was the only oxidase found in aerobic log-phase cells, was present in bacterial cells grown under various conditions; the amount present had no relation to the degree of cyanide resistance. On the other hand, cytochrome a(2) (and a(1)) was inducible, and a close relation was observed between the amount of cytochrome and resistance to cyanide. Spectrophotometric observations indicated that, among the cytochromes present in resistant cells, cytochrome a(2) could be oxidized most easily in the presence of cyanide and that cytochrome b(1) could be oxidized without the oxidation of cytochrome a(1). We concluded that cytochrome a(2) is a cyanide-resistant oxidase capable of catalyzing the oxidation of cytochromes in the presence of cyanide. Cytochrome a(2) is also resistant to azide, an inhibitor of cytochrome oxidase.     

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

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

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.     

Oxygen reactivity of both respiratory oxidases in Campylobacter jejuni: the cydAB genes encode a cyanide-resistant, low-affinity oxidase that is not of the cytochrome bd type

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K(m) = 0.8 microM) and a V(max) of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb'-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K(m) value of 40 nM and a V(max) of 6 to 9 nmol/mg/s. Low-temperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.     

The respiratory responses to cyanide of a cyanide-resistant Klebsiella oxytoca bacterial strain

The respiratory system of a cyanide-resistant Klebsiella oxytoca was analyzed by monitoring the changes in the cytochrome contents in response to various inhibitors in the presence of various concentrations of cyanide. The cells grown in the medium without cyanide (KCN) have two terminal oxidases, cytochrome d (Ki = 10(-5) M KCN) and o (Ki = 10(-3) M KCN). When cells were grown on medium with 1 mM KCN, the expression of both b-type cytochrome and cytochrome d in the plasma membranes of the cell decreased by more than 50%, while cytochrome o increased by 70%, as compared with the cells grown in the absence of KCN. Two terminal oxidases with Ki values of about 10(-3) M and 1.7 x 10(-2) M KCN were observed in the plasma membrane fractions of the cells growing on KCN enriched medium. 2-n-Heptyl-4-hydroxyquinoline-N-oxide markedly inhibited the oxidation of NADH by the plasma membranes from the cells grown in the medium without KCN, but not in those plasma membranes from KCN-grown cells. The NADH oxidases in plasma membranes of K. oxytoca grown with and without KCN were equally sensitive to UV irradiation. Adding freshly isolated quinone to the UV-damaged plasma membranes restored the NADH oxidase activity from both types of plasma membranes. From these results, we propose the presence of a non-heme type of terminal oxidase to account for the KCN resistance in K. oxytoca.     

Cyanide- and hydroxamate-resistant respiration in Neurospora crassa.

Strain inl-89601 of Neurospora crassa respires exclusively by means of the mitochondrial cytochrome chain. The respiration of this strain is entirely inhibited by cyanide or antimycin A, the classical inhibitors of cytochrome chain respiration. When this strain was grown in the presence of chloramphenicol, however, two additional terminal oxidases were detected. One of these oxidases is inhibited by substituted hydroxamic acids and has been described previously. The second oxidase was not inhibited by cyanide or hydroxamic acid but was inhibited by azide in the presence of both cyanide and hydroxamic acid. This azide-sensitive respiration was due to a single respiratory pathway with a Ki for azide of 200 micrometer. A small amount of azide-sensitive respiration was detected in mitochondrial fractions obtained from chloramphenicol-treated cells, and it is likely that the azide-sensitive oxidase is localized in the mitochondrion. The determinants for the azide-sensitive and hydroxamate-sensitive oxidases segregate in a Mendelian manner in crosses and are either unlinked or not closely linked to each other.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

Respiratory system of Gluconacetobacter diazotrophicus PAL5. Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases

In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q(10), Q(9) and PQQ in a 13:1:6.6 molar ratios. UV(360 nm) photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a(1)-CO adducts. Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 microM while cytochrome a(1) (442 nm) was not affected. A KCN-sensitive (I(50)=5 microM) cytochrome bb and a KCN-resistant (I(50)=450 microM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.     

Ferrous iron oxidation in moderately thermophilic acidophile Sulfobacillus sibiricus N1(T)

The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe2(+) to O? is terminated by 2 physiological oxidases: aa?-type cytochrome, which dominates in the early-exponential phase of growth, and bo?-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa? was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 μmol·L?1, respectively, compared with K(i) values for cytochrome bo?, which were 9.5 μmol·L?1 for cyanide and 7.0 μmol·L?1 for azide. This is the first evidence for the participation of a bo?-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b???.     

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.     

Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent.     

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.     

The Respiratory Chain of Plant Mitochondria. I. Electron Transport Between Succinate and Oxygen in Skunk Cabbage Mitochondria

The kinetics of oxidation of ubiquinone, flavoprotein, cytochrome c, and the cytochrome b complex in skunk cabbage (Symplocarpus foetidus) mitochondria made anaerobic with succinate have been measured spectrophotometrically and fluorimetrically in the absence of respiratory inhibitor and in the presence of cyanide or antimycin A. No component identifiable by these means was oxidized rapidly enough in the presence of one or the other inhibitor to qualify for the role of alternate oxidase. Cycles of oxidation and rereduction of flavoprotein and ubiquinone obtained by injecting 12 mum oxygen into the anaerobic mitochondrial suspension were kinetically indistinguishable in the presence of cyanide or antimycin A, implying that these 2 components are part of a respiratory pathway between succinate and oxygen which does not involve the cytochromes and does involve a cyanide-insensitive alternate oxidase. The cytochrome b complex shows biphasic oxidation kinetics with half times of 0.018 sec and 0.4 sec in the absence of inhibitor, which increase to 0.2 sec and 1 sec in the presence of cyanide. In the presence of antimycin A, the oxidation of the cytochrome b complex shows an induction period of 1 sec and a half-time of 3.5 sec. A split respiratory chain with 2 terminal oxidases and a branch point between the cytochromes and flavoprotein and ubiquinone is proposed for these mitochondria.     

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).     

Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex

The aerobic respiratory chain of Escherichia coli is branched and contains two terminal oxidases. The chain predominant when the cells are grown with low aeration terminates with the cytochrome d terminal oxidase complex, and the branch present under high aeration ends with the cytochrome o terminal oxidase complex. Previous work has shown that cytochrome d complex functions as a ubiquinol-8 oxidase, and that a minimal respiratory chain can be reconstituted in proteoliposomes with a flavoprotein dehydrogenase (pyruvate oxidase), ubiquinone-8, and the cytochrome d complex. This paper demonstrates that the cytochrome o complex functions as an efficient ubiquinol-8 oxidase in reconstituted proteoliposomes, and that ubiquinone-8 serves as an electron carrier from the flavoprotein to the cytochrome complex. The maximal turnover (per cytochrome o) achieved in reconstituted proteoliposomes is at least as fast as observed in E. coli membrane preparations. Electron flow from the flavoprotein to oxygen in the reconstituted proteoliposomes generates a transmembrane potential of at least 120 mV, negative inside, which is sensitive to ionophore uncouplers and inhibitors of the terminal oxidase. These data demonstrate the minimal composition of this respiratory chain as a flavoprotein dehydrogenase, ubiquinone-8, and the cytochrome o complex. Previous models have suggested that cytochrome b556, also a component of the E. coli inner membrane, is required for electron flow to cytochrome o. This is apparently not the case. It now is clear that both of the E. coli terminal oxidases act as ubiquinol-8 oxidases and, thus, ubiquinone-8 is the branch point between the two respiratory chains.     

Resistance of Candida parapsilosis to drugs

Several strains of Candida parapsilosis, isolated independently in our laboratory, had their resistance compared to a series of inhibitors which act either at the level of mitochondrial ribosomes (chloramphenicol, erythromycin, paromomycin) or at the level of mitochondrial respiration and oxidative phosphorylation (oligomycin, antimycin A, diuron, carbonylcyanide m-chlorophenylhydrazone). Cells were grown on glycerol media supplemented with one of these inhibitors, and it was demonstrated that the resistance of these yeasts to a large spectrum of antibiotics was due to several features: a resistance to oligomycin was found at the permeation level; the resistance to the other drugs was correlated to the relative insensitivity of cytochrome biosynthesis to the drugs; the cells developed, at the same time, two types of alternative pathways: the one branched at the ubiquinone level which drove electrons from Krebs cycle substrates to oxygen, and the other, antimycin A-insensitive but inhibited by amytal, salicylhydroxamic acid and high cyanide concentrations. This secondary mitochondrial pathway, driving reducing equivalents from cytoplasmic NADH to cytochrome c and then to cytochrome aa3 or to alternate oxidase, allowed the growth of Candida parapsilosis on a non fermentescible medium, supplemented with these drugs.