Alternative Terminal Oxidase Research Articles

Cell physiology of plants growing in cold environments

The life of plants growing in cold extreme environments has been well investigated in terms of morphological, anatomical, and ecophysiological adaptations. In contrast, long-term cellular or metabolic studies have been performed by only a few groups. Moreover, a number of single reports exist, which often represent just a glimpse of plant behavior. The review draws together the literature which has focused on tissue and cellular adaptations mainly to low temperatures and high light. Most studies have been done with European alpine plants; comparably well studied are only two phanerogams found in the coastal Antarctic. Plant adaptation in northern polar regions has always been of interest in terms of ecophysiology and plant propagation, but nowadays, this interest extends to the effects of global warming. More recently, metabolic and cellular investigations have included cold and UV resistance mechanisms. Low-temperature stress resistance in plants from cold environments reflects the climate conditions at the growth sites. It is now a matter of molecular analyses to find the induced genes and their products such as chaperones or dehydrins responsible for this resistance. Development of plants under snow or pollen tube growth at 0 degrees C shows that cell biology is needed to explain the stability and function of the cytoskeleton. Many results in this field are based on laboratory studies, but several publications show that it is not difficult to study cellular mechanisms with the plants adapted to a natural stress. Studies on high light and UV loads may be split in two parts. Many reports describe natural UV as harmful for the plants, but these studies were mainly conducted by shielding off natural UV (as controls). Other experiments apply additional UV in the field and have had practically no negative impact on metabolism. The latter group is supported by the observations that green overwintering plants increase their flavonoids under snow even in the absence of UV. Thus, their defense and antioxidant role dominates. Ultrastructural comparisons were unable to find special light adaptations in plants taken from polar regions vs. high alpine species. The only adaptation found at the subcellular level for most alpine and polar plants are protrusions of the chloroplast envelopes. They are seen as a demand for fast membrane transport requiring additional membrane surface area, whereby the increase in stroma volume may help to support carbohydrate formation. Plants forming such protrusions have to cope with a short vegetation time. These observations are connected to the question as to how photosynthesis works quite well even at or under zero temperatures. The interplay between plastids, mitochondria, and peroxisomes, known as photorespiration, seems to be more intense than in lowland plants. This organelle cooperation serves as a valve for a surplus in solar energy input under cold conditions. Additional metabolic acclimations are under investigation, such as the role of an alternative plastid terminal oxidase. Plants from cold environments may also be seen as ideal objects for studying the combined effects of high light plus cold resistance-from the molecular level to the whole plant adaptation. Modern instrumentation makes it possible to perform vital metabolic measurements under outdoor conditions, and research stations in remote polar and alpine areas provide support for scientists in the preparation of samples for later cellular studies in the home laboratory.

Respiration of Escherichia coli Can Be Fully Uncoupled via the Nonelectrogenic Terminal Cytochrome bd -II Oxidase

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

Acanthamoeba alternative oxidase genes: Identification, characterisation and potential as antimicrobial targets

Alternative oxidase (AOX) is a mitochondrial protein that acts as an alternative terminal oxidase to the conventional cytochrome oxidases. It is present in certain prokaryotes, plants, fungi and some protozoa but absent in mammals. AOX activity has previously been described in Acanthamoeba, although no genetic evidence has been reported. Herein, two AOX ( AcAOX) genes designated isoforms A and B, were obtained from Acanthamoeba castellanii by a combination of degenerate PCR from cDNA and a series of 5′ and 3′ rapid amplification of cDNA ends. The corresponding genomic sequences of these AcAOXs were also obtained. Each gene spans six exons over a region of 1607 and 1619 bp, respectively. Isoforms A and B have open reading frames of 1113 and 1125 bp, respectively. Each encodes a protein with a predicted molecular weight of 42 kDa. Each AcAOX protein has a predicted cleavable mitochondrial targeting sequence. The full-length AcAOX is functionally active as it complements hemL-deficient Escherichia coli and inhibited by the inhibitor of AOX, salicylhydroxamic acid (SHAM). SHAM is effective against A. castellanii and Acanthamoeba polyphaga only when used in conjunction with antimycin A, an inhibitor of the conventional cytochrome respiratory pathway. Transcripts for AcAOX are increased during the encystment process, indicating a possible role for alternative respiration during stress.

Dinitro- o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells

In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.

Terminal oxidases of cyanobacteria

The respiratory chain of cyanobacteria appears to be branched rather than linear; furthermore, respiratory and photosynthetic electron-transfer chains co-exist in the thylakoid membrane and even share components. This review will focus on the three types of terminal respiratory oxidases identified so far on a genetic level in cyanobacteria: aa3-type cytochrome c oxidase, cytochrome bd-quinol oxidase and the alternative respiratory terminal oxidase. We summarize here their genetic, biochemical and biophysical characterization to date and discuss their interactions with electron donors as well as their physiological roles.

The paradoxical cyanide-stimulated respiration of Zymomonas mobilis: cyanide sensitivity of alcohol dehydrogenase (ADH II).

The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacterium Zymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilized Z. mobilis cells, addition of 200 microM cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-course of inhibition of whole-cell respiration resembled that of NADH oxidase, yet the inhibition was almost complete, and was accompanied by an increase of intracellular NADH concentration. Cyanide did not significantly affect the activity of ADH I, the zinc-containing alcohol dehydrogenase isoenzyme. When an aerobic batch culture was grown in the presence of 200 microM cyanide, cyanide-resistant ADH II activity was observed, its appearance correlating with the onset of respiration. It is concluded that the membrane-associated respiratory chain, but not ADH II, is responsible for the whole-cell cyanide sensitivity, while the cyanide-resistant ADH II is needed for respiration in the presence of cyanide, and represents an adaptive response of Z. mobilis to cyanide, analogous to the induction of alternative terminal oxidases in other bacteria.

Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth.

The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa(3) is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa(3) are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa(3) or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa(3) is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa(3) or cytochrome aa(3), is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa(3) makes the strain sporulation deficient.

Mitochondrial sulfide oxidation in Arenicola marina. Evidence for alternative electron pathways.

Sulfide is oxidized in the mitochondria of the lugworm Arenicola marina. Mitochondrial sulfide oxidation is coupled with oxygen consumption and with an equimolar production of thiosulfate [Völkel, S. & Grieshaber, M. K. (1994) Mar. Biol. 118, 137-147]. Mitochondrial respiration in the presence of malate (or succinate) and ADP but without sulfide could be completely inhibited by rotenone, antimycin, cyanide, and sulfide. Only 40% inhibition was achieved by salicylhydroxamic acid. Sulfide oxidation (with sulfide as the only substrate) was fully inhibited by antimycin and by salicylhydroxamic acid but not by rotenone or sulfide. Moreover, sulfide oxidation was 3-4-fold less sensitive to cyanide as compared to normal respiration. The data indicate that sulfide oxidation in A. marina is linked to the respiratory electron transport chain. We suggest that electrons from sulfide enter the respiratory chain via ubiquinone or at the ubiquinol-cytochrome-c oxidoreductase. At sulfide concentrations higher than 10 microM, the cytochrome-c oxidase is blocked and electrons from sulfide are transferred to oxygen via an alternative terminal oxidase.

A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli

Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of fumarate reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of fumarate reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant fumarate reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that fumarate reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of fumarate reductase (1600 min-1), additional steric or electronic factors must accelerate its autoxidation.

Sulfide Tolerance and Detoxification inArenicola marinaandSipunculus nudus

SYNOPSIS. The lugworm Arenicola marina and the peanut worm Sipunculus nudus both inhabit intertidal flats. Sulfide concentrations in the pore water of the sediment can be as high as 340 μM in the habitat of A. marina . Sulfide levels in the burrow water and in the coelomic fluid increase during low tide. In contrast, S. nudus is only rarely exposed to sulfide. Neither A. marina nor S. nudus is able to exclude sulfide completely from its body. However, both worms accumulate only 15 to 40% respectively of the external sulfide concentration during hypoxic 1 mM sulfide incubations. During normoxia, sulfide accumulation is more than ten-fold lower than under hypoxic conditions in both species. When oxygen is available, sulfide is oxidized to thiosulfate. The oxidation rate is about 5 times higher in A. marina than in S. nudus . A major component of sulfide oxidation is due to the mitochondria of both species. Mitochondrial sulfide oxidation in the tissues of A. marina is insensitive to low cyanide and to high sulfide concentrations, but can be inhibited by salicylhydroxamic acid. The existence of an alternative terminal oxidase is suggested. When the sulfide level in the body of A. marina and S. nudus increases above a toxic level, both species survive by switching to an anaerobic metabolism.

An alternative cytochrome oxidase of Paracoccus denitrificans functions as a proton pump

Deletion of the genes encoding subunit I of cytochrome aa 3 in P. denitrificans yields mutants that lack cytochrome aa 3 but which are still capable of oxidizing ubiquinol by O 2 via the cytochrome bc 1 complex, as revealed by myxothiazol-sensitive respiration on succinate. An alternative terminal oxidase must be responsible for the myxothiazol-sensitive activity. The H +/e − ratio of proton translocation by mutant cells approached 3.0 for oxidation of succinate by O 2, while it was near 2.0 either with ferricyanide as electron acceptor (engaging the bc 1 complex only), or with O 2 as acceptor in the presence of myxothiazol (engaging the ubiquinol oxidase). However, proton translocation coupled to oxidation of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) plus ascorbate amounted to much less than 1.0 H +/e −. This may be due to interference by another TMPD-oxidising activity that is not engaged during electron flux to oxygen via the cytochrome bc 1 complex. We conclude that Paracoccus can express an alternative cytochrome b-containing terminal oxidase, which translocates protons at least when operating in conjunction with the cytochrome bc 1 complex.

Oxygen dependent sulfide detoxification in the lugwormArenicola marina

The lugwormArenicola marina L. oxidizes entering sulfide to thiosulfate. After 8 h of normoxic incubations with sulfide concentrations of 0.2 to 1.0 mmoll-1 thiosulfate in the coelomic fluid amounted up to about 4 mmoll-1 whereas sulfite concentrations were 100-fold lower and no accumulation of sulfate in the coelomic fluid was found. The sulfide oxidation was highly oxygen dependent. An increase of oxygen partial pressure (\(P_{O_2 }\)) in the medium was followed by enhanced thiosulfate production and by a decrease of sulfide concentration in the coelomic fluid. Under normoxia, the sulfide oxidation rate was sufficient to compensate the influx of sulfide into the coelomic fluid when the sulfide concentration in the medium was below 0.33 mmoll-1. When external sulfide was raised beyond this level, sulfide up to 5 μmoll-1 in the coelomic fluid appeared. Succinate in the body wall tissue was low as long as no sulfide appeared in the coelomic fluid, indicating the maintenance of an aerobic metabolism. The oxidation of sulfide to thiosulfate was localized in the mitochondria of the body wall tissue. The oxygen consumption of mitochondria was stimulated by the addition of sulfide. The mitochondrial sulfide oxidation rate depended on the amount of mitochondrial protein and followed a Michaelis-Menten kinetic. An apparentKm of 0.68±0.29 μmoll-1 and aVmax of 41.9±22.3 nmol min-1 mg-1 protein was calculated. Sulfide was stoichiometrically oxidized to thiosulfate with 1 mol sulfide consuming 1 mol oxygen. Sulfide oxidation was not inhibited by sulfide concentrations as high as 100 μmoll-1. At low concentrations of cyanide or azide, when respiration without sulfide was already inhibited, sulfide oxidation could still be stimulated, tentatively indicating the existence of an alternative terminal oxidase. Specimens examined in the present study were collected near St. Pol de Leon, France, from 1989 to 1992.

Has Sulfolobus an Archaic Respiratory System? Structure, Function and Genes of its Components

The electron transport system of Sulfolobus acidocaldarius has been shown to act as a respiration driven proton pump. Essentially it is composed of three functional units: the NADH-and substrate dehydrogenases, caldariella quinone as a pool of bound hydrogen, and one or two terminal oxidases catalyzing its reoxidation by molecular oxygen. The latter systems contain only FeS proteins, b - and a -type cytochromes; c -type cytochromes are absent. The cytochrome aa 3 from Sulfolobus is the first heme- a containing terminal oxidase shown to act as a quinol oxidase. Its redox centers were investigated by redox potentiometry, EPR- and Raman-resonance spectroscopy, revealing a typical binuclear heme- a /Cu center, however displaying unusual structural interaction between the formyl substituents and the protein environment; the redox potentials of low and high spin centers were titrated. A cytochrome a 587 atypical for any other species emerged to contain a low spin, low potential heme- a center, mimicing the function of a b -type cytochrome and likely to be the product of the SOX -C gene from the SOX operon ( Lübben et al., 1992). The aa 3 oxidase when reconstituted into liposomes is shown to generate a proton motive force. Though Sulfolobus contains no equivalent to the bc 1 complex of classical respiratory chains, in addition to the caldariella quinol oxidase a Rieske-type FeS protein could be detected in its plasma membrane; it was isolated and characterized by EPR, strongly suggesting a participation in respiratory electron transport. Its redox potential displays significant pH dependence revealing two distinct pKs. It remains to be established whether it interacts with the aa 3 oxidase or other electron carriers. A cytochrome b 562 appears to be present constitutively in small amount, while cytochrome- b 558 is the major heme- b containing compound in the membrane of Sulfolobus . A previously envisaged function as an alternate terminal oxidase is still questionable. Recently conditions were found allowing to modulate its expression dramatically, leading to almost complete suppression of cytochrome- b 558 . Based on differential spectroscopy and redox titrations a tentative scheme of the electron transport system from Sulfolobus is proposed. Sequence comparisons are discussed with regard to the question, as to whether it resembles a primitive “archaic” precursor form of more complex “modern” respiratory systems, or whether it evolved by aquisition and adaptation of “foreign” genes.

Properties of a copper-containing cytochrome ba3: a second terminal oxidase from the extreme thermophile Thermus thermophilus.

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

Identification of the alternative terminal oxidase of higher plant mitochondria

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed.

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.

Membrane-boundb-type cytochromes in Nitrobacter

Nitrobacter hamburgensis was shown to synthesize at least two distinct membrane-bound b-type cytochromes. One of these, a minor component detected during nitrite oxidation, was also found in the obligately autotrophic species Nitrobacter winogradskyi. During heterotrophic growth of N. hamburgensis a second (major) cytochrome b was detected, which we assume functions as an alternative terminal oxidase.

Biochemistry and physiology of aerobic carbon monoxide-utilizing bacteria

The use of CO as a growth substrate by aerobic CO-oxidizing (carboxydotrophic) bacteria requires some features not obvious in other bacteria. These are the presence of the enzyme CO dehydrogenase, a branched respiratory chain with an alternative CO-insensitive terminal oxidase (cytochrome b653) and formation of reduced pyridine nucleotides by a pmf-driven reversed electron transfer. Immunocytochemical localization studies revealed that CO dehydrogenase is attached to the inner aspect of the cytoplasmic membrane of Pseudomonas carboxydovorans. The enzyme is a molybdo iron-sulfur flavoprotein containing bactopterin as the organic portion of the molybdenum cofactor. Recent findings suggest that this novel pterin is universal to eubacterial molybdenum enzymes, whereas molybdopterin is universal to eukaryotic molybdoenzymes.

Biochemistry and physiology of aerobic carbon monoxide-utilizing bacteria

The use of CO as a growth substrate by aerobic CO-oxidizing (carboxydotrophic) bacteria requires some features not obvious in other bacteria. These are the presence of the enzyme CO dehydrogenase, a branched respiratory chain with an alternative CO-insensitive terminal oxidase (cytochrome b 653) and formation of reduced pyridine nucleotides by a pmf-driven reversed electron transfer. Immunocytochemical localization studies revealed that CO dehydrogenase is attached to the inner aspect of the cytoplasmic membrane of Pseudomonas carboxydovorans. The enzyme is a molybdo iron-sulfur flavoprotein containing bactopterin as the organic portion of the molybdenum cofactor. Recent findings suggest that this novel pterin is universal to eubacterial molybdenum enzymes, whereas molybdopterin is universal to eukaryotic molybdoenzymes.

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

1. 1. Titration of Trypanosorna cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. 2. 2. The inhibitions obtained at low cyanide concentrations (0–300 μM), taken together with cyanide effects on cytochrome aa 3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa 3 as T. cruzi main terminal oxidase. 3. 3. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was (a) active in both kinetoplastic and dyskinetoplastic (cytochrome aa 3-deficient) epimastigotes, and (b) azide- and antimycin-insensitive. 4. 4. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa 3, b, c 558 , 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. 5. 5. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. 6. 6. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa 3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.