Mechanism of nitrite inhibition of cellular respiration inPseudomonas aeruginosa

One of the principal mechanisms of nitrite inhibition of cellular respiration has been considered to be the interference with the action of iron-containing enzymes. In procaryotic systems, the effect of nitrite on cellular metabolism remains unclear. This study provides evidence which shows a direct inhibition by a low concentration of nitrite on a highly purified oxidase inPseudomonas aeruginosa. The inhibition pattern was observed and was consistent at cellular, electron-transport membranous, and enzymic (oxidase) levels. This implies that the mechanism of nitrite inhibition on bacterial respiration is due to a direct inhibition at the terminal site of oxygen reduction. The uncompetitive inhibition pattern shown by nitrite strongly suggested a mechanism quite different from those of classic cytochrome oxidase inhibitors such as cyanide, azide, and carbon monoxide.     

AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY OF A PURIFIED PREPARATION OF THE SUCCINATE AND DPNH OXIDASE SYSTEM

Electron micrographs of a purified succinate and DPNH oxidase system prepared from heart muscle reveal that it has a vesicular appearance and is membranous in nature. In keeping with its vesicular appearance is the fact that light scattering by this preparation shows marked changes as the molarity of the suspending medium is altered. Treatment of this preparation with 0.5 per cent deoxycholate solutions removes a large part of the lipide material, which comprises almost half of the dry weight of the preparation. The residue, which still contains the "core" of the cytochrome electron transmitter system, as shown by spectroscopic and enzymatic experiments, is still structured and is membranous in morphological appearance. It is concluded that the enzyme preparation is largely composed of fragmented mitochondrial membranes, and some of the consequences of the localization of the succinate and DPNH oxidase systems in or on these membranes are discussed.     

The interconversion between monomeric and dimeric bovine heart cytochrome c oxidase

Monomers and dimers of bovine heart cytochrome c oxidase (EC 1.9.3.1.) were separated by gel filtration chromatography on Ultrogel AcA 34 or by sucrose gradient centrifugation. Factors influencing the interconversion of the two aggregation states of this enzyme were analyzed. At very low ionic strength, in the presence of dodecyl maltoside, monomers were the main species. Salts appeared to stabilize the dimeric form, divalent cations being more efficient than monovalent. High enzyme concentrations favoured the formation of dimers, also at low ionic strength. The type of detergent had a strong influence on the monomer-dimer interconversion; in Triton X-100 and dodecyl maltoside (at high ionic strength) cytochrome c oxidase was homogenously dispersed in its dimeric form, while in Tween-80 gel filtration showed only large particles eluting in the void volume. In cholate monomers and aggregates were observed but no dimers. The aggregation state had an influence on the steady state kinetics of the ferrocytochrome c oxidase activity. Monomers showed linear Eadie-Hofstee plots, whilst the dimeric and aggregated enzyme gave nonlinear Eadie-Hofstee plots. Ionic strength, enzyme concentration and type of detergent were affecting the enzyme's kinetics in a way consistent with the molecular form obtained by the gel filtration or sedimentation analysis. The data support a negative cooperative mechanism for the interaction of cytochrome c with the dimeric enzyme, as proposed earlier (K.A. Na?ecz et al., (1983) Biochem. Biophys. Res. Commun., 114, 822-828).     

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.     

Absorption spectra and magnetic circular dichroism of heme-containing proteins in nonequilibrium states. VI. Cytochrome c-oxidase

Low temperature (77 degrees K) absorption spectra of nonequilibrium states of cytochrome c oxidase produced by reduction of oxidases form protein by thermolysed electrons at 77 degrees K was studied. During reduction of cytochrome oxidase water-glycerol solution by thermolysed electrons at 77 degrees K a nonequilibrium reduced protein is formed. Low temperature (77 degrees K) absorption spectra of the nonequilibrium cytochrome oxidase differs from those reduced by ditionite. It was shown that the oxidation state of cytochrome a3 or addition of cytochrom c have no influence on these spectral changes. It is assumed, that the observed effects are conditioned by structural differences of reduced and oxidased cytochrome oxidase active center. Similar spectral changes were observed for cytochrome oxidase, bound to the mitochondrial membrane. At temperature increasing the low temperature reduced protein is relaxed to a corresponding equilibrium state. The spectral properties of bacterial cytochrome oxidase M. lysodeicticus do not depend on the way of reduction (by dytionite or thermolysed electrons at 77 degrees K).     

Sulfhydryl oxidation, not disulfide isomerization, is the principal function of protein disulfide isomerase in yeast Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an essential protein folding assistant of the eukaryotic endoplasmic reticulum that catalyzes both the formation of disulfides during protein folding (oxidase activity) and the isomerization of disulfides that may form incorrectly (isomerase activity). Catalysis of thiol-disulfide exchange by PDI is required for cell viability in Saccharomyces cerevisiae, but there has been some uncertainty as to whether the essential role of PDI in the cell is oxidase or isomerase. We have studied the ability of PDI constructs with high oxidase activity and very low isomerase activity to complement the chromosomal deletion of PDI1 in S. cerevisiae. A single catalytic domain of yeast PDI (PDIa') has 50% of the oxidase activity but only 5% of the isomerase activity of wild-type PDI in vitro. Titrating the expression of PDI using the inducible/repressible GAL1-10 promoter shows that the amount of wild-type PDI protein needed to sustain a normal growth rate is 60% or more of the amount normally expressed from the PDI1 chromosomal location. A single catalytic domain (PDIa') is needed in molar amounts that are approximately twice as high as those required for wild-type PDI, which contains two catalytic domains. This comparison suggests that high (>60%) PDI oxidase activity is critical to yeast growth and viability, whereas less than 6% of its isomerase activity is needed.     

Two monoclonal antibody lines directed against subunit IV of cytochrome oxidase: a study of opposite effects

Two monoclonal lines of antibodies were isolated with specificities against the amino half of Subunit IV of beef heart cytochrome oxidase. The lines had nonoverlapping epitopes. Both bound to the matrix face of membranous oxidase, neither bound to the cytoplasmic face. One line (QA4/C4) stimulated electron transfer in soluble or membranous oxidase, while the other (QA4) inhibited that activity by both oxidase preparations. These effects on electron transfer activity were not altered by the inclusion or omission of detergent. ATP depressed the binding of either antibody to either soluble or membranous oxidase. In the absence of ATP, QA4/C4 stimulated electron transfer only in the high affinity phase of cytochrome c oxidation (with decreased KM and increased Vmax), causing slight inhibition in the low affinity phase (with decreased KM). In the presence of ATP, QA4/C4 abolished the high affinity phase, but did not alter the ATP influence on the low affinity phase. In the absence of ATP, antibodies of line QA4 abolished the low affinity phase, leaving a high affinity phase similar to that induced by ATP. In the presence of ATP, QA4 abolished the high affinity phase, leaving a low affinity phase similar to that seen with ATP alone. This behavior is consistent with the dissection of two catalytic sites for cytochrome c and more than one ATP affector site.     

Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617.

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-479]. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130,000 was determined by gel filtration. The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the alpha range. The prosthetic group is made of two hemes b, one high-spin (S = 5/2, gl = 5.9, g parallel approximately 2.0), the other low-spin (S = 1/2, gz = 2.94, gy = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 +/- 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2.min-1.mg protein-1 and commercially available coenzyme (101.74 nmol O2.min-1.mg protein-1). Exposure of the reduced enzyme to CO induced the collapse of alpha and beta bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.     

Effect of lipids on the structure and rheology of gels formed by canine submaxillary mucin

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G'(ω), and the dynamic complex viscosity, η^*(ω), are observed for lipid-containing CSM. TMs is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.     

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.     

Oxidation by superoxide of tocopherols dispersed in aqueous media with deoxycholate

alpha-Tocopherol dispersed in aqueous media with deoxycholate was found to be oxidized, at a physiological pH, by a xanthine-xanthine oxidase system. This reaction was completely inhibited by the addition of superoxide dismutase, whereas catalase and mannitol (scavenger of hydroxyl radical) did not affect the reaction. This finding indicates that the oxidation of alpha-tocopherol is caused by O2. The reaction product formed was identified as 8 alpha-hydroxy-alpha-tocopherone by thin-layer chromatography and ultraviolet spectroscopy. The product was found to change spontaneously to alpha-tocopherol quinone. beta-, gamma-, and delta-tocopherol dispersed with deoxycholate also reacted with O2. The reaction of tocopherols dispersed in the micellar form may be considered as a model of in vivo reaction of tocopherols, since tocopherols are present in tissues largely in the membranes, where O2 is known to be generated.     

Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.     

Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.     

Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm. Spectroscopic characterization of the steady-state species.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.     

ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).     

Multiple chromatographic forms of ATP citrate lyase from rat liver.

ATP citrate lyase is shown to exist as multiple forms in extracts of rat liver. DEAE-Sephadex ion-exchange chromatography of liver supernatants reveals two peaks of activity. A minor, basic, component, comprising 14% of the recovered activity, is eluted without retention, whereas the major, acidic, form is eluted by a KCl gradient. Gel filtration of similar extracts shows the presence of a high-Mr form of ATP citrate lyase (Mr around 10(7) in addition to the tetrameric enzyme (Mr 4.1 X 10(5). This associated state, which represents 10% of the total activity, is unstable, breaking down to the tetramer, and appears to be disrupted by Mg2+. The basic form changes in the partially purified state to give the acidic form. Most of the high-Mr enzyme is acidic in nature. No evidence could be found for an association of the enzyme with mitochondrial or microsomal membranes. ATP citrate lyase from rat brain also shows two peaks of activity on DEAE-Sephadex ion-exchange chromatography, but the activity is distributed between the peaks in almost equal proportions. However, only the tetrameric enzyme was observed on gel filtration.     

Transformation of revertant murine cells by 5-azacytidine results in rapid inhibition of lysyl oxidase expression

Lysyl oxidase (LO) is synthesized intracellularly as a proenzyme that is secreted and then processed extracellularly to a mature form. LO is expressed in NIH3T3 cells, but only very low levels are observed after NIH 3T3 is transformed by c-H-ras or one of several other oncogenes. LO functions as a tumor suppressor. Treatment of ras-transformed cells with interferon-alpha with or without retinoic acid results in their persistent reversion to a non-transformed state that is dependent on the restoration of LO expression. When such revertant cells are treated with 5-azacytidine (5-azaC), they undergo rapid morphological retransformation. Within one passage after addition of 5-azaC, there was a down regulation of LO mRNA and proenzyme protein. These data suggest a direct relationship between the transformed state and LO expression.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

Characteristics of enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain

The subcellular localization and character of the enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) were determined in rat brain. The aldehyde derivative of normetanephrine was produced in situ by monoamine oxidase, and two forms of aldehyde reductase were shown to metabolize the aldehyde to MOPEG. One form of the enzyme was found to have a low affinity for NADH and a higher affinity for NADPH as a cofactor, and was shown to be inhibited by pentobarbital and by high concentrations of 5-hydroxyindoleacetic acid. This enzyme form was localized primarily in the cytosol. The second aldehyde reductase had a high affinity for both NADH and NADPH, and was not inhibited to a great extent by either pentobarbital or 5-hydroxyindoleacetic acid. This second enzyme form was localized primarily in the mitochondrial fraction. The relative contribution of the two enzyme forms to MOPEG formation in homogenates was estimated, using the various inhibitors and cofactors.     

Dendrostilbella sp. (85-25), a New Source of Trichodermin

A sensitive kinetic determination of serum 5′-nucleotidase activity is described. Nucleoside oxidase catalyzes the oxidative coupling reaction of phenolic compounds and 4-aminoantipyrine to form colored substances in proportion of the amount of nucleoside. The present method relies on this phenomenon. Human serum 5′-nucleotidase was assayed using 5′-AMP as a substrate. When adenosine liberated was oxidized by nucleoside oxidase in the presence of phenolic compounds and 4-aminoantipyrine, a colored substance was formed at the same time. The rate of colored substance formation was proportional to the 5′-nucleotidase activity. The proposed method shows a low variation and a good correlation with the reference method. 3′-Nucleotidase was also assayed in a similar manner.