Tetramethyl-p-phenylenediamine Research Articles

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H 2O 2-producing side-reaction of respiratory complex I with O 2

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 O 2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O 2-affinity system ( K m app = 1.5 μM) was abolished by low cyanide concentration; it corresponded to cytochrome aa 3. The low-O 2-affinity system ( K m app = 120 μM) was resistant to cyanide. Similar O 2 saturation kinetics, using succinate or ascorbate–TMPD as electron donor, showed only the high-O 2-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 H 2O 2 (at least 10% of the total O 2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H 2O 2. Production of H 2O 2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O 2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O 2, producing H 2O 2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.

Mutations in Cytochrome Assembly and Periplasmic Redox Pathways inBordetella pertussis

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed.

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes.

N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I-P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I-P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b6/f complex, could prevent the quenching of I-P rise by TMPD. In control thylakoids, MV suppressed the I-P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I-P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O2 during re-oxidation of TMPD by PS I contributes towards an early decline in the I-P step of the OJIP fluorescence rise.

Radiolytically induced one-electron reduction of artemisinin in H2O/ethanol (1:1 v/v) solution: a pulse radiolysis study.

The aim was to obtain information on the one-electron reduction of the antimalarial natural drug artemisinin (ART). The pulse radiolysis of ART in H2O/ethanol (EtOH) (1:1 v/v) solution was studied in the absence and presence of the so-called redox indicators N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD), Fe(CN)64 − , 1,1′-dimethyl-4,4′-bipyridinium dichloride (methyl viologen, MV2 + ) and Fe(CN)63 − . In an argon-purged solution, ART reacts with solvated electrons (es−) with k = 4.4 × 109 dm3 mol − 1 s − 1 generating an absorption band rising in the ultraviolet region similar to the spectrum of the CH3•CHOH radical. The species originating from the reaction between ART and es− do not show any appreciable reactivity toward Fe(CN)64 − , TMPD, MV2 + and Fe(CN)63 − . The experiments performed in the presence of ART and MV2 + have provided strong support to the idea that the first species obtained from the addition of the electron, which is believed to occur at the endoperoxide group level, undergoes a rapid (k on the order of 108 s − 1 or higher) intramolecular rearrangement to give species, most likely carbon-centred radicals, that show some reactivity towards the methyl viologen radical cation (MV• + ).

Electron and Hole Transfer from Indium Phosphide Quantum Dots

Electron- and hole-transfer reactions are studied in colloidal InP quantum dots (QDs). Photoluminescence quenching and time-resolved transient absorption (TA) measurements are utilized to examine hole transfer from photoexcited InP QDs to the hole acceptor N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and electron transfer to nanocrystalline titanium dioxide (TiO2) films. Core-confined holes are effectively quenched by TMPD, resulting in a new approximately 4-ps component in the TA decay. It is found that electron transfer to TiO2 is primarily mediated through surface-localized states on the InP QDs.

Structural and functional alterations in mitochondrial membrane in picrotoxin-induced epileptic rat brain

Mitochondrial function is a key determinant of both excitability and viability of neurons. Present studies were carried out to decipher cerebral mitochondrial oxidative energy metabolism and membrane function in the chronic condition of generalized seizures induced by picrotoxin (PTX) in rats. PTX-induced convulsions resulted in decreased respiration rates (14–41%) with glutamate, pyruvate + malate, and succinate as substrate. The ADP phosphorylation rates were drastically reduced by 44–65%. An opposite trend was observed with ascorbate + N,N′, N′-tetramethyl- p-phenylenediamine (TMPD) as substrate. In general, uncoupling of the mitochondrial electron transport was observed after PTX treatment. Malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities were decreased by 20–80%; also, there was significant reduction in cytochrome b content after PTX treatment, while the F oF 1 ATPase (complex V) activity increased in basal and 2,4-dinitrophenol (DNP)-stimulated condition, indicating increased membrane fragility. The substrate kinetics analysis had shown that K m and V max of the higher affinity kinetic component of ATPase increased significantly by 1.2- to 1.4-fold in epileptic condition. Temperature kinetic analysis revealed 1.2-fold increase in energies of activation with decreased transition temperature. The total phospholipid (TPL) and cholesterol (CHL) contents decreased significantly with lowering of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), while lysophospholipid (lyso), sphingomyelin (SPM), and phosphatidylcholine components were found to be elevated. Brain mitochondrial membrane was somewhat more fluidized in epileptic animals. Possible consequences of mitochondrial respiratory chain (MRC) dysfunction are discussed. In conclusion, impairment of MRC function along with structural alterations suggests novel pathophysiological mechanisms important for chronic epileptic condition.

Equilibria and catalytic properties of a chloro-bridged Diimine copper(II) complex in the N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) oxidation

Some new copper(II) complexes with a tridentate imine ligand, bimpy (or 2-(benzimidazolyl)methylene-2-amino-1-ethylpyridine), were prepared, and had their catalytic activity in the N,N,N',N'-tetramethyl- p-phenylenediamine (TMPD) oxidation compared. Mononuclear species, [Cu(bimpy)(H2O)2](NO3 )2, (1) and [Cu(bimpy)Cl2]·H2O, (2), as well as the dinuclear species containing chloro-bridges, [Cu2(bimpy)2Cl2]X2·nH 2O as perchlorate 3, nitrate 4 and chloride 5 salts, respectively, were obtained and characterized by different techniques (UV/Vis, IR, EPR and X-ray crystallography). In non-aqueous solvents, the dinuclear species predominates; however, in aqueous solution or solvents containing water, equilibrium between dinuclear and correlated mononuclear species 1 was verified. Kinetic studies, in methanol solution, on the catalyzed TMPD oxidation indicated a first-order dependence of the initial rate with the catalyst concentration, and a saturation effect with increasing concentrations of the substrate. The dinuclear species was observed to be more reactive than the mononuclear one, and further experiments showed that the counter-ion chloride can significantly affect the kinetic parameters, mostly by shifting the mono-/dinuclear equilibrium, and stabilizing an ion-pair with the substrate.

Nitric oxide oscillations in Paracoccus denitrificans: the effects of environmental factors and of segregating nitrite reductase and nitric oxide reductase into separate cells

Nitric oxide is a denitrification intermediate which is produced from nitrite and then further converted via nitrous oxide to nitrogen. Here, the effect of low concentrations of the protonophore carbonylcyanide m-chlorophenylhydrazone on the time courses for dissolved gases was examined. While NO was found to oscillate, N 2O only increased gradually as the reduction of nitrite progressed. The frequency and shape of protonophore-induced NO oscillations were influenced by temperature and the concentration of electron donor N, N, N′, N′-tetramethyl- p-phenylene diamine (TMPD) in a manner compatible with the observed differential effects on the two involved enzyme activities. We demonstrated the existence of a pH interval, where [NO] oscillates even without uncoupler addition. Occurrence of nitric oxide oscillations in mixtures of a nitrite reductase mutant with a nitric oxide reductase mutant suggests that they cannot be due to a competition of the enzymes for redox equivalents from one common respiratory chain.

Effects of N-acylethanolamines on mitochondrial energetics and permeability transition

Effects of N-acylethanolamines (NAEs): N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine and N-palmitoylethanolamine, on energy coupling and permeability of rat heart mitochondria were investigated. In nominally Ca 2+-free media, these compounds exerted a weak protonophoric effect manifested by dissipation of the transmembrane potential and stimulation of resting state respiration. The strongest action was exhibited by N-arachidonoylethanolamine, followed by N-oleoylethanolamine, whereas N-palmitoylethanolamine was almost inactive. These protonophoric effects were resistant to cyclosporin A (CsA) and were much weaker than those of corresponding nonesterified fatty acids. In uncoupled mitochondria N-arachidonoylethanolamine and N-oleoylethanolamine partly inhibited mitochondrial respiration with glutamate and succinate but not with tetramethyl- p-phenylenediamine (TMPD) plus ascorbate as respiratory substrates. In mitochondria preloaded with small amounts of Ca 2+, NAEs produced a much stronger dissipation of the membrane potential and a release of accumulated calcium, both effects being inhibited by CsA, indicative for opening of the mitochondrial permeability transition pore (PTP). Again, the potency of this action was N-arachidonoylethanolamine> N-oleoylethanolamine> N-palmitoylethanolamine. However, in spite of making the matrix space accessible to external [ 14C]sucrose, N-arachidonoylethanolamine and N-oleoylethanolamine resulted in only a limited swelling of mitochondria and diminished the rate of swelling produced by high Ca 2+ load.

Changes in polyphasic chlorophyll a fluorescence induction curve upon inhibition of donor or acceptor side of photosystem II in isolated thylakoids

The action of various inhibitors affecting the donor and acceptor sides of photosystem II (PSII) on the polyphasic rise of chlorophyll (Chl) fluorescence was studied in thylakoids isolated from pea leaves. Low concentrations of diuron and stigmatellin increased the magnitude of J-level of the Chl fluorescence rise. These concentrations barely affected electron transfer from PSII to PSI as revealed by the unchanged magnitude of the fast component ( t 1/2=24 ms) of P700 + dark reduction. Higher concentrations of diuron and stigmatellin suppressed electron transport from PSII to PSI, which corresponded to the loss of thermal phase, the Chl fluorescence rise from J-level to the maximal, P-level. The effect of various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes S-state cycle and binds at the plastoquinone site on Q B, the secondary quinone acceptor PSII, on the Chl fluorescence rise was very similar to that of diuron and stigmatellin. Low concentrations of diuron, stigmatellin, or CCCP given on the background of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD), which is shown to initiate the appearance of a distinct I-peak in the kinetics of Chl fluorescence rise measured in isolated thylakoids [BBA 1607 (2003) 91], increased J-step yield to I-step level and retarded Chl fluorescence rise from I-step to P-step. The increased J-step fluorescence rise caused by these three types of inhibitors is attributed to the suppression of the non-photochemical quenching of Chl fluorescence by [S 2+ S 3] states of the oxygen-evolving complex and oxidized P680, the primary donor of PSII reaction centers. In the contrary, the decreased fluorescence yield at P step (J–P, passing through I) is related to the persistence of a “plastoquinone”-type quenching owing to the limited availability of photochemically generated electron equivalents to reduce PQ pool in PSII centers where the S-state cycle of the donor side is modified by the inhibitor treatments.

Ultrafast photoinduced electron transfer from N, N, N′, N′-tetramethyl- p-phenylenediamine and N, N, N′, N′-tetramethylbenzidine to dichloromethane

The photoinduced reaction of electron transfer (ET) from N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) and N, N, N′, N′-tetramethylbenzidine (TMB) to the dichloromethane solvent has been studied by picosecond transient absorption and time-resolved resonance Raman spectroscopy. In neat CH 2Cl 2, “static” quenching of the lowest excited singlet state (S 1) of these amines is demonstrated. For TMPD, the reaction shows two kinetic contributions ( τ 1<4 ps, τ 2=9.5 ps) that are ascribed to the fractions of excited molecules that react before and after they are vibrationally relaxed, respectively. A faster quenching reaction is observed for TMB ( τ<4 ps) although it is less favourable energetically than for TMPD. This result is ascribed to a stronger acceptor–donor electronic coupling in the case of TMB, principally due a larger delocalization of the S 1 state donor orbital. The observation of a partial back electron transfer (9% yield) is tentatively explained as resulting from an evolution of the reaction product from an initial [TMB + , CH 2Cl 2 − ] ion pair allowing charge recombination to a final form [TMB + , Cl −] inappropriate to back ET.

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.

Dynamics of positive charge carriers on Si chains of polysilanes.

The transient absorption of radical cations of a variety of substituted polysilanes is discussed quantitatively in terms of the molar extinction coefficient and oscillator strength by nanosecond pulse radiolysis. Oxygen-saturated polysilane solutions in benzene exhibit a strong transient absorption band ascribed to the polysilane radical cation. The transient species react with N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) to produce TMPD radical cations. On the basis of the molar extinction coefficient of the TMPD radical cation, the molar extinction coefficients for the radical cations of polysilanes are found to increase in the range 3.3 x 10(4) to 2.0 x 10(5) M(-)(1) cm(-)(1) with increasing polymer segment length. The stepwise increase in the total oscillator strength with an increase in the number of phenyl rings directly bonded to the Si skeleton suggests the delocalization of the positive polaron state and/or the SOMO state over the phenyl rings, indicating the importance of phenyl rings in intermolecular hole transfer processes.

Photoinduced interactions between oxidized and reduced lipoic acid and riboflavin (vitamin B2).

As a powerful natural antioxidant, lipoic acid (LipSS) and its reduced form dihydrolipoic acid (DHLA) exert significant antioxidant activities in vivo and in vitro by deactivation of reactive oxygen and nitrogen species (ROS and RNS). In this study the riboflavin (RF, vitamin B2) sensitized UVA and visible-light irradiation of LipSS and DHLA was studied employing continuous irradiation, fluorescence spectroscopy, and laser flash photolysis (LFP). Our results indicate that LipSS and DHLA quench both the singlet state (1RF*) and the triplet state (3RF*) of RF by electron transfer to produce the riboflavin semiquinone radical (RFH.) and the radical cation of LipSS and DHLA, respectively. The radical cation of DHLA is rapidly deprotonated twice to yield a reducing species, the radical anion of LipSS (LipSS.-). When D2O was used as solvent, it was confirmed that the reaction of LipSS with 3RF* consists of a simple electron-transfer step, while loss of hydrogen occurs in the case of DHLA oxidation. Due to the strong absorption of RFH. and the riboflavin ground state, the absorption of the radical cation and the radical anion of LipSS can not be observed directly by LFP. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and N,N,N',N'-tetramethyl benzidine (TMB) were added as probes to the system. In the case of LipSS, the addition resulted in the formation of the radical cation of TMPD or TMB by quenching of the LipSS radical cation. If DHLA is the reducing substrate, no formation of probe radical cation is observed. This confirms that LipSS.- is produced by riboflavin photosensitization, and that there is no oxidizing species produced after DHLA oxidization.

Probing reactive sites within the Photosystem II manganese cluster: Evidence for separate populations of manganese that differ in redox potential

When the oxygen evolving complex of Photosystem II is depleted of essential cofactor atoms (Ca2+ and Cl−) by a high ionic strength treatment that extracts 23 and 17 kDa extrinsic polypeptides, Mn2+ can be released by several large reductants (hydroquinone (H2Q), N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), methyl derivatives of hydroxylamine). This reactivity can be slowed if the enzyme is reconstituted with Ca2+. For TMPD, data from EPR and X-ray absorption spectroscopy indicate that Ca2+ reconstitution restricts initial Mn reduction to a site which contains a Mn4+ atom. Dimethylhydroxylamine (DMHA), a much smaller Mn reductant, is unable to reduce the cluster under these same conditions, even though DMHA and TMPD can generate Mn2+ from the Mn cluster in the absence of Ca2+. These reductants differ in redox potential by about 300 mV, and it is likely that the higher potential reductant, DMHA (+550 mV), is restricted to initially reacting with a Mn3+ species that is screened by the Ca2+ atom and is incapable, in the presence of Ca2+, of reducing lower potential atoms of the cluster that do react with TMPD. Reduction of the reactive Mn3+ species by DMHA in the absence of Ca2+, however, subsequently allows reduction of the remaining 3 Mn. Such an arrangement of metals and potentials in the cluster is in accord with models for the site of water oxidation and for the structure of the Ca–Mn4 cluster.

Hydrodynamics and Mass Transport in Wall-Tube and Microjet Electrodes: An Experimental Evaluation of Current Theory

The application of steady-state and fast-scan linear sweep voltammetry to a high-speed wall-tube electrode (HWTE) is reported in different solvents to investigate the response of the HWTE over a wide range of Reynolds' numbers (Re). Experiments are reported for the oxidation of N,N,N‘,N‘-tetramethyl-p-phenylenediamine (TMPD) in propylene carbonate (PC), water, butyronitrile (BN), acetonitrile (AN), and acetonitrile−water mixture solutions containing 0.10 M supporting electrolyte for a 24 μm radius platinum microdisk electrode housed within the HWTE using a range of average flow jet velocities from 0.03 to 19.8 m s-1 (corresponding to volume flow rates of 0.003−0.25 cm3 s-1 and center-line jet velocities from 0.05 to 39.5 m s-1). Fast scan linear sweep voltammetry is presented for the oxidation of TMPD in PC and of 9,10-diphenylanthracene (DPA) in AN. Theoretical results are derived using finite element methods for both one- and two-dimensional mass transport models. It is found that, for solvents with a k...

N, N, N′, N′-tetramethyl- p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids

Addition of N, N, N′, N′-tetramethyl- p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J–I–P phase of fluorescence rise and greatly retarded the I–P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence ( F v) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.

Excited Singlet (S1) State Resonance Raman Analysis of N,N,N‘,N‘-Tetramethylbenzidine and N,N,N‘,N‘-Tetramethyl-p-phenylenediamine

Vibrational data on the first excited singlet state (S 1 ) of N,N,N',N'-tetramethylbenzidine (TMB) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) have been obtained by using picosecond time-resolved Raman spectroscopyfor different isotopic derivatives and for the N,N,N',N'-tetraethyl analogues, TEB and TEPD. Reliable vibrational assignments are proposed. On this basis, the S 1 state structure and electronic configuration of these diamines are discussed and compared with the results previously reported for the ground state, the lowest excited triplet state, and the radical cation species.

Fast scan linear sweep voltammetry at a high-speed wall-tube electrode

The application of fast-scan linear sweep voltammetry methods to a high-speed wall-tube electrode (HWTE) is reported. Experiments are reported for the oxidation of N,N,N′ ,N′-tetramethyl- p-phenylenediamine (TMPD) in propylene carbonate solution containing 0.10 M tetrabutylammonium perchlorate for a 24 μm radius platinum microdisk electrode housed within the HWTE using a range of scan rates from 200 to 3000 V s −1 and average flow jet velocities from 0.24 to 9.4 m s −1 (corresponding to volume flow rates of 0.003–0.12 cm 3 s −1, and centre-line jet velocities from 0.5 to 18.9 m s −1). Linear sweep voltammograms (LSVs) are analysed for a simple electron transfer under high volume flow rates, by curve fitting. Analysis of the transient LSVs yielded values for k 0, α, and E f 0 for TMPD of (5.9±2.4)×10 −2 cm s −1, 0.46±0.08 and 0.217±0.019 V (vs. Ag), respectively. This is in good agreement with independent experiments conducted using the high-speed channel electrode which yielded the results: k 0=(6.3±0.4)×10 −2 cm s −1, α=0.52±0.01, and E f 0 =0.234±0.005 V (vs. Ag). The range of applicability of this method for measuring k 0 was also investigated and compared with existing channel electrode techniques.

In situ evidence of an alternative oxidase and an uncoupling protein in the respiratory chain of Aspergillus fumigatus

Aspergillus fumigatus is an unusual pathogen in immunocompetent individuals; its incidence has increased in the last decades in patients immunocompromised, like those with chronic granulomatosis disease and AIDS. The aim of this study was to identify differences between the respiratory chain of host and the fungus planning to use the later as a pharmacological target. We evaluated respiration, membrane potential and oxidative phosphorylation of mitochondria of the spheroplasts of A. fumigatus in situ, after permeabilization with digitonin. Firstly, a functional respiratory chain (complex I–V) was demonstrated: adenosine 5′-diphosphate (ADP) induced an oligomycin-sensitive transition from resting to phophorylating respiration in the presence of the oxidizable substrates malate, glutamate, α-ketoglutarate, pyruvate, dihydroorotate, succinate, N,N,N′ ,N′-tetramethyl- p-phenylenediamine (TMPD) and exogenous NADH. In addition, the ability of the fungus to oxidize exogenous NADH, as well as the insensitivity of its respiration to rotenone, in association with the sensitivity to flavone, indicate the presence of an alternative NADH–ubiquinone oxidoreductase; the partial sensitivity of respiration to antimycin A and cyanide, in association with the sensitivity to benzohydroxamic acid, indicates the presence of an alternative oxidase. The fatty acid-uncoupled respiration was partly reversed by bovine serum albumin (BSA) and guanosine 5′-triphosphate (GTP) and was insensitive to either carboxyatractyloside or ADP. These results, together with evidences obtained using antibodies raised against uncoupling protein (UCP) from potato, indicate in addition, the presence of an uncoupling protein in the respiratory chain of A. fumigatus.