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Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress-induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and corroborated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated nicotinamide adenine dinucleotide (NADH) and ubiquinol (QH2) levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH2 levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O2 at low O2 concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the electron transport system complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying workloads.

Doxorubicin is an anthracycline-based chemotherapeutic that causes myotoxicity with symptoms persisting beyond treatment. Patients experience muscle pain, weakness, fatigue, and atrophy, but the underlying mechanisms are poorly understood. Studies investigating doxorubicin-induced myotoxicity have reported disrupted mitochondrial function. Mitochondria are responsible for regulating both cellular energy status and Ca(2+) handling, both of which impact contractile function. Moreover, loss of mitochondrial integrity may initiate muscle atrophy. Skeletal muscle mitochondrial dysregulation may therefore contribute to an overall loss of skeletal muscle quality and performance that may be mitigated by appropriately targeted mitochondrial therapies. We therefore assessed the impact of doxorubicin on muscle performance and applied a multiplexed assay platform to diagnose alterations in mitochondrial respiratory control. Mice received a clinically relevant dose of doxorubicin delivered systemically and were euthanized 72 h later. We measured extensor digitorum longus and soleus muscle forces, fatigue, and contractile kinetics in vitro, along with Ca(2+) uptake in isolated sarcoplasmic reticulum. Isolated skeletal muscle mitochondria were used for real-time respirometry or frozen for protein content and activity assays. Doxorubicin impaired muscle performance, which was indicated by reduced force production, fatigue resistance, and sarcoplasmic reticulum–Ca(2+) uptake, which were associated with a substrate-independent reduction in respiration and membrane potential but no changes in the NAD(P)H/NAD(P)(+) redox state. Protein content and dehydrogenase activity results corroborated these findings, indicating that doxorubicin-induced mitochondrial impairments are located upstream of ATP synthase within the electron transport system. Collectively, doxorubicin-induced lesions likely span mitochondrial complexes I–IV, providing potential targets for alleviating doxorubicin myotoxicity.

Made available in DSpace on 2021-06-25T10:30:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-26 The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacterium Eikenella corrodens grown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N'-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2 without the bc1 complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitive cbb' cytochrome c oxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system of E. corrodens ATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD. Grupo de Investigación de Biomembranas (GIBIOM) Facultad Ciencias de La Salud Centro Seccional de Investigación (CIUL), Universidad Libre Campus Barranquilla Grupo de Micología Departamento de Microbiología Facultad de Medicina Universidad de Cartagena Campus Zaragocilla School of Agricultural and Veterinary Sciences Technology Department São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n School of Agricultural and Veterinary Sciences Technology Department São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n

Monte Carlo simulations, which are widely used for predicting the transport properties of semiconductors, use a large number of parameters, such as the effective masses, the non-parabolic constants, the deformation potential, the phonon energies, and the elastic constants. Most of these parameters are not very well known or have different reported computed or measured values. In this paper, we employ an uncertainty quantification technique that allows us to determine the effect of the uncertainty of parameter values on the transport properties computed using the Monte Carlo technique and establish the key parameters that strongly affect the results in contrast to the parameters whose wide variation has a small overall impact.

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be 9.384 A. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH → FDR6 → FDX4 → Gel16.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benznidazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel β-lapachone-derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC50/24 h in the range of 0.8–7.9 μM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled H2O2 release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydrogenase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.

Aerobic and anaerobic respiratory systems allow cells to transport the electrons to terminal electron acceptors. The quinone (ubiquinone or menaquinone) pool is central to the electron transport chain. In the majority of Gram-positive bacteria, vitamin K2 (menaquinone) is the sole quinone in the electron transport chain, and thus, the bacterial enzymes catalyzing the synthesis of menaquinone are potential targets for the development of novel antibacterial drugs. This manuscript reviews the role of vitamin K in bacteria and humans, and especially emphasizes on recent aspects of menaquinones in bacterial electron transport chain and on discoveries of inhibitor molecules targeting bacterial electron transport systems for new antibacterial agents.

Chronic kidney disease (CKD) leads to increased skeletal muscle fatigue, weakness, and atrophy. Previous work has implicated mitochondria within the skeletal muscle as a mediator of muscle dysfunction in CKD; however, the mechanisms underlying mitochondrial dysfunction in CKD are not entirely known. The purpose of this study was to define the impact of uremic metabolites on mitochondrial energetics. Skeletal muscle mitochondria were isolated from C57BL/6N mice and exposed to vehicle (DMSO) or varying concentrations of uremic metabolites: indoxyl sulfate, indole-3-acetic-acid, l-kynurenine, and kynurenic acid. A comprehensive mitochondrial phenotyping platform that included assessments of mitochondrial oxidative phosphorylation (OXPHOS) conductance and respiratory capacity, hydrogen peroxide production ( JH2O2), matrix dehydrogenase activity, electron transport system enzyme activity, and ATP synthase activity was employed. Uremic metabolite exposure resulted in a ~25–40% decrease in OXPHOS conductance across multiple substrate conditions ( P < 0.05, n = 5–6/condition), as well as decreased ADP-stimulated and uncoupled respiratory capacity. ATP synthase activity was not impacted by uremic metabolites; however, a screen of matrix dehydrogenases indicated that malate and glutamate dehydrogenases were impaired by some, but not all, uremic metabolites. Assessments of electron transport system enzymes indicated that uremic metabolites significantly impair complex III and IV. Uremic metabolites resulted in increased JH2O2 under glutamate/malate, pyruvate/malate, and succinate conditions across multiple levels of energy demand (all P < 0.05, n = 4/group). Disruption of mitochondrial OXPHOS was confirmed by decreased respiratory capacity and elevated superoxide production in cultured myotubes. These findings provide direct evidence that uremic metabolites negatively impact skeletal muscle mitochondrial energetics, resulting in decreased energy transfer, impaired complex III and IV enzyme activity, and elevated oxidant production.

The effects of CO2 elevation on sensitivity of photosynthetic electron transport system of wheat in relation to low temperature stress are unclear. The performance of photosynthetic electron transport system and antioxidant system in chloroplasts was investigated in a temperature sensitive wheat cultivar Lianmai6 grown under the combination of low temperature (2 days at 2/−1◦ C in the day/night) and CO2 elevation (800 µmol l−1). It was found that CO2 elevation increased the efficiency of photosynthetic electron transport in wheat exposed to low temperature stress, which was related to the enhanced maximum quantum yield for electron transport beyond QA and the increased quantum yield for reduction of end electron acceptors at the PSI acceptor side in plants under elevated CO2. Also, under low temperature, the activities of ATPases, ascorbate peroxidase, and catalase in chloroplasts were enhanced in wheat under elevated CO2. It suggested that the cold tolerance of photosynthetic electron transport system is enhanced by CO2 elevation.

Nanopesticides are widely applied in modern agricultural systems to replace traditional pesticides, which inevitably leads to their accumulation in soils. Nanopesticides based on copper oxide nanoparticles (CuO NPs) may affect the soil nitrogen cycle, such as the denitrification process; however, the mechanism remains unclear. Here, acute exposure experiments for 60 h were conducted to explore the effects of CuO NPs (10, 100, 500 mg kg−1) on denitrification. In this study, Cu speciation, activities of denitrifying enzymes, electron transport system activity (ETSA), expression of denitrifying functional genes, composition of bacterial communities and reactive oxygen species (ROS) were determined. In all treatments, Cu ions was the dominant form and responsible for the toxicity of CuO NPs. The results indicated that CuO NPs treatments at 500 mg kg−1 remarkably inhibited denitrification, led to an 11-fold increase in NO3− accumulation and N2O emission rates decrease by 10.2–24.1%. In the denitrification process, the activities of nitrate reductase and nitric oxide reductase reduced by 21.1–42.1% and 10.3–16.3%, respectively, which may be a reason for the negative effect of CuO NPs. In addition, ETSA was significantly inhibited with CuO NPs applications, which reflects the ability of denitrification to accept electrons. Denitrifying functional genes and bacterial communities composition were changed, thus further influencing the denitrification process. ROS analysis showed that there were no significant differences among NPs treatments. This research improves the understanding of CuO NPs impact on soil denitrification. Further attention should be paid to the nitrogen transformation in agricultural soils in the presence of nanopesticides.

This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (maxOCR) and spare respiratory capacity (SRC) in tumor cells. T98G and U-87MG glioma cells were titrated with the protonophore CCCP to induce maxOCR. The presence of oligomycin (0.3–3.0 µg/mL) led to underestimation of maxOCR and a consequent decrease in SRC values of between 25% and 40% in medium containing 5.5 or 11 mM glucose. The inhibitory effect of oligomycin on CCCP-induced maxOCR did not occur when glutamine was the metabolic substrate or when the glycolytic inhibitor 2-deoxyglucose was present. ATP levels were reduced and ADP/ATP ratios increased in cells treated with CCCP, but these changes were minimized when oligomycin was used to inhibit reverse activity of ATP synthase. Exposing digitonin-permeabilized cells to exogenous ATP, but not ADP, resulted in partial inhibition of CCCP-induced maxOCR. We conclude that underestimation of maxOCR and SRC in tumor cells when ATP synthase is inhibited is associated with high glycolytic activity and that the glycolytic ATP yield may have an inhibitory effect on the metabolism of respiratory substrates and cytochrome c oxidase activity. Under CCCP-induced maxOCR, oligomycin preserves intracellular ATP by inhibiting ATP synthase reverse activity.

Mitochondrial respiration and ATP production are compromised by hypoxia. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals and reduce metabolic rate in hypoxic environments; however, little is known regarding mitochondrial function during in vivo hypoxia exposure in this species. To address this knowledge gap, we asked whether the function of NMR brain mitochondria exhibits metabolic plasticity during acute hypoxia. Respirometry was utilized to assess whole-animal oxygen consumption rates and high-resolution respirometry and was utilized to assess electron transport system (ETS) function in saponin-permeabilized NMR brain. We found that NMR whole animal oxygen consumption rate reversibly decreased by ∼ 85% in acute hypoxia (4 hrs at 3% O2). Similarly, relative to untreated controls, permeabilized brain respiratory flux through the ETS was decreased by ∼ 90% in acutely hypoxic animals. Relative to FCCP-uncoupled total ETS flux, this functional decrease was observed equally across all components of the ETS except for complex IV (cytochrome c oxidase), at which flux was further reduced, supporting a regulatory role for this enzyme during acute hypoxia. The maximum enzymatic capacities of ETS complexes I-V were not altered by acute hypoxia; however, the mitochondrial H+-gradient decreased in step with the decrease in ETS respiration. Taken together, our results indicate that NMR brain ETS flux and H+ leak are reduced in a balanced and regulated fashion during acute hypoxia. Changes in NMR mitochondrial metabolic plasticity mirror whole animal metabolic responses to hypoxia.

Scleractinian corals host photosynthetic endosymbionts, making direct measurement of the host respiration rate via incubation methods based on O2 consumption impossible. We tested the use of the respiratory electron transport system activity (ETSA) for measuring host potential respiration. The applied method, modified from a previous study, is based on the re - duction of (4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazan. After the development of a protocol suitable for corals, the method was tested on 5 different spe- cies. Metabolism, including photosynthesis, dark respiration and light and dark calcification, was measured through incubation. Host and zooxanthellae fractions were separated and their ETSAs, protein contents and zooxanthellae densities were measured. Mean ETSA/dark respiration (ETSA/R) ratios for host corals ranged from 1.7 ± 0.2 to 3.5 ± 0.6, while ratios for zooxanthellae ranged from 3.7 to 7.8. The high ratios observed for zooxanthellae indicate that their respiration may be 5 times higher under light conditions than in the dark. Considering the obtained ratios, host respiration in light could increase at most by a factor of 3.5 compared with dark respiration rates. Ratios close to 1 were found for some specimens, which suggests that higher respiration rates under light compared with dark conditions are not possible. Therefore, increased respiration in light cannot explain the observed enhancement of calcification under light conditions. ETSA was correlated with zooxanthellae density, suggesting adaptation of the levels of host ETS enzymes to the amount of translocated photosynthetates under optimal conditions. Estimated dark host respiration was correlated with photosynthesis, which suggests that it is determined mainly by the amount of energy available but also the amount of electron transport system enzymes. This constrains the amount of ATP available for calcification. Hence, we propose a mechanism by which respiration limits the calcification rate.

CYP175A1 from Thermus thermophilus is a thermophilic cytochrome P450 and has great potential for industrial applications. However, a native electron transport system for CYP175A1 has not been identified. Here, an electron transport system for CYP175A1 was isolated from T. thermophilus HB27 by multistep chromatography, and identified as comprising ferredoxin (Fdx; locus in the genome, TTC1809) and ferredoxin-NAD(P)+reductase (FNR; locus in the genome, TTC0096) by N-terminal amino acid sequence analysis and MALDI-TOF-MS, respectively. Although TTC0096, which encodes the FNR, is annotated as a thioredoxin reductase in the T. thermophilus HB27 genome database, TTC0096 lacks an active-site dithiol/disulfide group, which is required to exchange reducing equivalents with thioredoxin. The FNR reduced ferricyanide, an artificial electron donor, in the presence of NADH and NADPH, but preferred NADPH as a cofactor (Km for NADH = 2440 +/- 546 microM; Km for NADPH = 4.1 +/- 0.2 microM). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The Tm value of the FNR was 99 degrees C at pH 7.4. With an electron transport system consisting of Fdx and FNR, CYP175A1 efficiently catalyzed the hydroxylation of beta-carotene at the 3-position and 3'-position at 65 degrees C, and the Km and Vmax values for beta-carotene hydroxylation were 14.3 +/- 1.6 microM and 18.3 +/- 0.6 nmol beta-cryptoxanthin min(-1) nmol(-1) CYP175A1, respectively. This is the first report of a native electron transport system for CYP175A1.

Small hibernators cycle between periods of torpor, with body temperature (T b) approximately 5 °C, and interbout euthermia (IBE), where T b is approximately 37 °C. During entrance into a torpor bout liver mitochondrial respiration is rapidly suppressed by 70 % relative to IBE. We compared activities of electron transport system (ETS) complexes in intact liver mitochondria isolated from 13-lined ground squirrels (Ictidomys tridecemlineatus) sampled during torpor and IBE to investigate potential sites of this reversible metabolic suppression. Flux through complexes I–IV and II–IV was suppressed by 40 and 60 %, respectively, in torpor, while flux through complexes III–IV and IV did not differ between torpor and IBE. We also measured maximal enzyme activity of ETS enzymes in homogenized isolated mitochondria and whole liver tissue. In isolated mitochondria, activities of complexes I and II were significantly lower in torpor relative to IBE, but complexes III, IV, and V did not differ. In liver tissue, only activity of complex II was suppressed during torpor relative to IBE. Despite the significant differences in both ETS flux and maximal activity, the protein content of complexes I and II did not differ between torpor and IBE. These results suggest that the rapid, reversible suppression of mitochondrial metabolism is due to regulatory changes, perhaps by post-translational modification during entrance into a torpor bout, and not changes in ETS protein content.

The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2-para (iodo-phenyl)-3(nitrophenyl)-5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2-6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

We report evidence for a modulation of Ehrlich cell plasma membrane redox activity by cytoplasmic pH. When Ehrlich cells were submitted to treatments leading to a slight decrease of cytoplasmic pH, there was a significant inhibition of plasma membrane ferricyanide reductase activity. However, those treatments which produced a slight alkalinization of the cytoplasm enhanced the activity of the plasma membrane redox system. Since it has been shown previously that plasma membrane redox activity controls cytoplasmic pH, it seems that plasma membrane redox activity and cytoplasmic pH mutually modulate each other.

TTC and INT-electron transport system activity tests were compared for assessing heavy metal inhibition of activated sludge. The median inhibitory concentrations (IC 50 s) of Cu 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Ni 2+ , Pb 2+ and Ag + measured via TTC test were lower than those measured via INT test, which indicates that the INT-electron transport system activity test was less sensitive to heavy metal toxicity than the TTC test. Tested heavy metals brought about similar decrease in TTC-electron transport system activity and COD removal rate, but less decrease in INT-electron transport system activity than COD removal rate, which suggests that the TTC-electron transport system activity is a better parameter for reflecting heavy metal inhibition of activated sludge than INT-electron transport system activity. The ranking of tested heavy metals in order of decreasing toxicity based on TTC test was Hg 2+ , Cd 2+ , Cu 2+ , Ag + , Zn 2+ , Ni 2+ and Pb 2+ , and the ranking based on INT test was Hg 2+ , Ag + , Cu 2+ , Cd 2+ , Zn 2+ , Ni 2+ and Pb 2+ .

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh , encoding NADH dehydrogenase II, and cyoABCDE , encoding the cytochrome bo -type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto-c. We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto-c electron transport pathway.

Tetrazolium reduction and enzyme kinetics were examined to estimate the ETS activity of decomposing reed (Phragmites australis /Cav./ Trin. ex Steud.) rhizome to collect information on the activity of microbial decomposers. Optimal incubation time was determined at 22 °C. For complete enzyme extraction, 4-6 min. of homogenization was necessary. The main substrates of the enzymatic reaction were NADH and NADPH. The reaction was fastest when 2-(-p-iodophenyl)-3-(-p-nitrophenyl)-5-phenyl tetrazolium chloride (INT ; 0.8 mM), NADPH (0.25 mM) and NADH (1.7 mM) were applied simultaneously. The optimal incubation time should be less than 20 minutes. The pH optimum of the enzyme reaction is 8.0-8.4. ETS activity of decomposing reed rhizome can be used to estimate potentially the oxygen consumption of microorganisms involved in decomposition and, indirectly, the rate of decomposition.

Electron transport system (ETS) activity was measured in amended and nonamended soil by measuring the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan), which can be easily extracted with methanol without interference from other compounds found in soil. A high correlation between ETS activity and oxygen consumption was observed. This technique allows rapid quantitative measurements of microbial ETS activity in soil.

In this communication, a novel, green, efficient and economically viable light mediated protocol for generation of Au-nanoparticles using most vital organelle, chloroplasts, of the plant system is portrayed. Thylakoids/chloroplasts isolated from Potamogeton nodosus (an aquatic plant) and Spinacia oleracea (a terrestrial plant) turned Au3+ solutions purple in presence of light of 600 µmol m−2 s−1 photon flux density (PFD) and the purple coloration intensified with time. UV-Vis spectra of these purple colored solutions showed absorption peak at ∼545 nm which is known to arise due to surface plasmon oscillations specific to Au-nanoparticles. However, thylakoids/chloroplasts did not alter color of Au3+ solutions in dark. These results clearly demonstrated that photosynthetic electron transport can reduce Au3+ to Au0 which nucleate to form Au-nanoparticles in presence of light. Transmission electron microscopic studies revealed that Au-nanoparticles generated by light driven photosynthetic electron transport system of thylakoids/chloroplasts were in range of 5–20 nm. Selected area electron diffraction and powder X-ray diffraction indicated crystalline nature of these nanoparticles. Energy dispersive X-ray confirmed that these nanoparticles were composed of Au. To confirm the potential of light driven photosynthetic electron transport in generation of Au-nanoparticles, thylakoids/chloroplasts were tested for their efficacy to generate Au-nanoparticles in presence of light of PFD ranging from 60 to 600 µmol m−2 s−1. The capacity of thylakoids/chloroplasts to generate Au-nanoparticles increased remarkably with increase in PFD, which further clearly demonstrated potential of light driven photosynthetic electron transport in reduction of Au3+ to Au0 to form nanoparticles. The light driven donation of electrons to metal ions by thylakoids/chloroplasts can be exploited for large scale production of nanoparticles.

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

Respiratory Electron Transport System activity (ETS) was determined in cell-free homogenates of mesozooplankton samples. Several assays were run to investigate possible improvements to the ETS method and to reduce sample processing time. The results showed that: (i) liquid nitrogen is a suitable method for preserving ETS activity for up to at least two months; (ii) the processed sample can be stored for up to 90 minutes in an ice-water bath without loss of activity; (iii) a glass-fibre filter, normally used in phytoplankton assays, is not necessary to achieve efficient enzyme extraction; (iv) centrifugation can be done prior to the assay without any significant loss in activity; and (v) the partitioned enzyme activities were different from the overall ETS activity, with NADH- dehydrogenase activity playing the major role (71 %) and succinate dehydrogenase the minor (

We examined the relationship between potential respiration rates, as measured by electron transport system (ETS) activity, standardized at a defined temperature (ETS2O). and nutrients and sestonic particles in a set of 101 Spanish reservoirs spannin a wide range of limnological characteristics. ETS activity ranged from 0.009 to 31 31 μmol e- 1−1 h−1 Among the nutrients, it was significantly correlated only with phosphorus, and only during the stratification period. During this period, the best regressor of ETS2O was the concentration of chlorophyll a (Chla), whereas during the mixing period the best regressor was particulate organic nitrogen (PON), suggesting a greater contribution of non-algal sestonic particles to total metabolism. Both the mean ETS20:PON ratio and the mean Chla:PON ratio increased systematically with increasing PON, indicating a lower relative contnbution of detrital particles to total seston as trophic state increased. In contrast, the mean ETS20:Chla ratio was constant across the range of Chla. Vertical profiles of ETS2O. Chla and PON were more coherent during the stratification period. when subsurface and metalimnetic peaks were frequent, than during the mixing period.

Anomalous choline metabolic patterns have been consistently observed in vivo using Magnetic Resonance Spectroscopy (MRS) analysis of patients with neurodegenerative diseases and tissues from cancer patient. It remains unclear; however, what signaling events may have triggered these choline metabolic aberrancies. This study investigates how changes in choline and phospholipid metabolism are regulated by distinct changes in the mitochondrial electron transport system (ETS). We used specific inhibitors to down regulate the function of individual protein complexes in the ETS of SH-SY5Y neuroblastoma cells. Interestingly, we found that dramatic elevation in the levels of phosphatidylcholine metabolites could be induced by the inhibition of individual ETS complexes, similar to in vivo observations. Such interferences produced divergent metabolic patterns, which were distinguishable via principal component analysis of the cellular metabolomes. Functional impairments in ETS components have been reported in several central nervous system (CNS) diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases.

The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane inGluconobacter oxydans (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW 23000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a number of cytochrome c species with very low midpoint potential difference.

Thermoluminescence is a burst of light emission by warming the leaf or chloroplasts which were illuminated by light. It results from the charge recombination between positively and negatively charged pairs created by photochemical reaction. The origin of four out of seven thermoluminescence bands was assigned: the A, Q, B1 and B2 bands arise from S3QA-, S2QA-, S3QB-·and S2QB- charge pairs, respectively. Thermoluminescence is applied to the analysis of oxidinzing and reducing side of photosystem II. The following facts were elucidated; 1) By removal of 33 kDa extrinsic protein, S3 to S4 transition is inhibited. 2) By Cl-·depletion, abnomal S2 state is formed. 3) Replacement of QB-quinone with quinone derivatives yields a modified thermoluminescence band. 4) Thermoluminescence is a powerful tool for the study of herbicides and the resistant mutants.

Accumulation of electrons under conditions of environmental stress produces a reduced state in the photosynthetic electron transport (PET) system and causes the reduction of O2 by PSI in the thylakoid membranes to produce the reactive oxygen species superoxide radical, which irreversibly inactivates PSI. This study aims to elucidate the molecular mechanism for the oxidation of reaction center Chl of PSI, P700, after saturated pulse (SP) light illumination of the cyanobacterium Synechococcus elongatus PCC 7942 under steady-state photosynthetic conditions. Both P700 and NADPH were transiently oxidized after SP light illumination under CO2-depleted photosynthesis conditions. In contrast, the Chl fluorescence intensity transiently increased. Compared with the wild type, the increase in Chl fluorescence and the oxidations of P700 and NADPH were greatly enhanced in a mutant (Δflv1/3) deficient in the genes encoding FLAVODIIRON 1 (FLV1) and FLV3 proteins even under high photosynthetic conditions. Furthermore, oxidation of Cyt f was also observed in the mutant. After SP light illumination, a transient suppression of O2 evolution was also observed in Δflv1/3. From these observations, we propose that the reduction in the plastquinone (PQ) pool suppresses linear electron flow at the Cyt b6/f complex, which we call the reduction-induced suppression of electron flow (RISE) in the PET system. The accumulation of the reduced form of PQ probably suppresses turnover of the Q cycle in the Cyt b6/f complex.

The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora. To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P. hydrogenovora by introducing an in-frame deletion into the hupC. The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain. Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.

Various drugs are inactivated by liver microsomes in the presence of NADPH and atomospheric oxygen (1). The enzymes catalyzing these reactions can activate molecular oxygen by a two-electron reduction so that one oxygen atom is introduced into the substrate leading to a hydroxylated product, whereas the second atom is reduced to water (1, 2). Recently the participation in these reactions of hemoprotein called cytochrome P-450 (3) as the oxygen-activating component (4-8) has been established (Fig. 1). The activity of drug-metabolizing enzymes of liver microsomes was altered by various factors, such as the administration of phenobarbital or methylcholanthrene (9, 10), thyroxine (11-13), anabolic hormone (11, 14, 15), carbon tetrachloride (11, 16) and morphine (11, 12), and adrenalectomy (11, 17), thyroidectomy (13), hepatectomy (18), starvation (19, 20), alloxan diabetes (11, 12) and low protein diet (16, 21, 22). It was also demonstrated that the activity of NADPH-linked electron transport system of liver microsomes was often altered in association with the alteration in the activity of drug-metabolizing enzymes under the above-given conditions (10, 13, 22, 26). Vitamin C is a well known component related to the control of oxido-reduction states of living cell, but detailed role of vitamin C has not been fully elucidated (27). On the other hand, Mitoma et al. (28), Tochino et al. (29) and more recently, Conney et al. (30) reported that the hydroxylation of acetanilide, hexobarbital and zoxazolamine was decreased in vitamin C deficient guinea-pigs. However, the studies on the mechanism of decreased hydroxylation activity in relation to the activity of NADPH-linked electron transport system has not yet been reported. The purpose of the present study, therefore, is to investigate whether or not the mechanism of decreased hydroxylation activity of liver microsomes from vitamin C deficient guinea-pigs is related to the decreased activity of NADPH-linked electron transport system.

The relationship between spleen and liver microsomal heme oxygenase activities and the microsomal electron transport system requiring NADPH and cytochrome P-450 has been demonstrated by immunochemical techniques. An antibody preparation to purified, homogeneous NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) was shown to inhibit concomitantly NADPH-cytochrome c reductase and heme oxygenase activities in rat liver and spleen and pig spleen microsomal preparations. Previous work demonstrated the requirement for this enzymic activity in the reduction of cytochrome P-450 associated with drug hydroxylation reactions and steroid metabolism by concomitant inhibition of these various activities with anti-NADPH-cytochrome c reductase γ-globulin. The levels of NADPH-cytochrome c reductase in both rat and pig spleen reported in the present study are less than 10% of those found in rat and pig liver. Even at these levels, however, the reductase activity is sufficient to maintain the electron flux required for heme oxygenase activity under conditions in which heme catabolism is maximal. Methemalbumin treatment of rats resulted in a 3.7-fold increase in hepatic heme oxygenase activity which was concomitantly inhibited by anti-reductase γ-globulin, indicating that the induced enzyme system maintained its requirement for the microsomal electron transport system. Comparison of electron paramagnetic resonance spectra and ethyl isocyanide difference spectra of liver microsomes from untreated and methemalbumin-treated rats failed to demonstrate the induction of a new cytochrome moiety as a result of in vivo administration of methemalbumin, a substrate for the heme oxygenase system. The administration of methemalbumin in vivo to rats did not result in an increase in the components of the hepatic microsomal electron transport system, i.e. NADPH-cytochrome c reductase activity or cytochrome P-450 content, indicating that the modulation and control mechanisms of the microsomal heme oxygenase system differ from those governing the microsomal electron transport system. Methemalbumin treatment resulted in noncompetitive inhibition of the binding of both Type I and Type II drugs in rat liver microsomal preparations. In addition to the antibody inhibition studies, the temporal relationship demonstrated among the decrease in magnitude of binding spectra of hexobarbital, the increase in heme oxygenase activity, the decrease in measurable cytochrome P-450 content, and the reversal of these parameters during the recovery phase following a single injection of methemalbumin strongly indicates the involvement of the same components of the microsomal electron transport system in both the drug metabolism and heme oxygenase systems in liver.

The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.

Hydrogenase activity and the H 2 -fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis , were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H 2 . Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H 2 was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum . Sulfhydryl compounds were not required for fumarate reduction by H 2 , but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline- N -oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H 2 when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H 2 to fumarate in B. fragilis is proposed.

The electron transfer system that transports electrons from formate to nitrogenase was examined in extracts of the obligate methanotroph Methylosinus trichosporium OB3b. By supplementing a crude nitrogenase extract with NAD, formate dehydrogenase, NADH-ferredoxin reductase and FMN (an FDH cofactor), an electron transport system was established that coupled formate oxidation to nitrogenase-dependent acetylene reduction. The ferredoxin dependence of this reaction was demonstrated by its severe inhibition by antibodies to ferredoxin. The reaction sequence is as follows: formate → formate dehydrogenase → NADH → NADH-ferredoxin reductase → ferredoxin → nitrogenase.

The study describes the inhibitory activity of natural piericidins and related compounds, including synthetic analogues, to the electron transport system of mitochondria. Consideration of the structure-activity relationships led to the proposing of a structural unit I that was essential to inhibitory activity.

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b5 reductase and cytochrome b5.

1. Aniline-induced difference spectrum in post-mortem human liver microsomes was recognized as type II similar to that in rat liver. Hexobarbital- and aminopyrineinduced P-450 difference spectra were also of type II in human, but they were different from those in rat liver microsomes.2. There was no correlation between enzyme activities of the NADPH-linked electron transport system in liver microsomes and aniline hydroxylase activity in human liver, which was similar to that of rat liver. The activity of hydroxylation. of hexobarbital, a type I compound, in human was lower than that in rat liver, and a similar result was observed when aminopyrine was used as substrate.

Components of the electron transport system involved in steroid 11β-hydroxylation in pig adrenal cortex are a flavoprotein (adrenodoxin reductase), a non-heme iron protein (adrenodoxin), and possibly cytochrome P450. Reconstitution of reduced nicotinamide adenine dinucleotide phosphate cytochrome P450 reductase was achieved from soluble components. Adrenodoxin contains 2 iron atoms and 2 moles of labile sulfide per mole, has a molecular weight of approximately 20,000, and has a high oxidation-reduction potential. The iron atoms, which are ferric in the oxidized form, play a dual role: as a chromophore and as a center of asymmetry. Upon reduction, the environment of the iron of adrenodoxin is changed greatly as judged by spectral and optical rotatory properties.