Respiratory Electron Transfer System Research Articles

Identification of proteins involved in intracellular ubiquinone trafficking in Saccharomyces cerevisiae using artificial ubiquinone probe

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ6, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ6-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ6-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.

Guanosine protects against behavioural and mitochondrial bioenergetic alterations after mild traumatic brain injury

Traumatic brain injury (TBI) constitutes a heterogeneous cerebral insult induced by traumatic biomechanical forces. Mitochondria play a critical role in brain bioenergetics, and TBI induces several consequences related with oxidative stress and excitotoxicity clearly demonstrated in different experimental model involving TBI. Mitochondrial bioenergetics alterations can present several targets for therapeutics which could help reduce secondary brain lesions such as neuropsychiatric problems, including memory loss and motor impairment. Guanosine (GUO), an endogenous neuroprotective nucleoside, affords the long-term benefits of controlling brain neurodegeneration, mainly due to its capacity to activate the antioxidant defense system and maintenance of the redox system. However, little is known about the exact protective mechanism exerted by GUO on mitochondrial bioenergetics disruption induced by TBI. Thus, the aim of this study was to investigate the effects of GUO in brain cortical and hippocampal mitochondrial bioenergetics in the mild TBI model. Additionally, we aimed to assess whether mitochondrial damage induced by TBI may be related to behavioral alterations in rats. Our findings showed that 24 h post-TBI, GUO treatment promotes an adaptive response of mitochondrial respiratory chain increasing oxygen flux which it was able to protect against the uncoupling of oxidative phosphorylation (OXPHOS) induced by TBI, restored the respiratory electron transfer system (ETS) established with an uncoupler. Guanosine treatment also increased respiratory control ratio (RCR), an indicator of the state of mitochondrial coupling, which is related to the mitochondrial functionality. In addition, mitochondrial bioenergetics failure was closely related with locomotor, exploratory and memory impairments. The present study suggests GUO treatment post mild TBI could increase GDP endogenous levels and consequently increasing ATP levels promotes an increase of RCR increasing OXPHOS and in substantial improve mitochondrial respiration in different brain regions, which, in turn, could promote an improvement in behavioral parameters associated to the mild TBI. These findings may contribute to the development of future therapies with a target on failure energetic metabolism induced by TBI.

Enhanced production of CoQ10 by constitutive overexpression of 3-demethyl ubiquinone-9 3-methyltransferase under tac promoter in Rhodobacter sphaeroides

Coenzyme Q10 (CoQ10), as an essential electron carrier in the aerobic respiratory electron transfer system, has been proven to be an effective compound in health care and disease prevention. With the aim of improving the production of CoQ10, the biosynthetic pathway of CoQ10 was engineered in Rhodobacter sphaeroides by overexpressing 3-demethyl ubiquinone-9 3-methyltransferase (UbiG), which catalyzes the two steps of O-methylations of the quinonoid ring. For expressing the UbiG, a constitutive expression vector harboring tac promoter and ubiG gene was firstly developed. After cultivated in dark conditions, the transcriptional level of ubiG of the recombinant R. sphaeroides without inducer, which approached to that of with inducer, was about 133 times that of the wild type. The production was correspondingly improved up to 65.8mg/L compared to 47.6mg/L for the wild type. In addition, it was found that isopropyl-beta-d-thiogalactopyranoside (IPTG) had a negative effect on CoQ10 production when added to the medium, although it showed no significant influence on the growth rate of the microbes.

Respiration predicted from an Enzyme Kinetic Model and the Metabolic Theory of Ecology in two species of marine bacteria

Respiratory oxygen consumption is the result of a cell's biochemistry. It is caused by enzymatic activity of the respiratory electron transfer system (ETS). However, in spite of this understanding, respiration models continue to be based on allometric equations relating respiration to body size, body surface, or biomass. The Metabolic Theory of Ecology (MTE) is a current example. It is based on Kleiber's law relating respiration ( R) and biomass ( M) in the form, R = C M 3 4 e − E a k T , where C is a constant, E a is the Arrhenius activation energy, k is the Boltzmann constant for an atom or molecule, and T is the temperature in Kelvin. This law holds because biomass packages the ETS. In contrast, we bypass biomass and model respiration directly from its causal relationship with the ETS activity, R = f (ETS). We use a biochemical Enzyme Kinetic Model (EKM) of respiratory oxygen consumption based on the substrate control of the ETS. It postulates that the upper limit of R is set by the maximum velocity, V max , of complex I of the ETS and the temperature, and that the substrate availability, S, modulates R between zero and this upper limit. Kinetics of this thermal-substrate regulation is described by the Arrhenius and Michaelis–Menten equations. The EKM equation takes the form R = E T S [ S ] e − E a R g T K + [ S ] where R g is the molar gas constant and K is the Michaelis–Menten constant. Here, we apply the EKM and the MTE to predict a respiration time-profile throughout the exponential, steady state, and nutrient-limited phases of the marine bacteria Pseudomonas nautica and Vibrio natriegens in acetate-based cultures. Both models were tested by comparing their output with the measured R O 2 time-profile. The MTE predicted respiration accurately only in the exponential growth phase, but not during the nutrient limitation part of the stationary phase. In contrast, the EKM worked well throughout both physiological phases as long as the modeled substrates fall with the declining carbon source. Results support the theoretical bases of the EKM. We conclude that the EKM holds promise for predicting respiration at the different physiological states and time-scales important to microbiological studies.

Exploring a first-principles-based model for zooplankton respiration

AbstractPackard, T. T., and Gómez, M. 2008. Exploring a first-principles-based model for zooplankton respiration. – ICES Journal of Marine Science, 65: 371–378. Oxygen consumption (R) is caused by the respiratory electron transfer system (ETS), not biomass. ETS is ubiquitous in zooplankton, determines the level of potential respiration (Φ), and is the enzyme system that ultimately oxidizes the products of food digestion, makes ATP, and consumes O2. Current respiration hypotheses are based on allometric relationships between R and biomass. The most accepted version at constant temperature (T) is R = i0M0.75, where i0 is a constant. We argue that, for zooplankton, a Φ-based, O2-consuming algorithm is more consistent with the cause of respiration. Our point: although biomass is related to respiration, the first-principles cause of respiration is ETS, because it controls O2 consumption. Biomass itself is indirectly related to respiration, because it packages the ETS. Consequently, we propose bypassing the packaging and modelling respiration from ETS and hence Φ. This Φ is regulated by T, according to Arrhenius theory, and by specific reactants (S) that sustain the redox reactions of O2 consumption, according to Michaelis–Menten kinetics. Our model not only describes respiration over a large range of body sizes but also explains and accurately predicts respiration on short time-scales. At constant temperature, our model takes the form: where Ea is the Arrhenius activation energy, Rg, the gas constant, and Km, the Michaelis–Menten constant.

Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants

Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.

Modeling physiological processes in plankton on enzyme kinetic principles

Many ecologically important chemical transformations in the ocean are controlled by biochemical enzyme reactions in plankton. Nitrogenase regulates the transformation of N2 to ammonium in some cyanobacteria and serves as the entryway for N2 into the ocean biosphere. Nitrate reductase controls the reduction of NO3 to NO2 and hence new production in phytoplankton. The respiratory electron transfer system in all organisms links the carbon oxidation reactions of intermediary metabolism with the reduction of oxygen in respiration. Rubisco controls the fixation of CO2 into organic matter in phytoplankton and thus is the major entry point of carbon into the oceanic biosphere. In addition to these, there are the enzymes that control CO2 production, NH4 excretion and the fluxes of phosphate. Some of these enzymes have been recognized and researched by marine scientists in the last thirty years. However, until recently the kinetic principles of enzyme control have not been exploited to formulate accurate mathematical equations of the controlling physiological expressions. Were such expressions available they would increase our power to predict the rates of chemical transformations in the extracellular environment of microbial populations whether this extracellular environment is culture media or the ocean. Here we formulate from the principles of bisubstrate enzyme kinetics, mathematical expressions for the processes of NO3 reduction, O2 consumption, N2 fixation, total nitrogen uptake.

Respiration and vertical carbon flux in the Gulf of Maine water column

The transport of carbon from ocean surface waters to the deep sea is a critical factor in calculations of planetary carbon cycling and climate change. This vertical carbon e ux can be calculated by integrating the vertical proe le of the seawater respiration rate but is rarely done because measuring seawater respiration is so dife cult. However, seawater respiratory oxygen consumption is the product of the combined activity of all the respiratory electron transfer systems in a seawater community of bacterioplankton, phytoplankton, and zooplankton. This respiratory electron transfer system (ETS) is the membrane bound enzymatic system that controls oxygen consumption and ATP production in all eukaryots and in almost all bacteria and archaea. As such, it represents potential respiratory oxygen consumption. Exploiting this, we measured plankton-community ETS activity in water column proe les in the Gulf of Maine to give the potential-respiration of the water column. To interpret these potentials in terms of actual seawater respiration we made use of previous measurements of respiratory oxygen consumption and ETS activity in the Gulf of Maine to calculate a ratio of respiratory potential to actual respiration. Armed with this ratio we calculated seawater respiration depth proe les from the ETS activity measurements. These proe les were characterized by: (1) high oxygen consumption rates in the euphotic zone; (2) subsurface maxima near the subsurface chlorophyll maxima (SCM); (3) rapid declines associated with thermoclines; (4) low declining rates below 50 m; (5) and elevated values occasionally near the bottom. Sea surface values ranged from 229 to 489 pmol O 2 min 2 1

Effects of inorganic mercury on the respiration and the swimming activity of shrimp larvae, Pandalus borealis

In order to test the sensitivity of respiration (physiological and potential) to mercury (Hg) contamination, larval shrimp Pandalus borealis were exposed to inorganic Hg (0–160 ppb) for 27 h in the laboratory. Oxygen consumption rates (RO 2), potential respiration (determined by respiratory electron transfer system activity, ETSA), protein content, and swimming activity for zoeae III and zoeae V stages were measured. For both zoeae stages, ETSA and protein content remained constant after 27 h exposure to 160 ppb Hg whereas RO 2 and swimming activity decreased. This study revealed the impact of different Hg levels and different exposure times on RO 2 of shrimp larvae. After 10 h exposure to 160 ppb Hg, the RO 2 decreased by 43 and 49% in zoeae III and zoeae V stages, respectively. Exposure time of 27 h to 80 ppb Hg and higher, induced paralysis in nearly 100% larvae. Surprisingly, the paralysed larvae displayed almost 50% of the control's RO 2. The results showed that Hg disturbs a part of the respiration process without modifying the maximum activity of the enzymes involved in the ETSA assay. Therefore, the ETSA assay can not be used as a sublethal bioanalytic probe to detect Hg in short-term exposures. The decline of the RO 2/ETSA ratios reported here, indicates an inability of contaminated larvae to adapt their metabolism to physiological stress caused by Hg.

Enhancement of microplankton respiratory activity in the Almería-Oran Front (Western Mediterranean Sea)

The activity of the respiratory electron transfer system (ETS) in micToplankton (<200 um cell size) was measured along depth profiles from the surface to 100 m in the Almeria-Oran Front (Eastern Alboran Sea). ETS activity gives an estimation of respiratory metabolism of plankton that can be compared with biomass and primary production values. Three regions, Frontal Water (FW), Mediterranean Surface Water (MSW) and Modified Atlantic Water (MAW), were sampled. Chloro- phyll a was also determined. The front appears as an enriched region, with chlorophyll concentration and ETS activity higher than in the adjacent waters. Changes in biomass and activity were strongly related to hydrographic conditions. Chlorophyll distributions were mostly influenced by depth, and ETS distribution by differences among the subregions. ETS activity showed a statistically significant correlation with chlorophyll in the whole region and in the three subregions. Depth-integrated ETS activity showed an increase of 62% in frontal versus Mediterranean water, whereas the chlorophyll concentration was only 40% higher, indicating an enhancement of biomass-specific activity. Depth- integrated respiration (estimated from ETS measurement) represents between 30 and 50% of depth- integrated primary production according to published data on this zone.

Psychrophilic and psychrotrophic respiratory metabolism in antarctic microplankton

The activity of the respiratory Electron Transfer System (ETS) was measured in total microplankt on (<200-μm size fraction) and nanoplankton (<20-μm size fraction) from the Bransfield Strait, during the ECOANTAR 1993–1994 cruise of the Spanish B.I.O.Hesperides in January 1994. Activity variation in response to temperature was measured at three stations belonging to three different water masses that showed in situ temperatures ranging from — 0.57 to 1.30°C. Subsamples from each station were assayed for ETS activity at 11 temperatures in the — 3 to 20°C range. The results showed a bimodal activity-temperature variation in plankton from the lower in situ temperatures, with a peak in activity at 0°C, and a minimum at 3°C, with subsequent continuous increase up to absolute maxima at 15°C. The water mass with higher than 0°C temperature did not show the 0°C activity peak. The results suggest the existence, in water masses with in situ temperature near or below 0°C, of psychrophilic microbial populations with a narrow temperature range of respiratory enzyme activity, coexisting with more numerous and widespread psychrotrophs, or cold-tolerant populations, whose ETSs showed a continuous increase in activity in the — 3 to 15°C temperature range. Arrhenius activation energies (Ea) of total microplankton ranged from 3 to 17 kcal mole−1, and the Q10 from 1.2 to 3.5. These facts point to the existence of differentiated biochemical adaptations and acclimations to low temperature in polar plankton, an issue that has been much discussed in the recent past.

Oxygen consumption in the marine bacterium Pseudomonas nautica predicted from ETS activity and bisubstrate enzyme kinetics

The respiratory O2 consumption in aerobic bacterial cultures has been modeled from the time profiles of the in vitro activity of the respiratory electron transfer system (ETS), the bacterial protein and the concentration of the carbon source in the cultures. The model was based on the concept of bisubstrate kinetic control of the ETS throughout the exponential, steady-state and senescent phases of the cultures. In the exponential phase, the measured rates of O2 consumption and the in vitro ETS activity were closely coupled, but in the senescent phase, they were uncoupled. The in vitro ETS activity remained high even after the culture's carbon source was exhausted, while the O2 consumption fell to low levels. Based on the hypothesis that this uncoupling was caused by limitation of the intracellular ETS substrates (NADH and NADPH), a semi-empirical model incorporating a bisubstrate enzyme kinetics algorithm was formulated and fitted to the observations of the experiments. The model predicted the rate of O2 consumption throughout the different phases of the cultures with an r2 > 0.92 (n = 9, P < 0.001) using physiologically realistic Michaelis and dissociation constants. These results suggest that plankton respiration in the field could be assessed more accurately than before by measuring the intracellular ETS substrates (NADH and NADPH), in addition to ETS activity, in plankton.

Potential impact of selected agricultural chemical contaminants on a northern prairie wetland: A microcosm evaluation.

An aquatic, multicomponent microcosm simulating a northern prairie wetland was used to assess the potential effects of six extensively used agricultural pesticides on this important wildlife habitat. Using a nested experimental design, 16 4-liter aquatic microcosms were treated with three concentrations of each of the pesticides carbofuran, fonofos, phorate, atrazine, treflan and trial-late. The microcosm units were incubated for 30 d in an environmental chamber, with a 16-h light:8-h dark cycle, maintained at 20°C. Specific limnological, biological and toxicological parameters were monitored over time by observing the interactions of water, animals, sediment and plants with the pesticides. The laboratory protocol was designed as an initial, rapid, economical screening test to determine the effect, but not the fate, of chemical contaminants in terms of toxicity, impaired productivity and community biochemical functions. Static acute toxicity tests with Daphnia magna and Chironomus riparius suggested that carbofuran, fonofos, phorate and triallate were very toxic to aquatic invertebrates. For D. magna the 48-h EC50 values were 48, 15, 19 and 57 μg/L, respectively. Invertebrate viability tests indicated rapid changes in the toxicological persistence of these pesticides after microcosm interaction. Populations of D. magna were established in the 10 μg/L test concentration of carbofuran, phorate, triallate and fonofos at 1, 1, 14 and 28 d, respectively. Preexposure of the wetland sediments to either triallate or fonofos did not appear to change the relative toxicological persistence of each compound in the water column. Changes in pH, alkalinity, conductivity, dissolved oxygen, total nitrogen and total phosphorus were also observed with different pesticide treatments. Atrazine significantly reduced gross primary productivity and inhibited algal and macrophytic growth. In general, there was no evidence of significant inhibition of microbial functions in the water or hydrosoil of the treated microcosms. The respiratory electron transfer system, phosphatase activity, oxygen consumption and mineralization of dissolved organic carbon were not significantly impacted by any of these pesticides in hydrosoils. However, the impact of atrazine, fonofos and triallate on invertebrates and plants in the microcosm-both key elements in wetland productivity-would suggest that caution be used in application of these pesticides in or near wetland habitats.

Purification of azotophore membranes containing the nitrogenase from [formula omitted

Azotophore membranes containing nitrogenase have been purified in high yield from A. vinelandii by differential and sucrose density gradient sedimentation. The purified preparations appeared as uniform vesicular membranes of 40–75 nm diameter containing the majority of the nitrogenase activity from these cells and were readily separated from the intracytoplasmic membranes containing the cytochromes of the respiratory electron transfer system. The yield and specific activity of azotophores from cells broken by mechanical or osmotic treatment were similar.

Studies on the electron-transfer systems in photosynthetic bacteria. I. The light-induced absorption-spectrum changes and the effect of phenylmercuric acetate.

Absorption-spectrum changes caused by illumination in photosynthetic purple bacteria, Rhodopseudomonas spheroides (wild type and blue-green mutant), Rhodospirillum rubrum (wild type and blue-green mutant) and Chromalium, were studied. Low-temperature spectrotootometry was advantageously used for the determination of heme components. Responses of the isolated chromatophores were compared with those of the intact cells. Heme proteins were found to be functioning in the photosynthetic and respiratory electron-transfer system. The effect of several inhibitors, especially phenylmercuric acetate, was studied. Light-induced responses of different types of cytochromes were analyzed. Absorbancy changes corresponding to carotenoids and bacteriochlorophyll were observed in certain strains and preparations. Carotenoids showed evidence that they had some correlation with other electron-transfer catalysts. A light-induced change in chlorophyll was observed in R. rubrum chromatophores. There was no evidence of appreciable chlorophyll-absorbancy change in the intact cells under weak infrared illumination. A scheme for the electron-transport path in the photo-synthetic and respiratory systems of photosynthetic bacteria was constructed from these data.