High Electron Flux Research Articles

Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells

Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative–nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells ( P ≤ 0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the αH195Q, αH195N, and αQ191K variant MoFe proteins (where His at position 195 in the α subunit is replaced by Gln/Asn or Gln at position α-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1s of initiating turnover under N2 with the αH195Q and αH195N variants, but not with the αQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, αH195Q or αH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the αQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze 57Fe Mössbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, αH195Q and αH195N MoFe proteins can bind N2, but αQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N2 reduction.

SCANNING ELECTRON MICROSCOPE IMAGES OF UNCOATED MICROFOSSILS: APPLICATIONS, PERSPECTIVES AND LIMITATIONS

We present a recently developed method using a field emission scanning electron microscope (FEG) to view and photograph microfossil specimens that are not coated by conductive material. The FEG microscope provides high electron flux and offers the option to capture images at low beam voltage. Balancing incident energy with absorbed energy from the detector leads to charge-free images of non-conductive material. As an example of the application, we show images of planktonic foraminifers and ostracods obtained with this method and compare them with those obtained on the same specimens after gold coating. The method is particularly useful for illustrating holotypes, neotypes and topotypes of microfossils when an environmental scanning electron microscope is not available.

Distribution and variability of accelerated electrons at Mars

We investigate accelerated electrons observed by Mars Global Surveyor (MGS), using data from the Electron Reflectometer (ER) instrument. We find three different types of accelerated electron events. Current sheet events occur over regions with weak or no crustal fields, have the highest electron energy fluxes, and are likely located on draped magnetotail fields. Extended events occur over regions with moderate crustal magnetic fields, and are most often observed on closed magnetic field lines. Localized events have the lowest energy fluxes, occur in strong magnetic cusp regions, and are the most likely kind of event to be found on open magnetic field lines. Some localized events have clear signatures of field-aligned currents; these events have much higher electron fluxes, and are preferentially observed on radially oriented open magnetic field lines. Electron acceleration events, especially localized events, are similar in many ways to events observed in the terrestrial auroral zone. However, physical processes related to those found in the terrestrial cusp and/or plasmasheet could also be responsible for accelerating electrons at Mars.

Origin of geosynchronous relativistic electron events

We have investigated the characteristics of solar wind and magnetospheric conditions associated with the occurrence of geosynchronous relativistic electron events. Most of the geosynchronous relativistic events for April 1999 to December 2002 are found to occur during prolonged (a number of days) quiet intervals following the appearance of high‐speed solar wind streams. In a typical relativistic event, the electron fluxes begin to increase by orders of magnitude when the solar wind density drops after reaching a sharp peak at the leading edge of a high‐speed stream. The increased fluxes stay at a high level until the quiet solar wind conditions cease. In addition, enhanced ULF wave activity and substorm injections of 10s to 100s keV electrons are observed at the time of the large flux increases in the events. We found that geosynchronous relativistic events can be observed only when both the solar wind and magnetospheric wave/substorm injection conditions are favorable regardless of whether or not a magnetic storm takes place. These observations suggest the following scenario for the occurrence of a geosynchronous relativistic electron event: (1) Quiet solar wind conditions (i.e., no strong solar wind pressure and large southward turnings of IMF Bz) can lead to stable and more dipole‐like magnetospheric configurations in which the geosynchronous orbit is located well inside the trapping boundary of the energetic electrons. (2) If a large population of MeV electrons is generated (by some acceleration process(es) involving enhanced ULF wave and substorm injections) in the inner magnetosphere, it can be trapped and effectively accumulated to a high intensity. (3) The high electron flux can persist for a number of days in the geosynchronous region as long as the solar wind conditions remain quiescent. The occurrence of a geosynchronous relativistic electron event requires not only the proper acceleration process and sufficient seed electrons but also no significant loss process that dominates over any acceleration/source.

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P 700 (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810–860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P 700. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P 700. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.

CHARACTERISTIC SOLAR WIND DYNAMICS ASSOCIATED WITH GEOSYNCHRONOUS RELATIVISTIC ELECTRON EVENTS

We have investigated characteristic solar wind dynamics associated with relativistic electron events at geosynchronous orbit. Most of the events for April, 1999 through December, 2002 are found to be accompanied by a prolonged solar quiet period which is characterized as low solar wind density, weak interplanetary magnetic field (IMF), and fast alfvenic fluctuations in IMF <TEX>$B_z$</TEX>. In a typical relativistic event, electron fluxes begin to increase by orders of magnitude when solar wind parameters drop to low values (e.g., <TEX>$n_{sw}∼5 cm^{-3}$</TEX> and ｜<TEX>$B_{IMF}$</TEX>∼5 nT) after sharp peaks. Then the elevated electron fluxes stay at the high level during the solar quiet period. This observation may suggest the following scenario for the occurrence of a geosynchronous relativistic event: (ⅰ) Quiet solar winds can yield a stable and more dipole-like magnetospheric configurations in which the geosynchronous orbit locates well inside the trapping boundary of the energetic electrons. (ⅱ) If a large population of MeV electrons are generated (by whatever acceleration process(es)) in the inner magnetosphere, they can be trapped and effectively accumulated to a high intensity. (ⅲ) The high electron flux can persist for a number of days in the geosynchronous region as long as the solar wind dynamics stays quiet. Therefore the scenario indicates that the occurrence of a relativistic event would be a result of a delicate balance between the effects of electron acceleration and loss. In addition, the sensitive dependence of a relativistic event on the solar wind conditions makes the prediction of solar wind variability as important as understanding of electron acceleration processes in the forecast of a relativistic event.

Aragats space-environmental centre: status and SEP forecasting possibilities

The Aragats Space Environment Center in Armenia provides real-time monitoring of cosmic particle fluxes. Neutron monitors operating at altitudes of 2000 m and 3200 m on Mt Aragats continuously gather data to detect possible abrupt enhancement of the count rates. Additional high precision detectors, measuring muon and electron fluxes, along with directional information have been put in operation on Mt Aragats in the summer of 2002. We plan to use this information to establish an early warning system against extreme solar energetic particle (SEP) events which pose danger to the satellite electronics and the space station crew. Solar ion and proton fluxes as measured by space-borne sensors on ACE and GOES satellites are used to derive expected arrival times of highest energy ions at 1 AU. The peaks in the time series detected by Aragats neutron monitors, coincided with these times, demonstrate the possibility of early detection of SEP events using the ground-based detectors.

Hydrogen production in anaerobic reactors during shock loads—influence of formate production and H 2 kinetics

In this article the role of hydrogen as a process monitoring tool in methanogenic systems was studied by considering the influence of several key system parameters. Hydrogen production was found to be influenced mainly by the inocula's source pH, and varied only slightly with external pH and HCO 3 − levels. When an inoculum adapted to above neutral conditions (pH>7) was shocked, reducing equivalents were selectively channelled through formate, while high hydrogen production was noticed with acidically (pH<6.5) adapted inocula. The results also revealed that the production of hydrogen or formate during shock loads was not strongly associated with microbial morphology (granules or flocs) as high electron fluxes were possible through either during acidogenesis. Shock load experiments in continuous reactors revealed that neither hydrogen nor formate accumulated to any significant degree, nevertheless digester recovery took a long time due to the slow kinetics of volatile fatty acid degradation. Selective formate production under neutral pH environments, coupled with high hydrogenotrophic activity, was found to be responsible for the dampened hydrogen response during the early phases of gradually shocked systems (step change). Based on these results it appears that the role of hydrogen as a process monitoring tool has been overemphasised in the literature.

Internal acceleration of relativistic electrons by large‐amplitude ULF pulsations

The flux of relativistic electrons in geostationary orbit exhibits a variability closely regulated by the solar wind, but the acceleration mechanism of relativistic electrons remains poorly understood. Recent observational evidence has shown that the intensification of relativistic electrons often takes place in a matter of several hours. The rapidity is difficult to reconcile with traditional diffusion‐based models which often take days to produce appropriate high‐energy electron fluxes and motivates us to search for alternative mechanisms of internal acceleration. Elaborating on the observation in an earlier paper of Rostoker et al. [1998], we propose that global oscillation of magnetosphere in the Pc4–5 range is capable of accelerating electrons under the catalysis of random pitch angle scattering. This view is developed theoretically and computationally in this paper. The most noteworthy result of the investigation is the demonstration that magnetic pumping by ULF waves can lead to the observed high relativistic electron flux in a time as short as a few hours under parameters appropriate for major magnetic storms. Further development and test of this theory are discussed.

An overview of the early November 1993 geomagnetic storm

This paper describes the development of a major space storm during November 2–11, 1993. We discuss the history of the contributing high‐speed stream, the powerful combination of solar wind transients and a corotating interaction region which initiated the storm, the high‐speed flow which prolonged the storm and the near‐Earth manifestations of the storm. The 8‐day storm period was unusually long; the result of a high‐speed stream (maximum speed 800 km/s) emanating from a distended coronal hole. Storm onset was accompanied by a compression of the entire dayside magnetopause to within geosynchronous Earth orbit (GEO). For nearly 12 hours the near‐Earth environment was in a state of tumult. A super‐dense plasma sheet was observed at GEO, and severe spacecraft charging was reported. The effects of electrons precipitating into the atmosphere penetrated into the stratosphere. Subauroral electron content varied by 100% and F layer heights oscillated by 200 km. Equatorial plasma irregularities extended in plumes to heights of 1400 km. Later, energetic particle fluxes at GEO recovered and rose by more than an order of magnitude. A satellite anomaly was reported during the interval of high energetic electron flux. Model results indicate an upper atmospheric temperature increase of 200°K within 24 hours of storm onset. Joule heating for the first 24 hours of the storm was more than 3 times that for typical active geomagnetic conditions. We estimate that total global ionospheric heating for the full storm interval was ∼190 PJ, with 30% ofthat generated within 24 hours of storm onset.

Investigation of CO binding and release from Mo-nitrogenase during catalytic turnover.

During enzymatic turnover in the presence of CO, Mo-nitrogenase has been shown to generate two different EPR signals termed lo-CO (PCO = 0.08 atm) and hi-CO (PCO = 0.5 atm). When the formation of hi-CO is monitored under the conditions of very low electron flux, a 2 min lag is observed prior to the initial detection of the signal followed by a near-linear rate of formation during which the S = 3/2 cofactor signal exhibits similar decay kinetics. Increasing the electron flux produces a significant increase in the rate of both the formation of hi-CO and the decay of the S = 3/2 cofactor. These results are interpreted in terms of a state of the enzyme (redox or structural) generated only during turnover which is needed to initially bind CO to the cofactor. Under high electron flux conditions, new EPR inflections are observed at g = 5.78, 5.15 and g = 1.95, 1.81 and tentatively assigned to S = 3/2 and 1/2 states of the CO-bound cofactor and 1 equiv of oxidized P cluster, respectively. Sudden removal of CO from the environment results in the slow decay (>10 min) of both the hi-CO signal and CO inhibition of acetylene reduction activity. The use of ethylene glycol to quench enzymatic activity strongly inhibits the decay of hi-CO (in the presence of CO) and the subsequent decay of lo-CO (after removal of CO) but does not prevent the reversible interconversion hi-CO left and right arrow lo-CO + CO.

Low-energy broad beam electron gun

A low-energy electron gun for the energy range of 5–100 eV providing a uniform and high electron flux over a circular surface area of several cm2 has been designed and tested. Focusing elements allow one to vary the beam diameter according to experimental needs. The uniform current densities make it suitable for the study of electron stimulated surface chemistry and thin-film production.

Analysis of Cellular Organelles and Macromolecular Assemblies by Electron Energy Loss Spectroscopy

Recent advances in electron energy loss spectroscopy (EELS) have significantly extended the range of applications for biological microanalysis. For example, EELS can now detect physiological concentrations of the important element calcium in rapidly frozen cells with a sensitivity greater than that achievable by energy-dispersive x-ray spectroscopy (EDXS). It can also detect small numbers of phosphorus atoms bound to macromolecular assemblies, and measure water distributions in frozen hydrated tissue. Here we discuss some of these developments in the context of detection limits and mapping techniques in the scanning transmission electron microscope (STEM) and energy-filtering transmission electron microscope (EFTEM).The useful information about elemental composition in EELS of biological specimens generally resides in weak core-edge signals corresponding to atomic concentrations in the 10−5−10−3(1–100 mmol/kg dry weight) range. For example, the Ca L2,3signal/background ratio is typically only 10−3and it is necessary to measure differences in signal that are only 104 of the background. Changes in low-loss fine structure corresponding to varying chemical composition are also very subtle; for example, detection of a 3% change in water content requires reliable measurement of a 0.1 eV shift in the low-loss intensity maximum. To extract such information requires efficient parallel detection of the energy loss spectrum and a high-brightness source to provide a sufficient number of incident electrons. The dedicated STEM is particularly well-suited for analyzing low concentrations of biological elements. If desired, the probe current can be reduced into the picoampere range for low-dose, high-resolution imaging prior to elemental analysis. The STEM’S field-emission source can then be used to deliver a current approaching 10 nA into a ~10 nm diameter probe. High electron flux conditions are ideal for spectrum-imaging applications where adequate counting statistics must be achieved within a limited pixel dwell time. The cold field-emission source of the STEM has the additional advantage of providing electrons with a narrow energy spread of &lt;0.5 eV which is important in fine structure studies.

Medium energy particle perspective from magnetopause to upstream region: Prognoz-10 data

We present typical examplesof Prognoz-10 ion (E>15 keV) and electron (E> keV) flux observations as the spacecraft crosses the low and mid-latitude magnetosheath and the foreshock. Both leakage from magnetosphere and bow shock acceleration contribute to magnetosheath populations. A statistical survey using 6 months of measurements reveals that the occurence of high ion and electron fluxes depends upon K p. For the same K p, the probability of high ion occurence is much higher in the magnetosheath than in the upstream region. In the upstream region, the occurence of high ion fluxes depends upon the distance to the bow shock and on the angle Θ Bn.

Combined electron and ion projection microscopy

We have designed a bright, atomic-size gas ion source for imaging partly transparent samples in a geometric projection mode. Combined with electron holography, this ion projection microscope supplements the holograms by generating a shadow image with ions. Helium ions are used to sputter carbon fibers down to only a few nanometers in diameter, which were subsequently imaged with electrons. Hydrogen ions are used to deposit charges on carbon fiber networks, which can then be imaged and neutralized with a high electron flux. Finally, we present preliminary data of magnetic structures, taking advantage of the different deflections due to the Lorentz force of ions and electrons, and obtain complementary image information on small magnetic particles.

A model for Co-operative electron transfer

Co-operative electron transfer through an electron transfer chain or an electron exchanging molecular complex, which has recently been introduced as a new mechanism of (photo)electrocatalysis, but could not unequivocally be verified by applying the slaving principle, is demonstrated for a slightly extended model. It involves a non-linear kinetic step prior to the feedback on electron transfer rate constants. An electron transfer state is found which is characterized by a high degree of co-operation of individual processes and a drastic reduction of activation energies resulting in a high electron flux. Such a co-operative electron transfer mechanism may be considered to be a simple model describing biological multicentre catalysts (nitrogen fixation, sulphate reduction, oxygen evolution). Introduction of a cubic non-linearity into the model results in a co-operative bistability which may act as a switch between a low conducting and a high conducting electron transfer state.

Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein.

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the 'Fe-protein cycle' are independent of the level of reduction of the MoFe-protein.

Fragmentation analysis of bradykinin by 252Cf-plasma desorption mass spectrometry

The 252Cf-plasma desorption (PD) mass spectrum of the nonapeptide bradykinin was studied in detail with particular emphasis on the fragmentation pattern resulting from fast metastable decay of the molecular ion. N-acetyl and O-methyl derivatives of bradykinin were used as mass shift species for assignment of N-terminal and C-terminal fragment ions. Thirty-seven sequence-specific fragment ions were identified, including those that are due to cleavage of peptide backbone as well as side chains bond (i.e., d n, v n, w n fragment ions). The degree of fragmentation correlates with what has been observed by fast atom bombardment tandem mass spectrometry using high energy collisional activation. The high degree of excitation of bradykinin molecules in 252Cf-PDMS is undoubtedly due to the special nature of excitation intrinsic to the 252Cf-PD process where electronic excitation occurs with a very high probability due to the high electron and photon flux surrounding the nuclear fission fragment track. These results offer further evidence that 252Cf-PDMS, in addition to providing molecular weight information, can be used to sequence peptides without the need for a second stage of molecular excitation.

Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide

The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.