Boris Epel

The mechanism of the oxidation of ascorbate and Mn2+ by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn2+ by isolated chloroplasts was reinvestigated. 2. 2. Our results suggest that ascorbate or Mn2+ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn2+ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin. 3. 3. In the absence of ascorbate or Mn2+ the superoxide formed dismutases either spontaneously or enzymatically producing O2 and H2O2. In the presence of ascorbate or Mn2+, however, the superoxide is reduced to H2O2 with no formation of O2. Consequently, in the absence of reducing compounds, in the reaction H2O to low potential acceptor one O2 (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn2+ or other suitable reductants up to three molecules O2 can be taken up per four electrons transported. 4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O2; the addition of ascorbate or Mn2+ to this system results in a two fold stimulation of O2 uptake, with no stimulation of NADPH oxidation. The stimulation of O2 uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O2 uptake in the system H2O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase. 5. 5. In Class II chloroplasts in the system H2O → NADP upon the addition of ascorbate or Mn2+ an apparent inhibition of O2 evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts. 6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP2e in this system (when the rate of electron flow is based on the rate of O2 uptake) should be revised in the upward direction.

HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings.

Inhibition of respiration and destruction of cytochrome a3 by light in mitochondria and cytochrome oxidase from beef heart

Abstract Irradiation of beef-heart mitochondria and of cytochrome oxidase purified from beef-heart mitochondria with blue light inhibited electron transport from substrate (succinate for the mitochondria and reduced cytochrome c for the cytochrome oxidase) to O2. The irradiation treatment also destroyed cytochrome a3 as assayed by the absorption band for the reduced cyanide-cytochrome a3 complex at 587 nm in the low-temperature absorption spectrum. Irradiation under anaerobic conditions was not inhibitory. Cytochrome a3 was protected against photodestruction if cyanide was present during the irradiation.

Absolute Oxygen R1e Imaging In Vivo with Pulse Electron Paramagnetic Resonance

Tissue oxygen (O2) levels are among the most important and most quantifiable stimuli to which cells and tissues respond through inducible signaling pathways. Tumor O2 levels are major determinants of the response to cancer therapy. Developing more accurate measurements and images of tissue O2 partial pressure (pO2), assumes enormous practical, biological, and medical importance.

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a3 components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a3 was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.

Comparison of 250 MHz electron spin echo and continuous wave oxygen EPR imaging methods for in vivo applications

PURPOSE The authors compare two electron paramagnetic resonance imaging modalities at 250 MHz to determine advantages and disadvantages of those modalities for in vivo oxygen imaging. METHODS Electron spin echo (ESE) and continuous wave (CW) methodologies were used to obtain three-dimensional images of a narrow linewidth, water soluble, nontoxic oxygen-sensitive trityl molecule OX063 in vitro and in vivo. The authors also examined sequential images obtained from the same animal injected intravenously with trityl spin probe to determine temporal stability of methodologies. RESULTS A study of phantoms with different oxygen concentrations revealed a threefold advantage of the ESE methodology in terms of reduced imaging time and more precise oxygen resolution for samples with less than 70 torr oxygen partial pressure. Above 100 torr, CW performed better. The images produced by both methodologies showed pO2 distributions with similar mean values. However, ESE images demonstrated superior performance in low pO2 regions while missing voxels in high pO2 regions. CONCLUSIONS ESE and CW have different areas of applicability. ESE is superior for hypoxia studies in tumors.

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I.

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F(X), F(A) and F(B), the last two are bound by the PsaC subunit. We have modelled the F(A) and F(B) binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F(A) and F(B). The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F(A)-maquette is -0.44+/-0.03 V and in the F(B)-maquette is -0.47+/-0.03 V. Both are close to that of F(A) and F(B) in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F(A) and F(B) were removed, and possibly participate in the light-induced electron transfer reaction in PS I.

A Dynamic Nuclear Polarization spectrometer at 95 GHz/144 MHz with EPR and NMR excitation and detection capabilities

A spectrometer specifically designed for systematic studies of the spin dynamics underlying Dynamic Nuclear Polarization (DNP) in solids at low temperatures is described. The spectrometer functions as a fully operational NMR spectrometer (144 MHz) and pulse EPR spectrometer (95 GHz) with a microwave (MW) power of up to 300 mW at the sample position, generating a MW B(1) field as high as 800 KHz. The combined NMR/EPR probe comprises of an open-structure horn-reflector configuration that functions as a low Q EPR cavity and an RF coil that can accommodate a 30-50 μl sample tube. The performance of the spectrometer is demonstrated through some basic pulsed EPR experiments, such as echo-detected EPR, saturation recovery and nutation measurements, that enable quantification of the actual intensity of MW irradiation at the position of the sample. In addition, DNP enhanced NMR signals of samples containing TEMPO and trityl are followed as a function of the MW frequency. Buildup curves of the nuclear polarization are recorded as a function of the microwave irradiation time period at different temperatures and for different MW powers.

Multifrequency EPR analysis of the dimanganese cluster of the putative sulfate thiohydrolase SoxB of Paracoccus pantotrophus

A detailed analysis of the EPR signatures at X-band and Q-band of an enzyme (SoxB) involved in sulfur oxidation from Paracoccus pantotrophus is presented. EPR spectra are attributed to an exchange-coupled dimanganese Mn2(II,II) complex. An antiferromagnetic exchange interaction of J=−7.0 (±1) cm−1 (H=−2JS1S2) is evidenced by a careful examination of the temperature dependence of the EPR spectra. The spin Hamiltonian parameters for a total spin of ST =1, 2 and 3 are obtained and an inter-manganese distance of 3.4 (±0.1) Å is estimated. The comparison with exchange coupling and inter-manganese distance data of other dimanganese proteins and model compounds leads to a tentative assignment of the Mn bridging ligands to bis(μ-hydroxo) (μ-carboxylato).

The effects of lipase on spinach and Chlamydomonas chloroplasts

Abstract Light-induced fluorescence yield changes and low-temperature changes were measured in spinach and Chlamydomonas reinhardi chloroplasts before and after treatment with pancreatic lipase. Lipase treatment destroyed the C-550 absorption band and eliminated the fluorescence of variable yield. In spinach chloroplasts the invariant fluorescence yield, after lipase treatment, was low whereas in Chlamydomonas chloroplasts it was high. Lipase treatment modified the cytochrome b559 in spinach chloroplasts so that it was no longer ascorbate reducible but was dithionite reducible. In Chlamydomonas chloroplasts some of the cytochrome b559 was destroyed by lipase treatment. After lipase treatment, cytochrome b559 could be reduced by Photosystem I activity if ascorbate was present as an electron donor and benzyl viologen present as an electron acceptor for Photosystem I. Lipase also eliminated the EPR Signal II.