Low-temperature Electron Paramagnetic Resonance Research Articles

Oxidation-Reduction Potentials of Bound Iron-Sulfur Proteins of Photosystem I

Digitonin - fractionated photosystem - I subchloroplasts were titrated potentiometrically between -450 and -610 mV at pH 10. Examination of the titrated subchloroplasts by low-temperature (13 degrees K) electron paramagnetic resonance spectroscopy revealed resonances centered at values of 2.05, 1.94, 1.92, 1.89, and 1.86 on the g-factor scale. The peak heights depended on the potentials at which the chloroplasts were poised. The resonances of at least three iron-sulfur centers can be recognized: one with lines at g = 2.05 and 1.94; one with lines at g = 2.05, 1.92, and 1.89; and one for which only a line at g = 1.86 has been resolved. The midpoint potentials of the iron-sulfur species fall into two distinctly separate regions: the titration profile of the g = 1.94 signal, the first segment of the g = 2.05 plot, and the rise phase of the g = 1.86 signal had a value of -530 +/- 5 mV; the upper segment of the g = 2.05 plot, the decrease phase of the g = 1.86 signal, and the g = 1.89 profile had a midpoint potential estimated to be [unk] -580 mV. The oxidation-reduction reaction of each of the bound iron-sulfur species, as represented by the changes of the electron paramagnetic resonance spectra, was reversible and apparently involved a two-electron change.Titration at pH 9 could only be carried to -560 mV, and essentially only the first half of the titration behavior as found at pH 10 was seen. At any given potential more positive than -560 mV, the part of the iron-sulfur protein that was not reduced electrochemically could be reduced photochemically, but only to the maximum extent reduced electrochemically at -560 mV. Whereas, chloroplasts illuminated at room temperature and then frozen while still being illuminated developed a signal similar to that produced by electrochemical reduction at -610 mV, illumination at 77 degrees K did not bring about photoreduction beyond that accomplished electrochemically at about -560 mV.Dithionite alone in the dark and under anaerobic conditions brought about a partial reduction to the extent of the first electrochemical reduction step. Dithionite plus illumination at room temperature or dithionite plus methyl viologen in the dark produced the maximum signal. Electron paramagnetic resonance spectra due to either light or electrochemically reduced iron-sulfur proteins showed no detectable decay for at least 3 days when samples were stored in the dark at 77 degrees K.

Electron spin resonance characterization of Chromatium D hemes, non-heme irons and the components involved in primary photochemistry

The combination of redox potentiometry with low temperature electron spin resonance (ESR) spectroscopy has led to further characterization of electron transfer components of Chromatium D. These include the readily buffer-soluble cytochromes c 553 and c′ and the high-potential iron-sulfur protein in the isolated state and associated with the chromatophore membrane. Buffer-insoluble cytochrome c 553, cytochro—me c 555, bacteriochlorophyll and the primary electron acceptor have been characterized both in the chromatophore membrane and also in a sodium dodecylsulfate detergent-solubilized subchromatophore preparation. Two iron-sulfur proteins have been revealed which are present in the chromatophore membrane but are released on treatment with sodium dodecylsulfate. They have central g values at 1.90 and 1.94 and have estimated midpoint potentials at pH 7.4 ( E m7·4) at +280 mV and −100 mV, respectively, when associated with the chromatophore. In the membrane associated state the apparent E m of cytochrome c′ is approximately 200 mV more positive than the E m values reported for the free state; this implies either that the reduced form of cytochrome c′ binds to the membrane (or to a component therein) to a degree which is > 10 3 times greater than that of the oxidized form or that the E m shift results from membrane solvation. In the case of the high-potential iron-sulfur protein however, its E m when associated with the chromatophore membrane is similar to that reported in the isolated state. The light-induced oxidation of the high-potential iron-sulfur protein at room temperature appears to be linked only to the oxidation of cytochrome c 555; it could serve as an electron pool in equilibrium with cytochrome c 555 in the cyclic electron flow system. The redox component defined in the reduced state by its g y = 1.82 and g x = 1.62 ESR spectrum satisfies the following criteria for its identification as the primary electron acceptor of P883. (a) The E m7·4 value of the g = 1.82 component is −120 ± 25mV. (b) At −70 mV, where the g = 1.82 component is mainly oxidized in the dark, brief illumination at low temperature which causes the irreversible oxidation of one cytochrome c 553 heme, also induces the permanent reduction of the g = 1.82 component; the extent of reduction after brief illumination, given by the g = 1.82 signal height, is the same as that induced chemically at −270 mV showing it to be fully reduced by the receipt of a single electron. (c) At more positive potentials where cytochrome c 553 is oxidized and is not involved in low-temperature reactions, the light-induced low-temperature kinetics of the g = 1.82 signal are reversible; the flash-induced g = 1.82 formation and subsequent dark decay are the same as those for the flash-induced P +883 ( g = 2) formation and dark decay. We suggest that until a full physical-chemical characterization is completed this g = 1.82 component be designated “photoredoxin”.

Low temperature electron paramagnetic resonance studies on two iron-sulfur centers in cardiac succinate dehydrogenase

At temperatures below 20°K, EPR signals from a new iron-sulfur center (designated here as Center S-2 or (Fe-S) S-2) in addition to the classical “g = 1.94 signal” (designated as Center S-1 or (Fe-S) S-1) were detected in purified, soluble succinate dehydrogenase, particulate succinate ubiquinone reductase (Complex II) and particulate succinate cytochrome c reductase from bovine heart. The measured half-reduction potential (E m7.4) of Center S-1 was 0 ± 10 mV, while E m7.4 of Center S-2 was −260 ± 15 mV in the membrane bound preparations. Upon solubilization of succinate dehydrogenase, the EPR behavior of Center S-2 became extremely labile similar to the characteristics of the reconstitutive activity of succinate dehydrogenase toward the rest of the respiratory chain.

Light-induced changes of bound chloroplast plastocyanin as studied by EPR spectroscopy: The role of plastocyanin in noncyclic photosynthetic electron transport

A low-temperature electron paramagnetic resonance (EPR) signal in spinach chloroplasts has been identified as originating from oxidized bound plastocyanin. The EPR parameters of the bound plastocyanin, which are the same as those of soluble plastocyanin, are: g ⊥ = 2.05, g ∥ = 2.23, and A ∥ = 0.006 cm −1. An estimation of the amount of bound plastocyanin based on the in situ EPR signal indicates a concentration of approximately 3 nmoles per mg chlorophyll, a value which agrees with the amount of plastocyanin determined by chemical assay after release from the chloroplast lamellae by sonication. Light-induced changes of plastocyanin in situ have been examined after illumination with monochromatic light at physiological temperatures. The bound plastocyanin undergoes a photoreduction in Photosystem II light which is sensitive to inhibitors of noncyclic electron transport. The presence of the NADP + acceptor system in Photosystem II light causes a shift in the steady-state level of plastocyanin providing direct evidence for the role of plastocyanin in noncyclic electron transport from water to NADP +. Phosphorylation cofactors and the uncoupler, NH 4Cl, also cause shifts in the plastocyanin steady-state level in red light. Photosystem I light is able to photooxidize reduced plastocyanin, while Photosystem II light is only capable of photooxidation in the presence of inhibitors of the photoreduction. These results of studies on plastocyanin photoreactions in situ are discussed in terms of different proposed roles for plastocyanin in the chloroplast noncyclic electron transport chain.

Evaluation of low-temperature electron paramagnetic resonance in analytical chemistry

An evaluation of low-temperature electron paramagnetic resonance has been completed and the possibility of enhanced quantitative lower limits of detection is discussed. By lowering the temperature of the cavity, the Q-factor is considerably increased producing a more efficient system. Also, the relaxation time is optimized by decreasing the temperature of the sample. Different temperatures for each system produced a maximum signal owing to a combination of these two factors. Thus, in order to improve the lower limits of detection for these metal ions, the analytical curves were run at the optimum temperature for each system. These data are compared to the data obtained at room temperature. Also, the instrumental parameter effects have been evaluated. The metal ions investigated are copper(II), manganese (II), titanium(III), vanadium(IV), chromium (III), gadolinium(III), and iron(III).

The radiation chemistry of liquid and glassy methanol

The methods of pulse radiolysis, low temperature optical and electron spin resonance spectroscopy and chemical analysis of stable products have been applied to an investigation of theγ-radiolysis of liquid and glassy methanol. Processes induced by generation of presumed intermediates by photoionization of solutes have been studied using the same techniques. Electrons are trapped in glassy methanol afterγ-irradiation at 77 K with a yieldG(e¯t)= 2.7 ± 0.3 and on warming are rendered mobile, ultimately forming an amount of hydrogen equal to1/2G(e¯t). In contrast, illumination of the irradiated sample at 77 K with visible light decomposes the trapped electron, forming hydrogen and CH2OH or CH2O-in yield equal toG(e¯t). The solvated electron in liquid methanol also reacts with the solvent, at a rate faster than the corresponding reaction in water. The addition of sodium methoxide to liquid methanol results in a marked increase in both the yield and lifetime of the solvated electron observed after 0.2μs irradiation pulse. The optical absorptions and decay parameters of CH2OH and CH2O-in liquid methanol were obtained; the ultraviolet absorption observed in irradiated methanol glass after removal of the trapped electrons by photo-bleaching is probably due to these two species. Solutions of methanol glass containing silver perchlorate exhibit complex optical and e. s. r. spectra, which provide evidence for several different species, but a complete analysis has not been possible. The radicals expected from the initial ionization event are considered. The results of this work together with published data are used to predict the reaction of the free radicals CH3O. , CH2OH, CH2O-, H. ande¯sor e¯tin the systems of interest and the relevance of these reactions to some aspects of the radiation chemistry of methanol is discussed.

The number of iron atoms in the paramagnetic center (G =1.94) of reduced putidaredoxin, a nonheme iron protein.

A number of oxidation-reduction proteins, containing iron and an acid-labile form of sulfide, have been shown to exhibit low-temperature electron paramagnetic resonance (EPR) signals near g = 1.94, on reduction.l-4 These proteins include oxidases of the xanthine type, ferredoxins, and nonheme-iron and iron flavoproteins from mitochondria and bacteria. By substitution with iron5 and sulfur6' 7 isotopes having nonzero nuclear spin, both elements were conclusively shown to participate in the paramagnetic center. The nuclear magnetic moments may couple to the moment of the unpaired electron of the center, producing small positive and negative incremental local fields which act to split or broaden the EPR spectrum compared to the unsubstituted cases. The effect depends, in the simplest case of isotropic hyperfine interaction, on three variables, viz., the enrichment in isotope of nonzero nuclear spin (Fe57 of S33 in the present case), the effective local field contribution or hyperfine splitting constant of each atom, and the number of atoms of each isotope involved with a single unpaired electron. If the enrichment can be estimated, trial spectra can be calculated for various hyperfine splittings and numbers of atoms. In favorable cases comparison to the observed spectrum will reveal the values of the variables that best fit hypothesis to experiment. In this way one can determine the number and kinds of atoms interacting with the unpaired electron under the assumption of predominantly isotropic hy

Electromagnetic Properties of Hemoproteins: II. THE EFFECT OF PHYSICAL STATES ON ELECTRON PARAMAGNETIC RESONANCE PARAMETERS OF HEMOPROTEINS

Abstract Low temperature electron paramagnetic resonance absorption spectra of randomly oriented preparations of cytochrome c peroxidase, ferrimyoglobin, ferrimyoglobin-azide, and ferrimyoglobin-fluoride are measured in various physical states. In general, the line width of resonance absorptions increases in relation to the physical state of preparation in the following order: concentrated solutions l wet polycrystalline suspensions l lyophilized preparations l dried polycrystalline preparations. The broadening of resonance absorptions caused by dehydration can be reversed by rehydration and dissolution of the dried preparation. When cytochrome c peroxidase and ferrimyoglobin-azide are dehydrated, their spin types are reversibly fixed in the state at dehydration and become temperature-independent until these compounds are rehydrated. The intensity of the high spin resonance absorption of ferrimyoglobin decreases by a factor of 10 to 20 on dehydration. The initial intensity can be restored by rehydration and dissolution of the dehydrated ferrimyoglobin. Resonance spectra of dehydrated ferrimyoglobin exhibit hitherto unreported absorptions in the high field region. These absorptions may represent a compound or compounds which may be responsible for the low spin characteristics of lyophilized ferrimyoglobin detected by Mossbauer absorption spectroscopy.

Environmental Factors Influencing the Hyperfine Structure of Manganous Low-Temperature Electron Paramagnetic Resonance Spectra

Hyperfine structure is observed in low temperature (T = -180 degrees C) EPR (electron paramagnetic resonance) spectra of a number of solutions containing Mn(++) ions 13, 15) which have characteristics in common with low temperature EPR spectra from biological substances such as mitochondria and microsomes (1-4). This investigation is an attempt to understand the features of these signals in terms of the molecular environment of the manganous ion, and a qualitative explanation for the observations reported here is advanced in terms of the amount of axial distortion of a manganese hydrate in different environments.

Diffuse reflectance spectroscopy of frozen samples as an adjunct to low-temperature electron paramagnetic resonance spectroscopy

A technique is described for applying diffuse reflectance spectrophotometry to samples frozen in round quartz tubes of small diameter as used in measurements of electron paramagnetic resonance. Limitations of the quantitative evaluation of the reflectance spectra are discussed.

Linewidth Studies in Electron Spin Resonance Spectra : The Para and Ortho Dinitrobenzene Anions

Large variations have been found among the linewidths of the different hyperfine lines in the low-temperature electron spin resonance spectra of the p- and o-dinitrobenzene anions generated electrolytically in N,N-dimethylformamide solutions. The magnitude of the variations in the para compound is so great that the spectrum is superficially uninterpretable. Detailed analysis of the spectrum of this radical shows, however, that most of the linewidth differences can be accounted for by modulation through molecular tumbling of the intramolecular anisotropic dipolar and g-tensor interactions. There may also be a small contribution from modulation of the isotropic proton hyperfine splittings, the mechanism that accounts for the alteranting linewidth phenomenon in a number of radicals, but an alternation of the widths is not observed in the p-dinitrobenzene anion spectrum because the contribution from this interaction is small. The sign of the isotropic nitrogen hyperfine splitting aN has been determined by a new method involving only pure dipolar interactions with the nitrogen nuclei and protons and not depending on any assumptions about the magnitudes of the components of the g tensor. A number of features of the relaxation matrix determining the linewidths are discussed, and the problems encountered when the components of a degenerate line have different widths are analyzed. The spectra obtained in dimethylformamide solutions are much better resolved than those previously reported in acetonitrile solutions. Changes of hyperfine splittings with both solvent and temperature are observed.

Low-Temperature Magnetic Properties of Dilute Mg-Mn, Al-Mn, Mg-Fe, and Al-Fe Alloys

Magnetic susceptibility measurements are reported on some dilute alloys of Mg-Mn and Al-Mn in the temperature range, room temperature -1.3\ifmmode^\circ\else\textdegree\fi{}K. Electron spin resonance (ESR) absorption has been observed in the magnesium alloy containing 0.6 at.% manganese and studied as a function of temperature. Room temperature magnetic susceptibility measurements and low temperature ESR experiments are reported for Mg-Fe and Al-Fe alloys. The concentration dependence of the Curie constant at high temperature suggests the presence of four unpaired electrons for the manganese atom dissolved in magnesium. Similar information concerning manganese dissolved in aluminum suggests one unpaired electron if a completely localized $d$ state is formed.The susceptibility of dilute Mg-Mn alloys shows departures from a Curie law, which could be interpreted as an increase in the spin state of the manganese ion in the region of the resistance minimum. This excess paramagnetism is discussed in terms of the possible mechanisms which could produce a resistance minimum and leads to the conclusion that if there is a ferromagnetic coupling between randomly distributed manganese pairs, the interaction mechanism operates over approximately 10 lattice spacings.The effective magneton number of Mn in the more dilute Mg-Mn alloys also increases with temperature at high temperatures. The susceptibility of the concentrated Mg-Mn alloy obeys a Curie law except in the region of the resistance maximum where both susceptibility and ESR data indicate an antiferromagnetic transition.The influence of the low-temperature transition, occurring in the magnesium alloy containing about 0.6 at.% manganese, on the electron spin resonance linewidth and magnetic field value has been measured as a function of temperature giving $g$ values of 2.01\ifmmode\pm\else\textpm\fi{}0.01 above 6\ifmmode^\circ\else\textdegree\fi{}K. The shift of the position of resonance field below 6\ifmmode^\circ\else\textdegree\fi{}K is consistent with antiferromagnetic behavior in Mg-Mn. The line shift is proportional to ${T}^{\ensuremath{-}1}$ in the antiferromagnetic region which would be consistent with a conduction electron superexchange mechanism for the antiferromagnetic manganese ion interaction. The linewidth is broadened in the region of the resistance minimum and attains a constant value as the temperature is lowered. This behavior is consistent with localized spin interactions, giving rise to larger averaged spin values in the resistance minimum region as suggested from the magnetic susceptibility studies of the more dilute alloys.

Air Lock for Changing Samples in Low Temperature Electron Spin Resonance Experiments

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