Cw Electron Paramagnetic Resonance Research Articles

The manganese center of oxygen-evolving and calcium-depleted photosystem II: a pulsed EPR spectroscopy study

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

A new ultra low-field ESR spectrometer

A CW electron spin resonance (ESR) spectrometer working at earth-field is described for the first time. The optimized sensitivity has allowed to reduce the size of the sample holder down to 100 mm3, a low value for such a low working frequency (1.845 MHz). At this frequency, only narrow ESR signals are significant (maximum sweep 125 μT or 100 A/m). The use of the Bloch configuration and the particular care to optimize the sensitivity lead to a design that partially compensates the lack of sensitivity due to low fields. Small material quantities are thus usable. Moreover, the spectrometer is low cost, portable, and easily reproducible with the announced performance. The main use of the spectrometer is to characterize materials for earth field magnetometry. For that purpose it has been necessary to define a figure of merit for magnetometry. Until this study, the only parameter discussed was the linewidth of the homogeneous ESR line (i.e., T2). The detailed discussion of the figure of merit, taking into account not only the material but the couple spectrometer material, considerably decreases the predominance of the linewidth and let emerge secondary factors such as packaging, susceptibility, ESR lineshape, T1 vs T2, etc. The figure of merit evaluated from the material parameters has been experimentally measured for different π radicals. Comparison with their performance in earth field magnetometry demonstrates the pertinence of the proposed figure of merit.

Electron spin resonance and electron-spin-echo study of oriented multilayers of L alpha-dipalmitoylphosphatidylcholine water systems

A detailed electron spin resonance (ESR) study of spin-labeled-oriented multilayers of L alpha-dipalmitoylphosphatidylcholine (DPPC) water systems for low water content (2-10% by weight) is reported with the purpose of characterizing the dynamical and structural properties of model membrane systems. Emphasis is placed on the value of combining such experiments with detailed simulations based on current slow-motional theories. Information is obtained regarding ordering and anisotropic rotational diffusion rates via ESR lineshape analysis over the entire motional range, from the fast motional region through the moderately slow and slow to the rigid limit. This includes the low-temperature gel phase, the liquid crystalline L alpha (1) phase and what appears to be a third high-temperature phase above the L alpha phase. Cholestane (CSL) and spin-labeled DPPC (5-PC, 8-PC, and 16-PC) have been used to probe different depths of the bilayer. While CSL and 5-PC both reflect the high ordering of the bilayer close to the lipid-water interface, CSL appears to be located close enough to the water for the nitroxide to be involved in hydrogen bonding with water molecules. 16-PC reflects the relatively low ordering near the tail of the hydrocarbon chain in the bilayer. Quantitative estimates of ordering and motion are obtained for these cases. The results from CSL indicate that close to the lipid-water interface the DPPC molecule is oriented approximately perpendicular to the bilayer in these low water-content systems. However, all three labeled lipid probes indicate that the hydrocarbon chain of DPPC may be bent away from the bilayer normal by as much as 30 degrees and this evidence is stronger at low temperatures. When cholesterol is added to the DPPC-water system at a concentration greater than or equal to 2.5 mol %, the ordering is greatly increased although the rotational diffusion rate remains almost unaffected in the gel phase. Electron spin echoes (ESE) are observed for the first time from oriented lipid-water multilayers. Results obtained from cw ESR lineshape analysis are correlated with data from ESE experiments, which give a more direct measurement of relaxation times. These results indicate that for detection of very slow motions (close to the rigid limit) ESE experiments are more sensitive to dynamics than continuous wave ESR for which inhomogeneous broadening becomes a major problem.