Using rapid-scan EPR to improve the detection limit of quantitative EPR by more than one order of magnitude.

X-band rapid-scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid-scan and continuous-wave EPR spectra were recorded for amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid-scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid-scan EPR results in signal-to-noise improvements by factors between 10 and 50. Rapid-scan EPR is thus capable of improving the detection limit of quantitative EPR by at least one order of magnitude. In addition, we provide a recipe for setting up and calibrating a conventional pulsed and continuous-wave EPR spectrometer for rapid-scan EPR.

CW and pulsed electrically detected magnetic resonance spectroscopy at 263GHz/12T on operating amorphous silicon solar cells.

Here we describe a new high frequency/high field continuous wave and pulsed electrically detected magnetic resonance (CW EDMR and pEDMR) setup, operating at 263GHz and resonance fields between 0 and 12T. Spin dependent transport in illuminated hydrogenated amorphous silicon p-i-n solar cells at 5K and 90K was studied by in operando 263GHz CW and pEDMR alongside complementary X-band CW EDMR. Benefiting from the superior resolution at 263GHz, we were able to better resolve EDMR signals originating from spin dependent hopping and recombination processes. 5K EDMR spectra were found to be dominated by conduction and valence band tail states involved in spin dependent hopping, with additional contributions from triplet exciton states. 90K EDMR spectra could be assigned to spin pair recombination involving conduction band tail states and dangling bonds as the dominating spin dependent transport process, with additional contributions from valence band tail and triplet exciton states.

Metastable defect formation at microvoids identified as a source of light-induced degradation in a-Si:H.

Light-induced degradation of hydrogenated amorphous silicon (a-Si:H), known as the Staebler-Wronski effect, has been studied by time-domain pulsed electron-paramagnetic resonance. Electron-spin echo relaxation measurements in the annealed and light-soaked state revealed two types of defects (termed type I and II), which can be discerned by their electron-spin echo relaxation. Type I exhibits a monoexponential decay related to indirect flip-flop processes between dipolar coupled electron spins in defect clusters, while the phase relaxation of type II is dominated by 1H nuclear spin dynamics and is indicative for isolated spins. We propose that defects are either located at internal surfaces of microvoids (type I) or are isolated and uniformly distributed in the bulk (type II). The concentration of both defect type I and II is significantly higher in the light-soaked state compared to the annealed state. Our results indicate that in addition to isolated defects, defects on internal surfaces of microvoids play a role in light-induced degradation of device-quality a-Si:H.

Atomic structure of interface states in silicon heterojunction solar cells.

Combining orientation dependent electrically detected magnetic resonance and g tensor calculations based on density functional theory we assign microscopic structures to paramagnetic states involved in spin-dependent recombination at the interface of hydrogenated amorphous silicon crystalline silicon (a-Si:H/c-Si) heterojunction solar cells. We find that (i) the interface exhibits microscopic roughness, (ii) the electronic structure of the interface defects is mainly determined by c-Si, (iii) we identify the microscopic origin of the conduction band tail state in the a-Si:H layer, and (iv) present a detailed recombination mechanism.

Bipolaron formation in organic solar cells observed by pulsed electrically detected magnetic resonance.

We report the observation of a spin-dependent dark transport current, exhibiting spin coherence at room temperature, in a π-conjugated polymer-fullerene blend using pulsed electrically detected magnetic resonance. The resonance at g = 2.0028(3) is due to polarons in the polymer, and exhibits spin locking at high microwave fields. The presence of an excess of fullerene, and the operating voltage (1 V) used, suppresses negative polaron formation in the polymer. It is concluded that spin-dependent transport is due to the formation of positive bipolarons.

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action.

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer.

The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending conventional EPR to high-field/high-frequency EPR, and highlights those details of molecular information that are obtained from high-field EPR in conjunction with genetic engineering and that are not accessible by "classical" spectroscopy. The importance of quantum-chemical interpretation of the experimental data by DFT and advanced semiempirical molecular-orbital theory is emphasized. A short description of the laboratory-built 95 GHz and 360 GHz EPR/ENDOR spectrometers at FU Berlin is also presented. The review concludes with an outlook to future opportunities and challenges of advanced bio-EPR in interdisciplinary research.

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P(700)(+z.rad;)A(1)(-z.rad;) by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz.

The radical-pair state of the primary electron donor and the secondary electron acceptor (P(700)(+z.rad;)A(1)(-z.rad;)) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 A resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C(2) symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.

[Contents and distribution of Fe, Cu, Zn, Ni, and Mn in fetuses, amniotic fluid, placenta, and uterus of rats]. / Gehalte and Verteilung von Fe, Cu, Zn, Ni und Mn in Foeten, Fruchtwasser, Placenta und Uterus von Ratten.

Fetuses, amniotic fluid, placenta, and uterus were taken from five gravid rats shortly prior to delivery, and the contents of Fe, Cu, Zn, Ni, and Mn were analyzed. Fe, Cu, Zn, and Mn exhibited a similar concentration in dry matter (DM) in all tested reproductive organs, whereby Fe-contents were five to ten times higher than Zn-contents and those five to ten times higher than Cu-contents, which amounted to 10-20 ppm. The Mn-contents range about a factor of ten below. Ni-concentration in DM of fetuses, placenta and uterus is below 1 ppm, whereas in contrast to this, Ni-concentration of 14 ppm in DM of amniotic fluid is 34 times higher than in fetuses and placenta. Uterus contained twice as much Ni as fetuses. Concerning the total content of the individual trace elements, Ni reacts also different as compared to Fe, Cu, Zn, and Mn. Its amniotic content exceeds the fetal content by 40%. Total excretion of Fe, Cu, Zn, Mn, and also DM is distributed differently as compared to Ni. Fetuses contain 60%-70% of these trace elements, placenta 15%-25%, uterus 6%-16%, and amniotic fluid only 1%-2%. Ni, however, is excreted mostly via amniotic fluid by about 35% of total excretion and only by about 25% via fetuses. These findings, concerning Ni, are discussed in detail.

[Alpha amylase and dehydrogenase activity in suboptimal Ni intake]. / Alpha-Amylase und Dehydrogenasenaktivität bei suboptimaler Ni-Versorgung.

Supplementary to studies on the activity of various enzymes during Ni deficiency, the present work was done to determine their response to suboptimal Ni supply. Live weight gain of rats was not affected by 60 versus 150 ppb Ni in the diet. The activity of alpha-amylase, however, was reduced by 43% in the case of 60 ppb dietary Ni and in relation to the change in this enzyme the activity of glucose-6-phosphate dehydrogenase by 56% and of the lactate dehydrogenase by 27%. These dehydrogenases responded with increased activities to the slight raise in Ni supply with the diet containing 150 ppb Ni as compared to the group given 60 ppb dietary Ni. Overall, it may be inferred that growth is not affected unless dietary Ni supply is below 50 ppb and that dietary Ni contents of 60 and 150 ppb must be regarded as suboptimal supply levels because of the diminished activity of various enzymes.

[Deficient nickel supply and the content of calcium, magnesium and phosphorus inthe bone of growing rats]. / Mangelnde Ni-versorgung und Ca-, Mg- und P-Gehalte in Knochen wachsender Ratten.

Based upon studies on Ni deficiency with marked changes in intermediary metabolism, the recent investigation examined the extent to which deficient Ni supply affects the content of calcium, magnesium and phosphorus in rat femurs. In Ni deficiency the content of calcium and phosphorus was reduced, in the case of calcium even more so than accounted for by the decrease in bone weight. The result was Ca : P ratio of 1.8 : 1 in Ni deficiency compared with 2.0 : 1 in control animals. In the case of magnesium, however, the Mg concentration was increased by 45% in the bone fresh matter of the deficient animals. The reduced Ca retention in the bone is accordingly contrasted by an enhanced deposition of magnesium.

[Effects of iron supply and lactation on iron availability in intermediate metabolism]. / Zum Einfluss der Fe-Versorgung und Laktation auf die Fe-Verfügbarkeit im intermediären Stoffwechsel.

The availability of iron in metabolism was determined in dependence on the performance after lactation had finished and on the level of Fe-supply of a total of 56 rats. The availability of iron of those animals which had reared young was, according to the increased requirement, high and decreased from 87 to 54% after increased doses of iron. The values of full-grown control animals were considerably lower and had a falling tendency from 52 to 36% after an increasing supply with iron. Because of the reduced utilization of iron after increased supply the gross requirement must be increased over-proportionally when the net requirement rises.

[Activity of proteases, leucine arylamidase and alpha-amylase in pancreatic tissue during nickel deficiency]. / Zur Aktivität der Proteasen, Leucinarylamidase und alpha-Amylase im Pankreasgewebe bei Nickelmangel.

In studies on the essentiality of nickel, important differences of enzyme activities and also of substrates were established. The results of the present paper show that these changes cannot be explained by a lowered reduction of dietary proteins, since the activity of the proteases rather increased during Ni deficiency. However, the digestion of the starch by alpha-amylase, being 57% lower, could have been partially responsible for the large differences in the activities of the hepatic enzymes and in the concentrations of the hepatic metabolites and also in the weight gains.

[Alkaline and acid phosphatase activity in the liver and serum during Ni versus Fe deficiency]. / Alkalische und Saure Phosphatase-Aktivität in Leber und Serum bei Ni-bzw. Fe-Mangel.

In previous studies on the essentiality of nickel, a reduced iron absorption causing anemia was observed. Since Ni deficiency also affects Zn metabolism, the different phosphatase activities were determined. Ni deficiency, however, resulted in an increased activity of the alkaline phosphatase in liver. On the other hand, the activity of the alkaline phosphatase was deduced by 59% during Fe deficiency. Similarly, the alkaline and acid phosphatases in serum were reduced during Fe deficiency. Consequently, determination of the activity of the alkaline phosphatase in serum, besides that of various liver enzymes, is suited well to differentiate between Fe and Ni deficiency.