Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60°rotational angle of C ␣-(C␤-C1)-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.anthrax ͉ electron paramagnetic resonance ͉ hydroxyurea ͉ N-methyl hydroxylamine ͉ bacterial growth inhibition

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The interactions between iron and neuromelanin (NM) have been studied by means of EPR spectroscopy. The variable temperature EPR spectral features of a specimen of NM extracted from normal human midbrains clearly indicate that iron is present as polynuclear oxy-hydroxy ferric aggregates as well as isolated Fe(III) centres. Ferric oxy-hydroxy phases are typical of the iron storage proteins ferritin and hemosiderin, but the comparison of the variable temperature EPR spectra of ferritin and NM highlights significant differences between the two iron(III)oxy-hydroxy domains. Moreover, further investigations on melanin models synthesised in the presence of either ferritin or a ferric salt as iron sources suggest that the same pathway of formation and inclusion of the polynuclear iron oxide is operating in NM and in the model systems, whatever is the source of iron.

Paramagnetic centers rigidly embedded in proteins serve as a probe in structural studies of macromolecular complexes. Pulse EPR at high frequencies allows the determination of not only the distance but also the relative orientation of these centers (see tyrosyl radicals; C gray, O red, N blue). The method has considerable potential for studying the assembly of protein complexes.

The di †usion and bonding of (x \ 1, 2) and Cu2`species in zeolites are reviewed, based mainly on our NOx own research. The molecular motion of adsorbed has been examined with EPR in several zeolite samples NO 2 and analyzed using the slow-motional EPR theory. In X-and Y-type zeolites the broadening of the spectra with temperature could be analyzed by simulations using a rotational di †usion model in agreement with earlier results in Vycor glass and Cu-metal. For the di †usion of in Na-mordenite and Na-ZSM-5 the NO 2 broadening of the spectra with increased temperature could be better simulated with the Heisenberg type of spin exchange model. The exchange was attributed to the interaction between molecules di †using along NO 2 the zeolite channels. In Na-ZSM-5 the spin exchange rate increased rapidly with an increasing Si/Al ratio of the zeolite. The e †ect was attributed to the hindrance against di †usion by Na`, the amount of which increases with a decreased Si/Al ratio. The rates increased with increasing number of molecules adsorbed on the H 2 O zeolite because of weaker interaction between and the surface. A more detailed slow-motional analysis NO 2 indicated that at each temperature a distribution of di †usion rates occurs. NO adsorbed on Na-A zeolite was found to be present as monomer at low pressure (\1 mbar) while a dimer characterised by a triplet state ESR spectrum involving and features became dominant at higher NO pressure ([100 mbar). *M s \ 1 *M s \ 2 The triplet was absent in calcium ion-exchanged A-type zeolite indicating that the NOÈNO species depends on Na`for its stability. The detailed structure of a Cu2`complex with water molecules in Cu-ZSM-5 zeolite was characterised with ENDOR at 4 K. The complex has an axial distorted octahedral structure with two water molecules at a longer distance in axial position and four molecules in the equatorial positions. The Cu2c omplexes with ammonia and deuterated ammonia have been investigated with electron nuclear double resonance (ENDOR). Simulations of the ENDOR spectra with 1H, 2H and 14N hyperÐne interactions have been undertaken. The analysis indicates that the complex has a square planar structure with four ammonia ligands.

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Lack of better understanding of nanoparticles targeted delivery into cancer cells calls for advanced optical microscopy methodologies. Here we present a development of fluorescence microspectroscopy (spectral imaging) based on a white light spinning disk confocal microscope with emission wavelength selection by a liquid crystal tunable filter. Spectral contrasting of images was used to localize polymer nanoparticles and cell membranes labeled with fluorophores that have substantially overlapping spectra. In addition, fluorescence microspectroscopy enabled spatiallyresolved detection of small but significant effects of local molecular environment on the properties of environment-sensitive fluorescent probe. The observed spectral shift suggests that the delivery of suitably composed cancerostatic alkylphospholipid nanoparticles into living cancer cells might rely on the fusion with plasma cell membrane.