The heart of the oxygen-evolving complex (OEC) of photosystem II is a Mn4OxCa cluster that cycles through five different oxidation states (S0 to S4) during the light-driven water-splitting reaction cycle. In this study we interpret the recently obtained 55Mn hyperfine coupling constants of the S0 and S2 states of the OEC [Kulik et al. J. Am. Chem. Soc. 2005, 127, 2392-2393] on the basis of Y-shaped spin-coupling schemes with up to four nonzero exchange coupling constants, J. This analysis rules out the presence of one or more Mn(II) ions in S0 in methanol (3%) containing samples and thereby establishes that the oxidation states of the manganese ions in S0 and S2 are, at 4 K, Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. By applying a "structure filter" that is based on the recently reported single-crystal EXAFS data on the Mn4OxCa cluster [Yano et al. Science 2006, 314, 821-825] we (i) show that this new structural model is fully consistent with EPR and 55Mn-ENDOR data, (ii) assign the Mn oxidation states to the individual Mn ions, and (iii) propose that the known shortening of one 2.85 A Mn-Mn distance in S0 to 2.75 A in S1 [Robblee et al. J. Am. Chem. Soc. 2002, 124, 7459-7471] corresponds to a deprotonation of a mu-hydroxo bridge between MnA and MnB, i.e., between the outer Mn and its neighboring Mn of the mu3-oxo bridged moiety of the cluster. We summarize our results in a molecular model for the S0 --> S1 and S1 --> S2 transitions.

55Mn pulse ENDOR experiments at 34 GHz (Q-band) are reported for the S0 and S2 states of the oxygen-evolving complex of photosystem II. Their numerical analysis (i) shows that in both states all four Mn ions are magnetically coupled, (ii) allows a refinement of the hyperfine interaction (HFI) parameters obtained earlier for the S2 state at X-band (Peloquin, J. M.; Campbell, K. A.; Randall, D. W.; Evanchik, M. A.; Pecoraro, V. L.; Armstrong, W. H.; Britt, R. D. J. Am. Chem. Soc. 2000, 122, 10926-10942), (iii) provides the first reliable 55Mn HFI tensors for the S0 state, and (iv) leads to the suggestion that the Mn oxidation states in S0 and S2 are Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. In addition, a Q-band EPR spectrum is reported for the S0 state, and inversion-recovery experiments at 4.5 K directly show that the electron spin-lattice relaxation for the S0 state is about 2 orders of magnitude faster than that for the S2 state.

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A versatile 250 MHz pulse electron paramagnetic resonance (EPR) instrument for imaging of small animals is presented. Flexible design of the imager hardware and software makes it possible to use virtually any pulse EPR imaging modality. A fast pulse generation and data acquisition system based on general purpose PCI boards performs measurements with minimal additional delays. Careful design of receiver protection circuitry allowed us to achieve very high sensitivity of the instrument. In this article we demonstrate the ability of the instrument to obtain three dimensional images using the electron spin echo (ESE) and single point imaging (SPI) methods. In a phantom that contains a 1 mM solution of narrow line (16 μT, peak-to-peak) paramagnetic spin probe we achieved an acquisition time of 32 seconds per image with a fast 3D ESE imaging protocol. Using an 18 minute 3D phase relaxation (T2e) ESE imaging protocol in a homogeneous sample a spatial resolution of 1.4 mm and a standard deviation of T2e of 8.5% were achieved. When applied to in vivo imaging this precision of T2e determination would be equivalent to 2 torr resolution of oxygen partial pressure in animal tissues.

ABSTRACT:A versatile control software for pulse EPR spectrometers is introduced.Common and task-specific problems are discussed and their solutions are described. The software provides the full spectrum of possibilities needed to perform arbitrary multidimensional pulse experiments. It allows for an easy interfacing of commonly used hardware components and enables straightforward modifications of the spectrometer. Good performance, configurability, and a number of unique features turn this software into an excellent tool for the operation of modern EPR spectrometers and upgrading old ones.

Purpose 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. Methods We present a fundamentally new technique to image pO2 in tumors and tissues with pulse electron paramagnetic resonance (EPR) imaging enabled by an injected, nontoxic, triaryl methyl (trityl) spin probe whose unpaired electron’s slow relaxation rates report the tissue pO2. Heretofore, virtually all in vivo EPR O2 imaging measures pO2 with the transverse electron spin relaxation rate, R2e, which is susceptible to the self-relaxation confounding O2 sensitivity. Results We found that the trityl electron longitudinal relaxation rate, R1e, is an order of magnitude less sensitive to confounding self-relaxation. R1e imaging has greater accuracy and brings EPR O2 images to an absolute pO2 image, within uncertainties. Conclusion R1e imaging more accurately determines oxygenation of cancer and normal tissue in animal models than has been available. It will enable enhanced, rapid, noninvasive O2 images for understanding oxygen biology and the relationship of oxygenation patterns to therapy outcome in living animal systems.

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W-band (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) spectroscopic techniques were used to determine the hyperfine couplings of different protons of Cu(II)-histidine complexes in frozen solutions. The results were then used to obtain the coordination mode of the tridentate histidine molecule and to serve as a reference for Cu(II)-histidine complexation in other, more complex systems. Cu(II) complexes with L-histidine and DL-histidine-alpha-d,beta-d2 were prepared in H2O and in D2O, and orientation-selective W-band 1H and 2H pulsed ENDOR spectra of these complexes were recorded at 4.5 K. These measurements lead to the unambiguous assignment of the signals of the H alpha, H beta, imidazole H epsilon, and the exchangeable amino, Ham, protons. The 14N superhyperfine splitting observed in the X-band EPR spectrum and the presence of only one type of H alpha and H beta protons in the W-band ENDOR spectra show that the complex is a symmetric bis complex. Its g parallel is along the molecular symmetry axis, perpendicular to the equatorial plane that consists of four coordinated nitrogens in histamine-like coordinations (NNNN). Simulations of orientation-selective ENDOR spectra provided the principal components of the protons' hyperfine interaction and the orientation of their principal axes with respect to g parallel. From the anisotropic part of the hyperfine interaction of H alpha and H beta and applying the point-dipole approximation, a structural model was derived. An unexpectedly large isotropic hyperfine coupling, 10.9 MHz, was found for H alpha. In contrast, H alpha of the Cu(II)-1-methyl-histidine complex where only the amino nitrogen is coordinated, showed a much smaller coupling. Thus, the hyperfine coupling of H alpha can serve as a signature for a histamine coordination where both the amino and imino nitrogens of the same molecule bind to the Cu(II), forming a six-membered chelating ring. Unlike H alpha the hyperfine coupling of H epsilon is not as sensitive to the presence of a coordinated amino nitrogen of the same histidine molecule.