Selective 2H-, 13C-, and 17O-isotope labeling of the tyrosine amino acid has been used to map the unpaired π-electron spin-density distribution of the UV-generated neutral l-tyrosine phenoxy radical in alkaline frozen solution. The use of 13C and 17O labels allowed accurate determination of the full spin-density distribution and provided more insight in the geometrical structure of the neutral tyrosine radical in vitro. Simulations of the X-band (9.2 GHz) and Q-band (34.8 GHz) EPR powder spectra yielded the principal components of the 1H-, 13C-, and 17O-hyperfine tensors. For the two β-methylene hydrogens, a static conformational distribution of the dihedral angles (90° < θ1 < 60° and 60° < θ2 < 30°) was taken into account. The major proton hyperfine interactions and the principal g values for the neutral tyrosine radical, obtained from selectively deuterated samples, are consistent with literature values. The spin density at the specifically labeled postitions (C1‘, C2‘, C3‘, C4‘, C5‘, O4‘) was evaluated from the anisotropy of the 13C- and 17O-hyperfine tensors. A quantitative analysis of the positions C3‘ and C5‘ provided evidence for a planar distortion of the aromatic ring at these positions. 17O enrichment of the phenol oxygen O4‘ of the tyrosine radical unambiguously showed that the spin density at this oxygen is 0.26 ± 0.01. From the relatively large delocalization of the spin density over the carbonyl group of the tyrosine aromatic ring system, it is concluded that the C4‘−O4‘ bond has a double-bond character. The experimentally determined spin-density distribution is compared with several computational calculated spin-density distributions found in the literature. The isotropic 13C-hyperfine interactions are discussed in the framework of the Karplus−Fraenkel theory. This theory proved to be accurate for the determination of sign and magnitude of the isotropic 13C- and 17O-hyperfine interactions.

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It was previously shown in the photosystem II membrane preparation DT-20 that photoxidation of the oxygen-evolving manganese cluster was blocked by 0.1 mM formate, unless 0.2 mM bicarbonate was present as well [Wincencjusz, H., Allakhverdiev, S. I., Klimov, V. V., and Van Gorkom, H. J. (1996) Biochim. Biophys. Acta 1273, 1-3]. Here it is shown by measurements of EPR signal II that oxidation of the secondary electron donor, YZ, is not inhibited. However, the reduction of is greatly slowed and occurs largely by back reaction with reduced acceptors. Bicarbonate is shown to prevent the loss of fast electron donation to . The release of about one or two free Mn2+ per photosystem II during formate treatment, and the fact that these effects are mimicked by Mn-depletion, suggests that formate may act by replacing a bicarbonate which is essential for Mn binding. Irreversible light-induced rebinding in an EPR-silent form of Mn2+ that was added to Mn-depleted DT-20 was indeed found to depend on the presence of bicarbonate, as did the reconstitution in such material of both the fast electron donation to and the UV absorbance changes characteristic of a functional oxygen-evolving complex. It is concluded that bicarbonate may be an essential ligand of the functional Mn cluster.

The relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz) EPR as a tool to probe for structural changes resulting from charge separation and stabilization and from replacing the native QA molecule with other quinones. We present a study of possible changes in QA-binding that might be responsible for the remarkably prolonged lifetime of the charge-separated state at cryogenic temperatures for RCs of Rhodobacter sphaeroides R26 cooled under illumination [Kleinfeld, D., et al. (1984) Biochemistry 23, 5780-5786]. It is shown that this effect is not caused by a major reorientation of the chromophores. Furthermore, we studied the effects of structurally different quinones functioning as primary electron acceptor in different purple bacteria. With simulations of ESP X-band spectra of the spin-polarized secondary radical pair P.+QA.+- in menaquinone-reconstituted, Zn(2+)-substituted RCs of Rb. sphaeroides R26, we show that quinone reconstitution is highly selective for site and orientation. Furthermore, we find that a very small exchange interaction between P.+ and QA.+- (magnitude of JPQ approximately 1 microT) is needed to account accurately for the observed relative line intensities at X-band, without affecting the accuracy of the simulations of reported ESP K-band spectra [Füchsle, G., et al. (1993) Biochim. Biophys. Acta 1142, 23-35; Van der Est, A., et al. (1993) Chem. Phys. Lett. 212, 561-568]. This pronounced influence of small values for JPQ on the X-band ESP line shape results from cancellation effects of absorptive and emissive contributions to the spectrum, such that small shifts can be observed. The exchange interaction has opposite sign for the native, ubiquinone-containing RC [viz. JP.UQ = (-0.8 +/- 0.2) microT] and the menaquinone-substituted RC [JP.MK = (+0.3 +/- 0.2) microT]. The implications of these observations for electron-transport theory are discussed.

The secondary photoinduced radical pair state P +• Q A -• in Zn-substituted and quinone-reconstituted photosynthetic reaction centers of Rhodobacter sphaeroides R26 is investigated with transient EPR spectroscopy at X-(9.3 GHz), Q-(34.7 GHz), and D-band (130.0 GHz) microwave frequencies. Novel D-band P +• Q A -• electron spin-polarized spectra are presented for three different reconstituted reaction centers. The shape of the electron spin-polarized P +• Q A -• EPR spectrum strongly depends on the lifetime and magnetic properties of its precursor radical pair P +• Φ A -• , whose lifetime was manipulated by replacing the native ubiquinone-10 (UQ-10) with duroquinone (DQ) or 2-ethyl anthraquinone (AQ). From spectral simulations incorporating the transfer of spin-correlation between the two radical pairs, information about the magnetic interactions and dynamics of the intermediate primary P +• Φ A -• radical pair was obtained. When the lifetime of P +• Φ A -• is longer than a few nanoseconds, the influence of the magnitude and sign of the exchange interaction J PΦ between P +• and Φ A -• on the shape of the observed ESP spectrum is significant. This effect is even more pronounced at the relatively high D-band microwave frequency, facilitating accurate determination of J PΦ ) -0.9 ( 0.1 mT, for both DQ and AQ-reconstituted RCs, a value not significantly different from that determined here for the UQ-10 reconstituted sample (J PΦ ) -0.7 ( 0.5 mT). The singlet and triplet rate constants (k S and k T ) for the intermediate radical pair P +• Φ A -• in the duroquinone-or 2-ethyl anthraquinone-reconstituted samples were (2 ( 1) × 10 7 s -1 and (2 ( 1) × 10 8 s -1 , respectively. The exchange interaction between P +• and Φ A -• is J PQ ) -0.5 ( 0.3 µT, -0.2 ( 0.1 µT, and -0.5 ( 0.2 µT for UQ-10, AQ, and DQ, respectively. X-band ESEEM experiments showed that the dipolar interaction between P +• and Φ A -• is -0.12

Improving authentication delay is a key issue for achieving seamless handovers across networks and domains. This paper presents an overview of fast authentication methods when roaming within or across IEEE 802.11 Wireless-LANs. Besides this overview, the paper analyses the applicability of IEEE 802.11f and Seamoby solutions to enable fast authentication for inter-domain handovers. The paper proposes a number of possible changes to these solutions (typically in terms of network architectures and/or required trust relationships) for inter-domain operation. In addition, the paper identifies the crucial research issues therein. Possible solutions and directions for future research include: update to security infrastructure, inter-layer communication and discovery of appropriate networks.

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