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The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD*-) and neutral (FADH*) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD*- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX=2.00429(3), gY=2.00389(3), gZ=2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso=2.00345(3). The gY-component of FAD*- from GO is moderately shifted upon deprotonation of FADH*, rendering the g-tensor of FAD*- slightly more axially symmetric as compared to that of FADH*. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH* radical into the anionic FAD*- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.

The pore-forming bacterial toxins of the colicin family undergo massive protein refolding, while attacking a target cell, to convert from the water-soluble conformational state to the membrane-associated state with subsequent insertion of helical hairpins into the cytoplasmic membrane. To explore the validity of proposed models for the mechanism by which the soluble channel-forming domain of colicin A turns inside out upon membrane association, five site-specific cysteine mutants of colicin A, each singly spin labeled with nitroxide side chains, were studied by 9.5 GHz (X-band) and 95 GHz (W-band) high-field EPR. By elucidating the mobility of the nitroxide side chains on one of the two hydrophobic helices and their accessibility to paramagnetic relaxer molecules in the membrane, as well as by measuring the gxx and Azz nitroxide tensor components, detailed information about conformational changes upon membrane association could be revealed. This information on the channel-forming domain of colicin A goes ...

The flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form, FADH•, was investigated by high-frequency/high-field continuous-wave electron paramagnetic resonance at 360 GHz. The data presented are the first flavin radical spectra where the full rhombic symmetry of the g-tensor is resolved. A fit of the spectrum yields accurate principal values of g, which show only a small anisotropy: gX = 2.004 31(5), gY = 2.003 60(5) and gZ = 2.002 17(7). The hyperfine splitting observed in the gY region could be assigned to a hyperfine tensor component of the H(5) proton in the 7,8-dimethyl isoalloxazine moiety of FADH•. From a comparison of this effective hyperfine coupling with the principal value obtained from pulsed (Davies) electron−nuclear double resonance, the orientation of the g-tensor principal axes with respect to the H(5) hyperfine principal axes could be derived. Remaining ambiguities in the sign of the angle between the principal axes of g and the molecular axes are discussed by ta...

Davies-type pulsed H electron nuclear double resonance (ENDOR) measurements were performed at a magnetic field of 3.4 T and a microwave (MW) frequency of 95 GHz (W-band). By taking advantage of the increased electron Zeeman interaction at high field, the small g-anisotropy of the semiquinone anion radicals could be resolved in frozen solutions. Hence, the W-band ENDOR spectra could be taken at the well-separated canonical peaks of the powder electron paramagnetic resonance (EPR) spectra, thereby becoming highly orientation-selective with respect to the relative orientation of the radicals to the external magnetic field. The measurements were performed on various randomly oriented semiquinone anion radicals in frozen alcoholic solution and on the primary ubiquinone anion radical, Q, frozen photosynthetic bacterial reaction centers (RCs) of Rhodobacter sphaeroides in which the Fe was replaced by Zn (ZnRC). A simulation program was used to obtain magnitudes and orientations of the hyperfine tensors. The W-band ENDOR spectra of the immobilized radicals show not only hyperfine couplings (HFC) of local protons of the semiquinones, but also those of protons from the environment. These are, for example, involved in hydrogen bonds between the amino acid surrounding and the quinone carbonyl groups. For Q in ZnRCs, a particularly large H-bond HFC was obtained from which direction and distance of the H-bond could be estimated. These data were compared with those measured for the respective ubisemiquinone radical, UQ-10, in protonated and deuterated 2-propanol, where two H-bonds of comparable strength but different directions could be detected. Comparison of H ENDOR spectra of the 2,3,5,6-tetra-methyl-1,4-benzoquinone anion radical in frozen solution, performed both at W-band and at X-band frequency, demonstrated the limitations of achieving orientation selected ENDOR spectra from X-band experiments on systems with small g-anisotropy.

The spin-polarized EPR spectra at 95 GHz (W-band), 24 GHz (K-band), and 9 GHz (X-band) of the radical pair in highly purified photosystem I particles are presented. The spectra are analyzed to obtain both the magnetic parameters of the radical pair as well as the relative orientation of the two species. From the analysis, the g-tensor of is found to be gxx = 2.0062, gyy = 2.0051, and gzz = 2.0022, and it is shown that A1 is oriented such that the carbonyl bonds are parallel to the vector joining the centers of and . The anisotropy of the g-tensor is considerably larger than that obtained for chemically reduced phylloquinone in frozen 2-propanol solution. Possible reasons for this difference and their implications for the A1 binding site are discussed. The relative orientation of and is compared with earlier estimates obtained using less accurate g-values for . A comparison with the spectra of in bacterial reaction centers (bRCs) of Rhodobacter sphaeroides R-26 in which the nonheme iron has been replaced b...