A high-spin ground-state donor-acceptor conjugated polymer.

Interest in high-spin organic materials is driven by opportunities to enable far-reaching fundamental science and develop technologies that integrate light element spin, magnetic, and quantum functionalities. Although extensively studied, the intrinsic instability of these materials complicates synthesis and precludes an understanding of how fundamental properties associated with the nature of the chemical bond and electron pairing in organic materials systems manifest in practical applications. Here, we demonstrate a conjugated polymer semiconductor, based on alternating cyclopentadithiophene and thiadiazoloquinoxaline units, that is a ground-state triplet in its neutral form. Electron paramagnetic resonance and magnetic susceptibility measurements are consistent with a high-to-low spin energy gap of 9.30 × 10-3 kcal mol-1. The strongly correlated electronic structure, very narrow bandgap, intramolecular ferromagnetic coupling, high electrical conductivity, solution processability, and robust stability open access to a broad variety of technologically relevant applications once thought of as beyond the current scope of organic semiconductors.

Electron spin echo envelope modulation of molecular motions of deuterium nuclei.

Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study of hyperfine interactions between an unpaired electron and nearby nuclei in solids, and is employed in quantitative structural studies. Here, we describe the use of ESEEM to study the slow motion of deuterium nuclei using their nuclear quadrupole resonance (NQR) line shapes. Two ESEEM techniques were employed: the conventional three-pulse ESEEM experiment, π/2 - τ - π/2 - T- π/2 - τ - echo, and the four-pulse ESEEM, π/2 - τ - π/2 - T/2 - π - T/2 - π/2 - τ - echo, with the time variable T scanned in both cases. The nitroxide free radical 4-tert-butyliminomethyl-2,2,5,5-tetramethyl(d12)-3-imidazoline-1-oxyl with four deuterated methyl groups was investigated in a glassy ortho-terphenyl matrix over a wide temperature range. It was shown that four-pulse ESEEM allowed measurement of the nearly pure (2)H NQR line shape. Between 90K and 120K, the ESEEM spectra change drastically. At low temperatures, four-pulse ESEEM spectra show a Pake-like pattern, which evolves into a single line at higher temperatures, which is typical for NQR of rotating methyl CD3 groups. Comparison with literature data on NQR allows estimation of the reorientation rate, k. At ∼100K, where the spectral changes are most pronounced, k was found to be ∼10(5)s(-1). The spectral linewidths for the three-pulse ESEEM were found to decrease similarly with increasing temperature; so the three-pulse technique is also capable to detect motion of this type. The ESEEM approach, along with site-directed spin labeling, may be useful for detection of motional transitions near the spin labels in biological systems, when information on motion is required in a wide temperature range.

Hyperfine interactions of narrow-line trityl radical with solvent molecules.

The electron nuclear dipolar interactions responsible for some dynamic nuclear polarization (DNP) mechanisms also are responsible for the presence formally in CW EPR spectra of forbidden satellite lines in which both the electron spin and a nuclear spin flip. Such lines arising from (1)H nuclei are easily resolved in CW EPR measurements of trityl radicals, a popular family of DNP reagents. The satellite lines overlap some of the hyperfine features from (13)C in natural abundance in the trityl radical, but their intensity can be easily determined by simple simulations of the EPR spectra using the hyperfine parameters of the trityl radical. Isotopic substitution of (2)H for (1)H among the hydrogens of the trityl radical and/or the solvent allows the dipolar interactions from the (1)H on the trityl radical and from the solvent to be determined. The intensity of the dipolar interactions, integrated over all the (1)H in the system, is characterized by the traditional parameter called reff. For the so-called Finland trityl in methanol, the reff values indicate that collectively the (1)H in the unlabeled solvent have a stronger integrated dipolar interaction with the unpaired electron spin of the Finland trityl than do the (1)H in the radical and consequently will be a more important DNP route. Although reff has the dimensions of distance, it does not correspond to any simple physical dimension in the trityl radical because the details of the unpaired electron spin distribution and the hydrogen distribution are important in the case of trityls.

The solution conformation of triarylmethyl radicals.

Hyperfine coupling tensors to 1H, 2H, and natural abundance 13C were measured using X-band pulsed electron nuclear double resonance (ENDOR) spectroscopy for two triarylmethyl (trityl) radicals used in electron paramagnetic resonance imaging and oximetry: methyl tris(8-carboxy-2,2,6,6-tetramethyl-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) and methyl tris(8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl). Quantum chemical calculations using density functional theory predict a structure that reproduces the experimentally determined hyperfine tensors. The radicals are propeller-shaped with the three aryl rings nearly mutually orthogonal. The central carbon atom carrying most of the unpaired electron spin density is surrounded by the sulfur atoms in the radical and is completely shielded from solvent. This structure explains features of the electron spin relaxation of these radicals and suggests ways in which the radicals can be chemically modified to improve their characteristics for imaging and oximetry.

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex.

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.

Intensity of cross-peaks in hyscore spectra of S = 1/2, I = 1/2 spin systems.

The cross-peak intensity for a S = 1/2, I = 1/2 spin system in two-dimensional HYSCORE spectra of single-crystals and powders is analyzed. There is a fundamental difference between these two cases. For single crystals, the cross-peak intensity is distributed between the two (+, +) and (+, -) quadrants of the hyperfine sublevel correlation (HYSCORE) spectrum by the ratio c(2):s(2) (C. Gemperle, G. Aebli, A. Schweiger, and R. R. Ernst, J. Magn. Reson. 88, 241 (1990)). However, for powder spectra another factor becomes dominant and governs cross-peak intensities in the two quadrants. This factor is the phase interference between modulation from different orientations of the paramagnetic species. This can lead to essentially complete disappearance of the cross-peak in one of the two (+, +) or (+, -) quadrants. In the (+, +) quadrant, cross-peaks oriented parallel to the main (positive) diagonal of the HYSCORE spectrum are suppressed, while the opposite is true in the (+, -) quadrant where cross-peaks nearly perpendicular to the main (negative) diagonal of HYSCORE spectra are suppressed. Analytical expressions are derived for the cross-peak intensity profiles in powder HYSCORE spectra for both axial and nonaxial hyperfine interactions (HFI). The intensity is a product of two terms, one depending only on experimental parameter (tau) and the other only on the spin Hamiltonian. This separation provides a rapid way to choose tau for maximum cross-peak intensity in a region of interest in the spectrum. For axial HFI, the Hamiltonian-dependent term has only one maximum and decreases to zero at the canonical orientations. For nonaxial HFI, this term produces three separate ridges which outline the whole powder lineshape. These three ridges have the majority of the intensity in the HYSCORE spectrum. The intensity profile of each ridge resembles that observed for axial HFI. Each ridge defines two principal values of the HFI similar to the ridges from an axial HFI.

Electron nuclear quadruple resonance for assignment of overlapping spectra.

Multiple resonance methods are important tools in EPR for revealing the network of hyperfine levels of free radicals and paramagnetic centers. The variations of electron nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM) techniques help to correlate nuclear frequencies with each other. These methods have limited utility when there is extensive overlap or suspected overlap in the EPR spectrum between different species or different orientations. In the ENDOR spectrum, overlap and second-order shifts of lines also leads to ambiguity in assignment and interpretation. A new electron nuclear multiple resonance method is presented here that is based on population transfer ENDOR. It is a quadruple resonance method that correlates ENDOR lines and reveals the network of hyperfine levels in samples with unoriented paramagnetic species and in samples with overlapping EPR or ENDOR lines.

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis.

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.

EPR properties of mixed-valent mu-oxo and mu-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K.

Radiolytic reduction at 77 K of oxo/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S = 1/2 ground state with small g-anisotropy as characterized by the uniaxial component (gz-gav/2 < 0.03) observable at temperatures as high as approximately 100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (gz-gav/2 > 0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins.

Measurement of bioreduction rates of cells with distinct responses to ionizing radiation and cisplatin.

A low frequency electron paramagnetic resonance (EPR) spectrometer has been used to measure the bioreduction rate of an exogenously added nitroxide free radical species. Measurements have been made in a well controlled, in vitro system using an X-ray and cisplatin sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and partial revertants which display wild-type levels of sensitivity to X-rays but retain xrs-5 levels of cisplatin sensitivity. The xrs-5 cells reduce this radical species at a rate which is approx. 50% that of the wild-type CHO cell line, K1. The partial revertants maintain this defect in bioreduction despite their decrease in radiosensitivity. However, the bioreduction rate observed in these cells correlates with their sensitivity to the chemotherapeutic drug cisplatin. Low frequency EPR allows measurements and imaging of living tissue and may be of value as a predictive assay of human tumor response to chemotherapy.

Direct measurement of nitroxide pharmacokinetics in isolated hearts situated in a low-frequency electron spin resonance spectrometer: implications for spin trapping and in vivo oxymetry.

The pharmacokinetics of two nitroxides were investigated in isolated rat hearts situated in a low-frequency electron spin resonance spectrometer. The spin labels 2,2,3,3,5,5-hexamethyl-1-pyrrolidinyloxy and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy were chosen for their physiochemical analogy to the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and its corresponding spin-trapped adduct, 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH). The bioreductive rates of the two nitroxides were measured during constant perfusion as well as during ischemia and are discussed in terms of a two-compartment pharmacokinetic model. These data provide information necessary to the design and application of spin traps to detect oxy radicals during reperfusion of ischemic tissue and suggest the feasibility of monitoring free-radical processes in intact, functioning mammalian tissues by using a low-frequency electron spin resonance spectrometer.

Electron spin resonance of charge carriers in chlorophyll a/water micelles.

Chlorophyll a/water micelles (P740) prepared in hydrocarbon media have been shown by small-angle neutron scattering to consist of hollow cylinders whose surface is formed of a monolayer of chlorophyll crosslinked by water. The micelles can be reversibly oxidized or reduced to generate highly mobile holes or electrons that undergo rapid, one-dimensional transport along the chains of chlorophyll macrocycles comprising the surface of the micelles. Large pi-pi overlap within the chains facilitates the one-dimensional charge transport and is expected to do the same for energy transport. Structural defects in the micelle surface act as boundaries for charge transport, confining the spins to one-dimensional domains of approximately 200 macrocycles. The one-dimensional transport within the limited domains results in motionally narrowed electron spin resonance lines with some residual inhomogeneous broadening. Although the chlorophyll a incorporated in micelles is more easily oxidized than is monomeric chlorophyll a, it is much more resistant to chemical alteration.

Bacteriopheophytin g: Properties and some speculations on a possible primary role for bacteriochlorophylls b and g in the biosynthesis of chlorophylls.

Bacteriopheophytin g and small amounts of bacteriochlorophyll g have been obtained in high purity from the recently discovered photosynthetic bacterium Heliobacterium chlorum. Preparative methods and precautions in handling these sensitive compounds are described. The compounds have been characterized by californium-252 plasma desorption mass spectrometry, HPLC, visible absorption, and electron spin resonance spectroscopy. Our results agree with the structure of bacteriochlorophyll g advanced by H. Brockmann and A. Lipinski [(1983) Arch. Microbiol. 136, 17-19], with the exception that we find the esterifying alcohol to be farnesol and not geranylgeraniol as originally suggested. Zero field splitting parameters of triplet state bacteriopheophytin g and the ESR properties of the cation free radical of bacteriochlorophyll g are reported. The photoisomerization of the subject compounds has been studied. Bacteriopheophytin g undergoes photo-isomerization in white light to pheophytin a with a half-time of approximately 42 min. We suggest that all of the chlorophylls are biosynthesized from a common intermediate containing an ethylidine group, [unk]CH-CH(3), such as is present in bacteriochlorophylls b and g.

Heliobacterium chlorum: cell organization and structure.

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.

Magnetic characterization of the primary state of bacterial photosynthesis.

The results of reaction yield-detected magnetic resonance (RYDMR) experiments carried out on modified bacterial photosynthetic reaction centers (RCs) are interpreted in terms of a model that assigns the initial charge-separated radical ion-pair state, P(F), as the carrier of the spectrum. The radical pair theory, which has been invoked to explain magnetic field effects in RCs, was significantly expanded to take into consideration the electron dipole-dipole interaction. It is shown that this is the largest interaction between the components of the radical ion pair. Quantum statistical calculations are described simulating the RYDMR spectra and low-field effects in quinone-depleted RCs. The experimental data on which the simulations are based are (i) the magnitude of the field effect at 3,000 G, (ii) the field at which 0.5 of the maximal field effect is observed, (iii) the P(F) population as a function of time at zero magnetic field, (iv) the RYDMR linewidth for low microwave field strength, (v) the RYDMR intensity and width as a function of microwave field, and (vi) the maximum RYDMR intensity at H(I) approximately 2J. With this information it was found possible to characterize P(F) in terms of four parameters, two containing structural information and two with kinetic implications. These are the dipole-dipole interaction, D = -47 +/- 10 x 10(-4) cm(-1); the exchange interaction, J = -7.5 +/- 1.9 x 10(-4) cm(-1); and the inverse rate constants of the decay of the radical pair states with singlet and triplet spin functions, respectively, k(S) (-1) = 15 +/- 4 nsec and k(T) (-1) = 1.8 +/- 0.2 nsec. The structural and dynamic implications of these parameters are discussed.

Magnetic resonance spectroscopy of the primary state, P, of bacterial photosynthesis.

We have obtained the magnetic resonance spectrum of the radical pair state P(F) by using reaction yield detected magnetic resonance spectroscopy. The magnetic resonance spectrum is quite sensitive to the local environment of P(F). The data place limits on the lifetime of triplet P(F) and the distance of charge separation.