Nuclear Double Resonance Research Articles

Reverse dynamic nuclear polarisation for indirect detection of nuclear spins close to unpaired electrons.

Polarisation transfer schemes and indirect detection are central to magnetic resonance. Using the trityl radical OX063 and a pulse electron paramagnetic resonance spectrometer operating in the Q-band (35 GHz, 1.2 T), we show here that it is possible to use pulsed dynamic nuclear polarisation (DNP) to transfer polarisation from electrons to protons and back. The latter is achieved by first saturating the electrons and then simply using a reverse DNP step. A variable mixing time between DNP and reverse DNP allows us to investigate the decay of polarisation on protons in the vicinity of the electrons. We qualitatively investigate the influence of solvent deuteration, temperature, and electron concentration. We expect reverse DNP to be useful in the investigation of nuclear spin diffusion and envisage its use in electron-nuclear double-resonance (ENDOR) experiments.

High-Frequency Electron Nuclear Double-Resonance Spectroscopy Studies of the Mechanism of Proton-Coupled Electron Transfer at the Tyrosine-D Residue of Photosystem II

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive the catalytic oxidation of water. Photosystem II contains two symmetrically placed tyrosine residues, YD and YZ, one on each subunit of the heterodimeric core. The YZ residue is kinetically competent and is proposed to be directly involved in the proton-coupled electron transfer reactions of water oxidation. In contrast, the YD proton-coupled electron transfer redox poises the catalytic tetranuclear manganese cluster and may electrostatically tune the adjacent monomeric redox-active chlorophyll and β-carotene in the secondary electron transfer pathway of photosystem II. In this study, we apply pulsed high-frequency electron paramagnetic resonance (EPR) and electron nuclear double-resonance (ENDOR) spectroscopy to study the photochemical proton-coupled electron transfer (PCET) intermediates of YD. We detect the "unrelaxed" and "relaxed" photoinduced PCET intermediates of YD using high-frequency EPR spectroscopy and observe an increase of the g anisotropy upon temperature-induced relaxation of the unrelaxed intermediate to the relaxed state as previously observed by Faller et al. [(2002) Biochemistry 41, 12914-12920; (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8732-8735]. This observation suggests the presence of structural differences between the two intermediates. We probe the possible structural differences by performing high-frequency (2)H ENDOR spectroscopy experiments. On the basis of numerical simulations of the experimental (2)H ENDOR spectra, we confirm that (i) there is a significant change in the H-bond length of the tyrosyl radical in the unrelaxed (1.49 Å) and relaxed (1.75 Å) PCET intermediates. This observation suggests that the D2-His189 residue is deprotonated prior to electron transfer at the YD residue and (ii) there are negligible changes in the conformation of the tyrosyl ring in the unrelaxed and relaxed PCET intermediates of YD.

Electron−Nuclear and Electron−Electron Double Resonance Spectroscopies Show that the Primary Quinone Acceptor QA in Reaction Centers from Photosynthetic Bacteria Rhodobacter sphaeroides Remains in the Same Orientation Upon Light-Induced Reduction

Reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides R-26 exhibit changes in the recombination kinetics of the charge-separated radical-pair state, P(·+) Q(A)(·-), composed of the dimeric bacteriochlorophyll donor P and the ubiquinone-10 acceptor Q(A), depending on whether the RCs are cooled to cryogenic temperatures in the dark or under continuous illumination (Kleinfeld et al. Biochemistry 1984, 23, 5780-5786). Structural changes near redox-active cofactors have been postulated to be responsible for these changes in kinetics and to occur in the course of light-induced oxidation and reduction of the cofactors thereby assuring a high quantum yield. Here we investigated such potential light-induced structural changes, associated with the formation of P(·+) Q(A)(·-), via pulsed electron-nuclear double resonance (ENDOR) at Q-band (34 GHz) and pulsed electron-electron double resonance (PELDOR) at W-band (95 GHz). Two types of light excitation have been employed for which identical RC samples were prepared: (a) one sample was frozen in the dark and then illuminated to generate transient P(·+) Q(A)(·-), and (b) one was frozen under illumination which resulted in both trapped and transient P(·+) Q(A)(·-) at 80 K. The hyperfine interactions between Q(A)(·-) and the protein were found to be the same in RCs frozen in the dark as in RCs frozen under illumination. Furthermore, these interactions are completely consistent with those observed in RC crystals frozen in the dark. Thus, QA remains in its binding site with the same position and orientation upon reduction. This conclusion is consistent with the result of our orientation-resolving PELDOR experiments on transient P(·+) Q(A)(·-) radical pairs. However, these findings are incompatible with the recently proposed ~60° reorientation of Q(A) upon its photoreduction, as deduced from an analysis of Q-band quantum-beat oscillations (Heinen et al. J. Am. Chem. Soc. 2007, 129, 15935-15946). Such a large reorientation appears improbable, and our objections against this proposition are substantiated here in detail. Our results show that Q(A) is initially in an orientation that is favorable for its light-driven reduction. This diminishes the reorganization requirements for fast electron reduction and high quantum efficiency.

Primary Oxidation Products of 5-Methylcytosine: Methyl Dynamics and Environmental Influences

The primary oxidation product in X-irradiated single crystals of 5-methylcytosine hemihydrate and 5-methylcytosine hydrochloride has been studied at 10 K, using electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) spectroscopies. The radical is characterized by large couplings to the methyl protons and appears to be deprotonated at N1 in both crystal systems. In the hydrochloride crystal the methyl group is completely frozen at 10 K, whereas in the hemihydrate crystal it undergoes tunneling rotation. For the hemihydrate crystal, four ENDOR lines associated with transitions within the A and E rotational states were followed in three planes of rotation. Large ENDOR shifts as measured by saturation of the high- and low-field parts of the EPR spectrum indicate that the rotation is rather slow. Sidebands due to mixing of A and E rotational states are expected for slow rotation and were observed in both the EPR and the EIE spectra. The ENDOR shifts and the sideband frequencies indicate a tunneling splitting between 40 and 60 MHz. Estimates of the barrier to rotation in both crystalline systems were calculated using cluster and single-molecule density functional theory methods, and the results are consistent with those obtained by analysis of the experimental results.

Formation of Interacting Spins on Flavosemiquinone and Tyrosine Radical in Photoreaction of a Blue Light Sensor BLUF Protein TePixD

Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.

One‐Electron‐Reduced and ‐Oxidized Stages of Donor‐Substituted 1,1,4,4‐Tetracyanobuta‐1,3‐dienes of Different Molecular Architectures

A series of monomeric and oligomeric donor-substituted 1,1,4,4-tetracyanobuta-1,3-dienes (TCBDs) with various topologies have been synthesized by means of thermal [2+2] cycloaddition between tetracyanoethylene (TCNE) and donor-substituted alkynes, followed by retro-electrocyclization. One-electron-reduced and -oxidized stages of the donor-substituted TCBDs were generated by chemical methods. The obtained radical anions and radical cations were studied by using electron paramagnetic resonance/electron nuclear double resonance (EPR/ENDOR) spectroscopy, supported by density functional theory (DFT) calculations. The extent of pi-electron delocalization in the paramagnetic species was investigated in terms of the EPR parameters. Despite favorable molecular orbital (MO) coefficients, the EPR results suggest that in radical anions the spin and charge are confined to the electron-withdrawing TCBD moieties on the hyperfine EPR timescale. The observed spin localization is presumably caused by an interplay between the nonplanarity of the studied pi systems, limited pi-electron conjugation, and very likely counterion effects. In radical cations, an analogous spin and charge localization confined to the electron-donating N,N-dialkylaniline moieties was found. In this case, an efficient electron delocalization is disabled by small MO coefficients at the joints between the donor and acceptor portions of the studied TCBDs.

Three Different Tyrosyl Radicals Identified in l-Tyrosine HCl Crystals upon γ-Irradiation: Magnetic Characterization and Temporal Evolution

High-frequency electron paramagnetic resonance (EPR) and X-band electron-nuclear double resonance (ENDOR) spectroscopies were used to investigate the effect of gamma-irradiation on single crystals of L-tyrosine hydrochloride at room temperature. The oxidation product is the tyrosyl radical formed by hydrogen abstraction from the phenolic group; interestingly, on freshly irradiated crystals, two tyrosyl radicals were identified, characterized by slightly different magnetic parameters. In particular, one of the two radicals, with a gxx value of 2.00621, has its phenoxyl oxygen strongly hydrogen-bonded to one or more donors; to our knowledge, this is the lower gxx value reported for tyrosyl radicals. These two oxidation radicals are found to evolve very slowly to a third, single more stable radical conformation. To interpret the experimental data, a possible molecular scenario is presented, where the process of radical formation can be seen as a hydrogen atom transfer or a proton-coupled electron transfer. These processes seem to be controlled by the specific network of hydrogen-bond interactions present in the crystal. The results are discussed in relation to their relevance for the interpretation of EPR spectra of tyrosyl radicals in biological systems.

Low-Temperature Interquinone Electron Transfer in Photosynthetic Reaction Centers fromRhodobacter sphaeroidesandBlastochloris viridis: Characterization of QB-States by High-Frequency Electron Paramagnetic Resonance (EPR) and Electron−Nuclear Double Resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.

The17O Hyperfine Interaction in V17O(H217O)52+and Mn(H217O)62+Determined by High Field ENDOR Aided by DFT Calculations

The 17O hyperfine interaction of the water ligands and the V=O oxygen in the vanadyl aquo complex and of the water ligands in the Mn2+ aquo complex in a frozen solution were determined by W-band (95 GHz) electron-nuclear double resonance (ENDOR). Orientation selective ENDOR spectra of the vanadyl complex exhibited two distinct signals assigned to the vanadyl oxygen and the water ligands. The assignment of the signals was done based on the orientation of the principal axis system of the hyperfine interaction and through comparison with the hyperfine interaction predicted by DFT calculations. The latter showed good agreement with the experimental values thus providing clear evidence that the vanadyl oxygen is exchangeable. The interaction of the vanadyl oxygen, especially its anisotropic part, was significantly larger than that of the water oxygens due to a relatively large negative spin density on the oxygen p orbitals. The 17O hyperfine interaction of the water ligand in the Mn2+ complex was found to be similar to that of the water ligand in the vanadyl complex and was in good agreement with earlier single-crystal data. Here, due to the large thermal polarization, it was also possible to determine the absolute sign of the hyperfine coupling by selecting different EPR transitions.

Unambiguous Determination of theg-Matrix Orientation in a Neutral Flavin Radical by Pulsed Electron−Nuclear Double Resonance at 94 GHz

The recent observation of photoinduced radical pairs comprising a flavin radical and an oxidized amino acid residue in various blue-light-sensitive proteins has highlighted the need to gain a more complete understanding of the electronic structure of flavin radicals. In particular, precise knowledge of the anisotropy of the Zeeman interaction quantified by the g-tensor is necessary for attaining an unambiguous identification of flavin radicals by electron paramagnetic resonance (EPR). In a recent study of a protein-bound neutral flavin radical, we have determined the principal values of the g-tensor using high-frequency/high magnetic field EPR performed at 360 GHz/12.8 T. However, in those experiments, the orientation of the principal axes of g could not be unambiguously established with respect to the molecular frame of the isoalloxazine moiety. In this contribution we resolve this ambiguity by pulsed electron-nuclear double resonance (ENDOR) at 95 GHz/3.5 T (W-band). At such high values of the microwave frequency and the magnetic field, the g anisotropy provides improved spectral resolution compared to an ENDOR experiment performed at conventional 9.5 GHz/0.35 mT (X-band). This enables one to utilize Zeeman magnetoselection to obtain single-crystal-like data from disordered samples in frozen solution. Experiments exploiting this orientation selection have allowed us to use the hyperfine coupling of the methyl protons at C(8alpha) of the isoalloxazine ring to determine the angle between the molecular frame and the principal axes of g. Quite surprisingly, the g-tensor in FADH* is not oriented as one would have expected for a 1,3-semibenzoquinone radical. For the latter, the X-axis of g commonly bisects the smaller angle between the two axes along the C=O bonds. In FADH*, the large spin density on N(5) and C(4a) apparently contributes to a significant (44 degrees ) reorientation of the g-tensor axes.

Structure of Radical Cations of Saturated Heterocyclic Compounds with Two Heteroatoms As Studied by Electron Paramagnetic Resonance, Electron−Nuclear Double Resonance, and Density Functional Theory Calculations

The radical cations of piperazine, morpholine, thiomorpholine, and thioxane were investigated by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy in a solid Freon matrix. Optimized geometry and magnetic parameters of the radical cations were calculated using a density functional theory (DFT)/Perdew-Burke-Ernzerhof (PBE) method. Both experimental and theoretical results suggest that all the studied species adopt chair (or distorted chair) conformations. No evidence for the boat conformers with intramolecular sigma-bonding between heteroatoms were obtained. In the cases of morpholine and thioxane, the oxygen atoms are characterized by relatively small spin populations, whereas a major part of spin density is located at N and S atoms, respectively. The thiomorpholine radical cation exhibits nearly equal spin population of N and S atoms. In most cases (except for thioxane), the calculated magnetic parameters agree with the experimental data reasonably well.

Enzyme Control of Small-Molecule Coordination in FosA as Revealed by31P Pulsed ENDOR and ESE-EPR

FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, which contains a phosphonate group. The active site of this enzyme consists of a high-spin Mn(2+) ion coordinated by endogenous ligands (a glutamate and two histidine residues) and by exogenous ligands, such as substrate fosfomycin. To study the Mn(2+) coordination environment of FosA in the presence of substrate and the inhibitors phosphonoformate and phosphate, we have used (31)P pulsed electron-nuclear double resonance (ENDOR) at 35 GHz to obtain metrical information from (31)P-Mn(2+) interactions. We have found that continuous wave (CW) (31)P ENDOR is not successful in the study of phosphates and phosphonates coordinated to Mn(2+). Parallel studies of phosph(on)ate binding to the Mn(2+) of FosA and to aqueous Mn(2+) ion disclose how the enzyme modifies the coordination of these molecules to the active site Mn(2+). Through analysis of (31)P hyperfine parameters derived from simulations of the ENDOR spectra we have determined the binding modes of the phosph(on)ates in each sample and discerned details of the geometric and electronic structure of the metal center. The (31)P ENDOR studies of the protein samples agree with, or improve on, the Mn-P distances determined from crystal structures and provide Mn-phosph(on)ate bonding information not available from these studies. Electron spin echo electron paramagnetic resonance (ESE-EPR) spectra have also been recorded. Simulation of these spectra yield the axial and rhombic components of the Mn(2+) (S = (5)/(2)) zero-field splitting (zfs) tensor. Comparison of structural inferences based on these zfs parameters both with the known enzyme structures and the (31)P ENDOR results establishes that the time-honored procedure of analyzing Mn(2+) zfs parameters to describe the coordination environment of the metal ion is not valid or productive.

Intermediates in P450 catalysis

Cytochromes P450 catalyse the insertion of one O2-derived oxygen atom in unactivated C-H bonds, and as such, are potent oxidants. A significant amount is known about the P450 catalytic cycle owing partly to the single heme group at the active site that provides spectroscopic handles in tracking various intermediates. A sophisticated array of electron paramagnetic, electron double nuclear resonance, and more traditional absorption spectroscopies have been able to identify key intermediates, while crystallography has defined the structure of the substrate-free, -bound, and oxy-complexes. What has remained elusive is the Fe(IV)=O intermediate, thought to be the active hydroxylating agent. Here, theory and especially density functional calculations have provided valuable insights.

Pulsed ENDOR Study of Water Coordination to Gd3+Complexes in Orientationally Disordered Systems

In orientationally disordered systems, the ENDOR (electron−nuclear double resonance) spectra of high-spin ions having weak crystal field interaction (cfi) contain nuclear transition lines (often overlapped) that belong to several different electron spin manifolds, and the transition lines for each manifold are distorted by the effect of the cfi. In this work, we have shown that, although the latter distortions can be quite considerable in a general case, the statistical distributions of the cfi parameters and orientations of the cfi axes in glassy samples result in ENDOR line shapes similar to the usual powder pattern determined by the hyperfine interaction only, which greatly simplifies their analysis. We have also shown that the two-dimensional Mims ENDOR technique can be used to disentangle the nuclear transitions that belong to different electron spin manifolds. The results of the analysis were applied to study the Gd3+ aquo complex and the Gd3+-based MRI contrast agent GdHPDO3A in frozen glassy water...

The electronic structure of the photoexcited triplet state of free-base (tetraphenyl)porphyrin by time-resolved electron-nuclear double resonance and density functional theory.

The photoexcited triplet states of free-base porphyrin (H(2)P) and free-base tetraphenylporphyrin (H(2)TPP) have been investigated by time-resolved electron paramagnetic resonance and electron-nuclear double resonance in a toluene glass at 80 K. Both the zero-field splitting parameters, D and E, and the proton A(zz) hyperfine coupling tensor components could be determined. D is about 13% larger in H(2)P than in H(2)TPP. In contrast, however, the A(zz) hyperfine coupling tensor components showed differences of less than 2%. To aid the understanding of these results, the electronic structures of H(2)P and H(2)TPP have been modeled using density functional theory. The geometrical structures of both molecules in their lowest triplet states were calculated using the Becke3 Lee-Yang-Parr composite exchange correlation functional and the 6-31G* basis set. Hyperfine couplings for these structures were calculated using the same functional but with the extended EPR-II basis set. These allow unambiguous assignment of the experimentally determined couplings. The theoretical values for H(2)P and H(2)TPP agree with the experimental values in that the presence of the phenyl groups has only a small effect on the unpaired electron spin-density distribution. The difference in sensitivity of the zero-field splitting parameters and the hyperfine couplings to mesophenyl substitution is discussed in terms of the wave functions of the four frontier orbitals of porphyrins introduced by Gouterman.

Electron paramagnetic resonance and electron–nuclear double-resonance study of Ti3+ centres in KTiOPO4

Electron paramagnetic resonance and electron–nuclear doubleresonance have been used to characterize four Ti3+centres in undoped crystals of potassium titanyl phosphate (KTiOPO4 or KTP).These 3d1defects (S = 1/2)are produced by ionizing radiation (either 60 kV x-rays or 355 nm photons from atripled Nd:YAG laser), and form when the regular Ti4+ions in the crystal trap an electron. Two of these trapped-electroncentres are only observed in hydrothermally grown KTP and the othertwo are dominant in flux-grown KTP. Both of the Ti3+ centresin hydrothermally grown crystals have a neighbouring proton (i.e. an OH−molecule). In the flux-grown crystals, one of the Ti3+centres is adjacent to an oxygen vacancy and the other centre istentatively attributed to a self-trapped electron (i.e. a Ti3+centre with no stabilizing entity nearby). Theg matrixand phosphorus hyperfine matrices are determined for all four Ti3+ centres, andthe proton hyperfine matrix is determined for the two centres associated with OH− ions. TheseTi3+centres contribute to the formation of the grey tracks often observed in KTPcrystals used to generate the second harmonic of high-power, near-infrared lasers.

Magnetic field compensation for field-cycling NMR Relaxometry in the ULF band

In setting up field-cycling experiments aimed to study physical phenomena in the low-field region, magnetic field contributions from external sources (earth’s field, environment, other magnets, etc.) become important. Indeed, a compensation of these contributions has successfully been used for the application of field-cycling methods to nuclear magnetic relaxation and double resonance experiments. This feature becomes relevant in samples where local fields are stronly averaged due to motional narrowing, on the ground that relaxation experiments can therefore be extended to lower fields. Compensation of external contributions is also crucial for the study of kinky processes related to internal local fields. In this article we outline NMR field-cycling experiments aimed to detect and quantify the external net magnetic field sensed by the spin-system. Both parallel and normal components with respect to the high-field Zeeman axis can be determined separately by using different experimental protocols.

Alanine Radicals. 2. The Composite Polycrystalline Alanine EPR Spectrum Studied by ENDOR, Thermal Annealing, and Spectrum Simulations

Radiation-induced free radical formation in the amino acid l-α-alanine has been studied using powder and single-crystal X-, K-, and Q-band electron paramagnetic resonance (EPR) spectroscopy, X-band powder electron−nuclear double resonance (ENDOR), thermal annealing, and EPR spectrum simulations. The spectra obtained after room temperature irradiations are composite, consisting of resonances from mainly three radicals denoted R1, R2, and R3. R1 is the well-known, stable room-temperature species formed by deamination from a protonated alanine anion radical. On the basis of simulations of EPR spectra obtained at X-, K-, and Q-bands, the room-temperature EPR spectrum seems to consist of about 55% of R1. Upon thermal annealing, the R1 resonance disappears faster than those of the other two components. The R2 species is presumably formed in the oxidative chain of radiation-induced events by net H-abstraction from the central alanine carbon atom. Q-band EPR was used to determine the g-tensor of R2. This species contributes about 35% to the resonance recorded at room temperature. Upon thermal annealing this radical decays slower than R1, resulting in the predominance of R2 in spectra obtained after prolonged warming at 480 K. Powder ENDOR was used to verify that the dominating species remaining after thermal annealing at this temperature indeed is R2 and not a successor species of either of the room-temperature radicals. The R3 species was previously assigned to an N-deprotonated version of R2 being additionally protonated at the carboxyl group. Detailed spectral data for this resonance are missing but a set of parameters based on available data and otherwise estimated using literature values for similar products was constructed. Simulations indicated that 5−10% of the room-temperature resonance could be ascribed to R3. R3 is more heat-resistant than the R1 and R2 radicals, and after prolonged annealing at 480K it was estimated that the resulting resonance consisted of about 51% R2 and 43% R3. The remaining part (about 6%) of the resonance was due to R1. These numbers must, however, be considered as tentative because of the lack of precise spectral data for R3.

Pulsed ENDOR Studies of Short-Lived Spin-Correlated Radical Pairs in Photosynthetic Reaction Centers

Time-resolved electron−nuclear double resonance (TR-ENDOR) performed on the spin-correlated radical pair in frozen solution samples of PSI shows a superposition of the ENDOR lines expected for the individual radicals and . Presented here are TR-ENDOR spectra taken over a range of EPR field positions. The spectra are unusual because they show both absorptive and emissive contributions and a strong dependence on the field. The nature of the spectra is explored using the theory of spin-correlated radical pairs. It is found that the electron spin polarization in the radical pair is responsible for the absorptive/emissive nature of the TR-ENDOR spectra and that the mixed electronic eigenstates give rise to transitions in addition to those usually found in ENDOR. Computer simulations based on a model containing two coupled electrons and a single spin-1/2 nucleus are able to reproduce the spectra and their field dependence. It is also shown that TR-ENDOR spectra from a spin-correlated radical pair are sensitiv...

The Process of Mn(II) Incorporation into Aluminophosphate Zeotypes through High-Field ENDOR Spectroscopy and DFT Calculations

High-field EPR and pulsed electron−nuclear double resonance (ENDOR) spectroscopies were used to investigate the formation of Mn−AlPO4-11, Mn−AlPO4-5, and Mn−SAPO-5. Samples recovered from reaction mixtures quenched at different times were subjected to EPR, ENDOR and X-ray diffraction (XRD) measurements, and the variations in the 31P and 1H hyperfine couplings, which are sensitive probes to the Mn−P interaction and the Mn(II) hydration, respectively, were followed. The intensity of the 1H ENDOR signal decreased with reaction time, showing that the amount of both water ligands and solvent water in the Mn(II) vicinity decreased. A relatively large isotropic 31P hyperfine coupling (Aiso(31P) ≈ 7 MHz), confirming the formation of Mn(II) framework sites, was found in all final products, whereas a smaller Aiso(31P), 4−5 MHz, was detected in samples quenched at early stages of the reaction. The latter was assigned to Mn(II) incorporated into a network of disordered aluminophosphate precursors. These precursors are formed prior to the detection of an XRD pattern, and are gradually transformed to the final three-dimensional crystalline structures. The changes in Aiso(31P) were attributed to transformations occurring both in the bonding topology and in the coordination sphere of Mn(II), where water ligands are gradually replaced by −O−P linkages. This interpretation was supported by the decrease in the intensity of the 1H ENDOR signals, and by a series of DFT cluster model optimizations on intermediates of the form [Mn(H2O)x(OP(OH)3)y]2+, where x + y = 6, 5 or 4, followed by calculations of hyperfine coupling constants. Although the theoretical hyperfine values were overestimated with respect to the experimental ones, a satisfactory correlation was found between the trends within the calculated Aiso(55Mn,31P), and the experimental trends observed during the molecular sieves formation.