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Troponin (Tn) is a protein that consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT), and Tn controls cardiac muscle contraction by calcium ion binding and phosphorylation. The Ca2+-binding site is the E–F hand motif (C helix–loop–D helix) in the N-terminal domain of TnC, and the structural transition induced by Ca2+ is the opening of these helices and the interaction with TnI, probably at the A and B helices. In this paper, we studied structural changes in the TnC–TnI binary complex on Ca2+ binding by double quantum coherence (DQC) distance measurements. We used a binary complex of the cardiac troponin C and I (cTnC and cTnI) complexes, chose four positions of nitroxide spin label at helices A, B, C, and D in the N-terminal domain and chose the E helix in the C-terminal domain as the reference position to study the structural changes on Ca2+ addition. The label positions were (A22C/S98C), (M47C/S98C), (Q58C/S98C), and (C84/S98C) for the A, B, C, and D helices, respectively. The effects of phosphorylation of the cardiac-specific N-terminal region of cTnI were studied using a phosphomimetic cTnI mutant. Analysis of the modulation of the DQC echo signals provided the distribution of the spin–spin distance. The distances averaged over the distribution showed that the labels on the A, B, and C helices decreased, i.e., moved to the E helix, on Ca2+ binding, while the distance of the label on the D helix showed almost no change. Shoulders and/or small separate peaks were observed in the shape of the distribution and were analyzed as the sum of a few Gaussian functions. The Gaussian functions were grouped into two components, components 1 and 2, at the longer and shorter distances, respectively, separated by 0.7–1.5 nm. The fractions of component 2 were ca. 0.1–0.2 in the Ca2+-free state and increased by 0.2–0.3 on Ca2+ addition, suggesting that the increase in component 2 is related to physiological control of cardiac muscle contraction. The phosphomimetic-modification effects on the Ca2+-induced changes of the fraction of components and the distances of the C- and D-helix labels are small. On the other hand, in the A and B helices, there are significant effects on the Ca2+-induced changes in the distances of the components. The different behaviors of A/B and C/D helices support the current model of the phosphorylation effects in which both N-terminal region and regulatory domain of cTnI interact with the A and B helices of cTnC.

Polymer chain morphology and its response to annealing were investigated in terms of g-tensor anisotropy in poly(3-hexylthiophene) (P3HT)/fullerene blend films fabricated by dip coating. The g-anis...

Mobius aromatic molecules have attracted great attention as new functional materials because of their π-orbital cyclic conjugations lying along the twisted Mobius topology. To elucidate the electronic character of the lowest excited triplet (T1) state of a Mobius aromatic [28]hexaphyrin, we employed a time-resolved electron paramagnetic resonance (TREPR) method with applied magnetophotoselection measurements at 77 K. Analyses of the EPR parameters have revealed that the T1 state possesses intramolecular charge-transfer (CT) character together with local excitation character residing at one side in the Mobius strip ring. We have also demonstrated that the CT character between orthogonal unpaired orbitals triggers quick triplet deactivation by spin-orbit coupling. This deactivation can be an important barometer to represent the “antiaromaticity” because of a connection between the orthogonal CT character and instability by a weakened spin-spin exchange coupling in the T1 state.

We studied the effects of isotopic substitution (namely, deuterium substitution in solvents and 15N substitution in a spin label) on improving the sensitivity of distance measurements based on double quantum coherence (DQC) electron paramagnetic resonance (EPR) using cardiac troponin C. Deuteration of solvents increased the phase memory time, Tm, by a factor of only 1.7, whereas the DQC amplitude increased significantly at long pulse intervals. The two-pulse echo amplitude was 1.7 times larger for the 15N spin label than it was for the 14N label because of the limited bandwidth of the microwave pulses. In the case of DQC, the echo is 2.7 times greater for the 15N label than it is for the 14N label due to the accumulated effects of the finite bandwidth. The combination of the two isotopic substitutions is expected to increase the sensitivity by about a factor of 6 when measuring distances longer than 4–5 nm.

The lowest excited triplet state of free-base 5,10,15-trispentafluorophenylcorrole (H3Cor) was studied in rigid glass by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. Triplet sublevels were experimentally determined by using magnetophotoselection and liquid crystal. Quantum chemical calculations of zero-field splitting parameters D and E were used for the sublevel assignment. The out-of-plane sublevel Tz was found to be the lowest; namely, D is positive for H3Cor, in contrast to previous reports. The origin of the difference is discussed in detail. Preliminary TR-EPR experiments on rhodium corrole, accompanied by the quantum chemical calculations, emphasize the important contribution of spin-orbit couplings.

In alkaline aqueous solutions, 3,4-diaminobenzoate (H 2 ( 2 L PDA ) − ) reacts with Pt II to form a 1:2 (Pt:L) complex that intensely absorbs near-infrared (NIR) light at 713 nm ( e = 8.0 × 10 4 M −1 cm −1 ). The absorption disappeared at pH II ( 1 L ISQ ) 2 ] 2− , containing the analogous phenylenediamine ligand [( 1 L ISQ ) 2− = o -diiminobenzosemiquinonate radical], the complex can be formulated as [Pt II ( 2 L ISQ ) 2 ] 2− . The assignment of the entity was consistent with the redox and spectroelectrochemical behaviors and electronic spin resonance (ESR) spectroscopy. First, one-electron oxidation of [Pt II ( 2 L ISQ ) 2 ] 2− formed an ESR-silent complex assignable to the dimeric complex [{Pt II ( 2 L ISQ )( 2 L IBQ )} 2 ] 2− [( 2 L IBQ ) = o -iminobenzoquinone form] in which the two radical centers at ( 2 L ISQ ) 2 - were antiferromagnetically coupled. Second, the one-electron reduced complex of [Pt II ( 2 L ISQ ) 2 ] 2− exhibited an ESR signal attributed to [Pt II ( 2 L ISQ )( 2 L PDA )] 3− ; 34% of the electronic spin was located at the Pt II center rather than on the ( 2 L ISQ ) 2 - moiety. The pH-responsive switching-off of the NIR absorption was thus rationally explained by oxidation of [Pt II ( 2 L ISQ ) 2 ] 2− to [{Pt II ( 2 L ISQ )( 2 L IBQ )} 2 ] 2− by the increase of the rest potential of the solution in the lower pH region.

Multiple-frequency (X-band and W-band) time-resolved electron paramagnetic resonance spectra of Zn-tetraphenylporphyrin (ZnTPP) triplet states coordinated to two different stable nitroxide moieties are presented and discussed. The position of the pyrimidyl nitroxide relative to the ZnTPP plane is the only structural change made between the two complexes, from ortho to para. This changes the angle between the interacting orbitals of the three-spin system with only a minor change in the distance between the unpaired electron of the nitroxide and the TPP ring system. Changes in the electron spin polarization patterns at the two different frequencies of observation are discussed in terms of a radical–triplet pair spin-state mixing model, including the intersystem crossing processes. It is determined that the inclusion of an additional spin-selective relaxation process to the computational model for the spectral shapes gives adequate reproduction of the experimental results using the same parameters at each frequency.

The synthesis of [14]triphyrin(2.1.1) compounds is described. In contrast with conventional subporphyrins, which consistently contain a central boron atom, free-base heteroaromatic compounds can be formed. A modified Lindsey method was used to prepare a range of different [14]triphyrins(2.1.1) in yields of up to 35% based on the reaction of diethylpyrrole (1a) and fused pyrroles of bicyclo[2.2.2]octadiene (BCOD) (2a-e) and dihydroethanonaphthalene (4a) with various aryl aldehydes. The concentration of BF(3)·OEt(2) catalyst plays the key role in determining the yield of the [14]triphyrin(2.1.1) macrocycle relative to the conventional tetrapyrrole porphyrin product. Retro-Diels-Alder reactions of 2a-e and 4a result in the formation of [14]tribenzotriphyrin (2.1.1) (3a-e) and [14]trinaphthotriphyrin(2.1.1) (5a). The effects of exocyclic ring annulation on the electronic structure are examined in detail based on optical spectroscopy, theoretical calculations, and electrochemical measurements. The availability of free-base compounds enables the formation of [Re(I)(CO)(3)(triphyrin)] (6a) and [Ru(II)(CO)(2)Cl(triphyrin)] (7a) complexes based on a modified retro-Diels-Alder reaction. X-ray structures are reported for 4a and 6a.

The oxidation of a redox-active tyrosine residue Y(Z) in photosystem II (PSII) is coupled with proton transfer to a hydrogen-bonded D1-His190 residue. Because of the apparent proximity of Y(Z) to the water-oxidizing complex and its redox activity, it is believed that Y(Z) plays a significant role in water oxidation in PSII. We investigated the g-anisotropy of the tyrosine radical Y(Z)(•) to provide insight into the mechanism of Y(Z)(•) proton-coupled electron transfer in Mn-depleted PSII. The anisotropy was highly resolved by electron paramagnetic resonance spectroscopy at the W-band (94.9 GHz) using PSII single crystals. The g(X)-component along the phenolic C-O bond of Y(Z)(•) was calculated by density functional theory (DFT). It was concluded from the highly resolved g-anisotropy that Y(Z) loses a phenol proton to D1-His190 upon tyrosine oxidation, and D1-His190 redonates the same proton back to Y(Z)(•) upon reduction.

The electronic structure of a water-soluble near-infrared (NIR)-absorbing complex was determined to be singlet diradical [PtII(LISQ)2]2– [(LISQ)2–· = o-diiminosulfobenzosemiquinonate radical] by X-ray diffraction and 1H NMR spectroscopy. Cyclic voltammetry (CV) showed redox reactions of [PtII(LISQ)2]2– to give [PtII(LISQ)(LIBQ)]– (E1 = –0.26 V vs. Fc+/Fc) and [PtII(LISQ)(LPDI)]3– (E2 = –1.46 V) [(LIBQ)– = o-diiminosulfobenzoquinonate, (LPDI)3– = o-phenylenediiminesulfonate]. The monoradical structure of the latter was confirmed by electron paramagnetic resonance (EPR) spectroscopy. Splitting of the reduction wave of [PtII(LISQ)(LIBQ)]– in CV and the weak EPR signal suggested the formation of a dimer. Because the rest potential of the solution exceeds Epa1 at pH 4.0, the decrease in the NIR absorption at pH

The lowest excited triplet (T(1)) states of magnesium and zinc tetraphenylporphines (MgTPP and ZnTPP) were studied by time-resolved (TR) high-frequency/high-field W-band electron paramagnetic resonance (hf-EPR) spectroscopy in rigid glasses at low temperatures. Inspections of the TR-hf-EPR spectra of the spin-polarized triplets revealed that the zero field splitting (ZFS) parameters, D and E, for MgTPP and ZnTPP triplets were nearly the same. At the same time, their g-tensors were found to be different. These results are interpreted quantitatively in terms of spin-orbit couplings (SOCs) and angular momenta among the excited states, giving a magnitude of SOC in the T(1) state of ZnTPP. For the first time, both the TR-hf-EPR spectra and corresponding time profiles were acquired on the ZnTPP's triplet at room temperature in liquid paraffin solution with the populations of the electron spin states being in Boltzmann equilibrium. Because of relatively fast paramagnetic relaxation in rotating triplet at room temperature, the spectra and time profiles were free from the effects of microwave saturation that allowed for the direct measurement of the absolute intersystem crossing ratios P(x):P(y):P(z) 0.085:0.085:0.83. All of these results have demonstrated advantages and new perspectives of the W-band EPR spectroscopy.

Four different types of polynuclear manganese(II) compounds with bhnq2− bridges were obtained from the reaction of Mn(CH3COO)2·4H2O with the flexible hinge-like ligand H2bhnq (H2bhnq = 2,2′-bi(3-hydroxy-1,4-naphthoquinone)) by controlling the reaction solvent: [Mn3(bhnq)3(H2O)2]·10.5H2O (1), [Mn2(bhnq)2(DMSO)4]·2DMSO·CHCl3 (2), {[Mn(bhnq)(H2O)2]·2H2O}n (3), and {[Mn(bhnq)(DMF)2]}n (4). All complexes are neutral with the same Mn/bhnq 1:1 formulation ratio. Compounds 1 and 2 are discrete systems, while compounds 3 and 4 are one-dimensional ones. All compounds were characterized by X-ray diffraction and show different coordination modes for the bhnq2− ligand.

A highly time-resolved high-frequency/high-field W-band electron paramagnetic resonance (EPR) (ν ~ 94 GHz) is a powerful technique to determine small g anisotropies of transient paramagnetic species. We applied this method to studies of the lowest excited triplet (T1)3 ππ* states in metal complexes such as a platinum (Pt) diimine complex and metal (Zn and Mg) porphines in rigid glasses. From the analyses of time-resolved EPR spectra, g anisotropies were obtained as g z = 2.0048, g x = g y = 2.0035 for Pt(b-iq)(CN)2 (b-iq = 3,3′bi-isoquinoline) and g z = 1.9968, g x = g y = 2.0022 for zinc tetraphenylporphine (ZnTPP). No measurable anisotropies were found for magnesium (Mg) TPP. The g values of the Pt complex are larger than g e (=2.0023, g value of free electron) and that g z of ZnTPP is smaller than g e. These results were interpreted in terms of the nature of the perturbed states: the higher triplet ππ′* state mixes with T1(ππ*) via spin–orbit coupling in ZnTPP. In contrast, the higher triplet dπ* state is involved in this coupling for the Pt complex. Thus, the nature of the perturbed state can be distinguished from the anisotropic g values of the T1(ππ*) state.

We reported determination of the loaded quality factor (Q) of highly overcoupled (dielectric, loop-gap, and cavity) resonators used in time-domain electron paramagnetic resonance. We introduced a microwave absorber into resonators and achieved critical-coupling. Due to the deep “Q-dip” of critical-coupling, we can easily determine the loaded Q as low as 10. The loaded Q of resonators with and without the microwave absorber was examined under various overcoupling conditions. We found that the radiation Q (Q r) can be calculated from the loaded Q of the resonator that contains the microwave absorber. We proposed a simple model that represents the loaded Q of the overcoupled resonator in terms of two parameters, Q 0 and Q r. Q 0 is the effective unloaded Q of the resonator determined for the critically coupled resonator without the microwave absorber and is independent of a degree of coupling. The model can be applied to overcoupling in which the coupling parameter (Q 0/Q r) is in the range of 1 to ca. 20.

Ordered microporous carbon that was synthesized in the nanochannels of zeolite Y is characterized by an extremely large surface area, surprisingly uniform micropores and a long-range periodicity originating from the parent zeolite. However, the molecular structure of this zeolite templated carbon (ZTC) has been completely unknown. In this study, an attempt was made to construct a possible molecular model for ZTC. The proposed model is made up of buckybowl-like nanographenes assembled into a three-dimensionally regular network, which reflects all the experimental results obtained from Raman spectroscopy, electron energy-loss spectroscopy, and previously obtained analyses with several other means. Starting from this idealized model, possible forms of defects that would be included in the actual ZTC were also investigated. Moreover, the amount and the type of oxygen functional groups were analyzed and, as per the results, some functional groups were bound to the edge sites of each buckybowl unit in the molecular model. The elemental composition, pore curvature, pore size and pore volume and surface area estimated from such oxygen-containing model agree well with the corresponding experimentally obtained results. The present model can be considered as a reasonable starting point for future refinements of the structure of this quite novel carbon material.

We propose a novel formation model of an intermolecular hydrogen bond between radicals and a diamagnetic matrix in the condensed phase. W-band and multi-frequency ESR studies revealed that the poly...

Neuronal PAS protein 2 (NPAS2), a heme-binding transcriptional regulatory factor, is involved in circadian rhythms. Period homologue (Per) is another important transcriptional regulatory factor that binds to cryptochrome (Cry). The resultant Per/Cry heterodimer interacts with the NPAS2/BMAL1 heterodimer to inhibit the transcription of Per and Cry. Previous cell biology experiments indicate that mouse Per2 (mPer2) is also a heme-binding protein, and heme shuttling between mPer2 and NPAS2 may regulate transcription. In the present study, we show that the isolated PAS-A domain of mPer2 (PAS-A-mPer2) binds the Fe(III) protoporphyrin IX complex (hemin) with a heme:protein stoichiometry of 1:1. Optical absorption and EPR spectroscopic findings suggest that the Fe(III)-bound PAS-A-mPer2 is a six-coordinated low-spin complex with Cys and an unknown axial ligand. A Hg (2+) binding study supports the theory that Cys is one of the axial ligands for Fe(III)-bound PAS-A-mPer2. The dissociation rate constant of the Fe(III) complex from PAS-A-mPer2 (6.3 x 10 (-4) s (-1)) was comparable to that of the heme-regulated inhibitor (HRI), a heme-sensor enzyme (1.5 x 10 (-3) s (-1)), but markedly higher than that of metmyoglobin (8.4 x 10 (-7) s (-1)). As confirmed by a Soret absorption spectral shift, heme transferred from the holo basic helix-loop-helix PAS-A of NPAS2 to apoPAS-A-mPer2. The Soret CD spectrum of the C215A mutant PAS-A-mPer2 protein was markedly different from that of the wild-type protein. On the basis of the data, we propose that PAS-A-mPer2 is a heme-sensor protein in which Cys215 is the heme axial ligand.

EPR spectra of the excited quartet and doublet molecular states of (tetraphenylporphinato)zinc(II)covalently bounded to 3-( N -nitronyl-notroxide) pyridine stable radical are modeled in terms of the spin-Hamiltonian given by the sum of the contributions from the radical and triplet moieties, and the interaction between them. The later is represented by anisotropic point dipolar and isotropic exchange electron spin–spin interactions. It is shown that the high field (W-band) EPR spectra depend on energy separation between the electronic doublet (D) and quartet (Q) states. This dependence was utilized to estimate the upper limit of the intensity of exchange interaction between the radical and porphyrin moieties.

Product analyses and nanosecond time-resolved spectroscopy on laser flash photolysis were studied for the photoinduced electron-transfer reaction of 3,4-di(alpha-styryl)furan (6a). A combination of these results, kinetic, density functional theoretical (DFT), and time-dependent DFT analyses enabled assignment of the absorption to the tetramethyleneethane (TME)-type radical cation (7a*+, lambda(max) = 392 nm) and the corresponding singlet biradical ((1)7a**, lambda(max) = 661 nm). These two intermediates were mechanistically linked to each other with a facile back electron-transfer reaction. The present studies provide a new method for the generation of aryl-substituted TME-type intermediates.

A doubly protonated hydrochloride salt of a saddle-distorted dodecaphenylporphyrin (H 2 DPP), [H 4 DPPP]Cl 2 , forms a porphyrin nanochannel (PNC). X-ray crystallography was used to determine the structure of the molecule, which revealed the inclusion of guest molecules within the PNC. Electron-donating molecules, such as p-hydroquinone and p-xylene, were selectively included within the PNC in sharp contrast to electron acceptors, such as the corresponding quinones, which were not encapsulated. This result indicates that the PNC can recognize the electronic character and steric hindrance of the guest molecules during the course of inclusion. ESR measurements (photoirradiation at 2> 340 nm at room temperature) of the PNC that contains p-hydroquinone, catechol, and tetrafluorohydroquinone guest molecules gave well-resolved signals, which were assigned to cation radicals formed without deprotonation based on results from computer simulations of the ESR spectra and density functional theory (DFT) calculations. The radicals are derived from photoinduced electron transfer from the guest molecules to the singlet state of H 4 DPP 2+ . Transient absorption spectroscopy by femtosecond laser flash photolysis allowed us to observe the formation of 1 (H 4 DPP 2+ )*, which is converted to H 4 DPP +. by electron transfer from the guest molecules to 1 (H 4 DPP 2+ )*, followed by fast disproportionation of H 4 DPP +. , and charge recombination to give diamagnetic species and the triplet excited state 3 (H 4 DPP 2+ )*, respectively.

The coordination of small molecules and ions at the sixth position of ferric myoglobins (Mb) from the heart muscle of two species of shark,Lamna ditropis (LD) andPrionace glauca (PG), was studied in frozen solution by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. The results were compared with those of horse heart Mb (EQMb). From our preliminary studies of the absorption spectra and partial amino acid sequences of PGMb and LDMb, we expected PGMb to have glutamine (Gln E7) in place of the distal His E7 that is highly conserved in most animals and present in LDMb. Here, we have used proton ENDOR to show that a water molecule coordinates to the ferric ion in both LDMb and PGMb at pH 6.3. The observed proton hyperfine coupling constant of the coordinating water molecule of PGMb is smaller than those of LDMb and EQMb, which suggests weaker coordination of the water molecule in PGMb, consistent with the absent distal His (E7). Coordination of several exogenous ligands at the distal site was examined by EPR. PGMb showed a coordination behavior different from that of LDMb and EQMb for 1-methylimidazole, 4-methylimidazole and SCN−, suggesting that its distal site is less sterically hindered. On the basis of the ENDOR and EPR results, we assigned the distal residues of PGMb and LDMb as Gln and His, respectively.

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. We report the Raman spectra for the dark state of PYP whose chromophore is isotopically labeled with 13C at the carbonyl carbon atom or at the ring carbon atoms. Spectra have been also measured with PYP in D2O where the exchangeable protons are deuterated. Most of the observed Raman bands are assigned on the basis of the observed isotope shifts and normal mode calculations using a density functional theory. We discuss the implication for the analysis of the infrared spectra of PYP. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in PYP by vibrational spectroscopy.

Magnetic control over excited states of molecules presents interest for many applications. Here we show for the first time that visible room temperature phosphorescence in multichromophoric donor-acceptor systems can be modulated by weak magnetic fields (1 T) via magnetic field effects (MFE) on the spin dynamics in photogenerated radical pairs (RPs). The studied compounds comprise Pt porphyrin (PtP)-Rosamine B (RosB) dyads, which possess strong visible absorption bands and phosphoresce at room temperature. The observed MFE is unique in that it occurs upon direct excitation of the PtP in the dyads, whereby ultrafast quantitative formation of the local PtP triplet state precedes the occurrence of radical intermediates. A model explaining the effect is proposed, which is based on reversible electron transfer between the local triplet state and a long-lived RP. External magnetic field modulates spin dynamics in the RP, affecting contribution of the singlet RP recombination channel and thereby influencing phosphorescence.

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

A computational model to simulate electron spin polarization in the three-spin-1/2 system composed of the molecular excited triplet state of (tetraphenylporphinato)zinc(II) (ZnTPP) and the doublet ground state of the 3-(N-nitronyl-notroxide) pyridine (3-NOPy) stable radical is proposed. The model is based on numerical solutions of the stochastic Liouville equation for the diffusively rotating system where the magnetic dipolar, isotropic Heisenberg exchange, and anisotropic Zeeman electron spin interactions are taken into account in a full measure, whereas the intersystem crossing processes between the singlet and triplet states of ZnTPP are considered in terms of kinetic equations for the relevant spin density matrices. Additional longitudinal and transversal paramagnetic relaxation caused by relative rotation motions of the ZnTPP and 3-NOPy moieties is taken into consideration in the form of the generalized relaxation operator.

Bis-(quinoline-TEMPO)-yttrium-tetraphenylporphyrin complexes having potassium and silver counter cations have been studied by cw-EPR in their ground states and by time-resolved (TR) EPR and two-dimensional (2D) nutation spectroscopy in the excited states after laser excitation. Simulations of the TR-EPR spectra at 30 K and at room temperature were made, yielding zero-field splitting parameters D and E, and g values. The parameters obtained together with the nutation frequencies clearly assigned the excited states of the bis-radical complexes having counter cations of potassium and silver as quartet and quintet states, respectively. This result is explained by modulation of radical–triplet interactions by the counter ion due to different sizes of metal ions.

The interplanar interactions for fully overlapped (FO) and partially overlapped (PO) carbazole dimers were investigated using zero field splitting (ZFS) parameters D and E in the excited triplet (T1) states. The observed D value decreases from the monomer to the dimers due to electron delocalization in the T1 dimers. The highest electron delocalization was observed within the two planes of the FO dimer. The interplanar interactions were analysed in terms of exciton (EX) and charge resonance (CR) types interactions by comparing the D values of monomer and the dimers. It is found that about 90% of excitation is delocalized over the two carbazole units in the T1 state of the FO dimer whereas for PO this contribution is 40%. In a model system, DCR and ECR were calculated using the two approximations, point charge and half point charge, for the evaluation of the ZFSs obtained.

Multifrequency (X-, Q-, and W-band) electron spin resonance (ESR) spectroscopy has been used to characterize the phenoxyl radical produced from alpha-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone, which is a new spin-trapping reagent. The X-band measurement did not resolve the powder-pattern ESR spectrum. Because of its higher resolution with g value, the Q-band ESR study revealed that the g factor has an axial-like symmetry and that the observed hyperfine structure in the Z-direction is caused by the nitrogen nucleus at the para-position. Furthermore, the results of the W-band ESR experiment more clearly distinguished the perpendicular components from the parallel component, resolving the perpendicular components into x and y components. The X-band powder spectrum was similar to the X-band ESR spectrum of the radical in a frozen solution of toluene. The computer simulation spectra performed using the obtained parameters fitted the experimental spectra well. A comparison of the amplitude of g( perpendicular)(gx, gy) with that of gz showed that the unpaired electron is delocalized over the pi-conjugated framework. Considering the hyperfine coupling constant, it was concluded that about 16% of the unpaired electron distributed over the nitrogen nucleus at the para-position. This study thus showed the significant potential of a multifrequency ESR approach to a powder sample radical in terms of its high resolution with g value.

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50--Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.

Double coaxial carbon nanotubes with nitrogen (N)-doped and boron (B)-doped multiwalls possess composite Raman characteristics, originating not only from the outer N-doped but also from inner B-doped layers. Both N and B dopings result in substantial shifts of the characteristic D band and G band of sp(2) carbon constituting nanotube walls but in different ways. The downshift of the G band is correlated with the decreases of electrical resistivity of carbon nanotubes regardless of N or B doping.

Some of 1,6-(N-substituted) aza-[60] fulleroids (1) and their corresponding photochemical reaction products, 1,2-(N-substituted) aziridino-[60] fullerenes (2) showed peculiar transient absorption peaks depending on the N-substituents. Although these bands are considered to concern with very slow photochemical rearrangements 1 → 2 in the excited triplet state, no definitive evidence has been obtained yet. We report on the character of the excited triplet states in these compounds by means of a time-resolved electron paramagnetic resonance technique. Definitive evidence on the involvement of intra-molecular charge transfer states is shown in this Letter.

Influence of axial ligands, MeCN, H2O, py, and piperidine (pip), on distortion of (2,3,5,10,12,13,15,20-octaphenylporphinato)chromium(III), [Cr(OPP)]+, was investigated by X-ray crystallography and UV–vis and ESR spectroscopies. In crystal structures of [Cr(OPP)(MeCN)(H2O)]ClO4 · MeCN and [Cr(OPP)(H2O)2]ClO4 · 3THF, the OPP2− ligand had a planar structure. On the other hand, crystal structures of [Cr(OPP)(pip)2]ClO4 · 2CH2Cl2 and [Cr(OPP)(py)2]ClO4 exhibited a waved structure and a saddle-shaped structure with ruffling, respectively. In the UV–vis spectrum of [Cr(OPP)(py)2]ClO4 in CH2Cl2, the large red shift of the Soret band was observed. Furthermore, the small D value of 0.10 cm−1 was obtained for [Cr(OPP)(py)2]ClO4 from the ESR spectrum in frozen 1,2-dichloroethane solution at 5 K. These results suggest that the OPP2− ligand is distorted both in solid and in solution, and that the axial ligand would exert some effects on the porphyrin distortion.

The blue light receptor photoactive yellow protein (PYP) displays a photocycle that involves several intermediate states. Here we report resonance Raman spectroscopic investigations of the short-lived red-shifted intermediate denoted PYP(L). We have found that the Raman bands of the carbonyl C=O stretching mode nu(11) as well as the C=C stretching mode nu(13) for the chromophore can be resolved into two peaks, and the ratio of the two components varies as a function of pH with pK(a) approximately 6. The isotope effects on the resonance Raman spectra have confirmed a deprotonated cis-chromophore for the two components. The results indicate the presence of two conformations in the active site of PYP(L). The normal coordinate calculations based on the density functional theory provide a structural model for the two conformations, where the low pH form is possibly an active structure for the protonation reaction generating a following intermediate in the photocycle.

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --Ala, Tyr42 --Phe, Glu46 --Gln, and Thr50 --Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.

The electron paramagnetic resonance (EPR) signals of photoexcited quartet (Q1) states for zinc(II) tetra-tert-butyl-phthalocyanine (ZnPc) ligated by 3- and 4-(N-nitronyl-nitroxide) pyridine radicals (3-NOPy, 4-NOPy) were observed in toluene solution at room temperature by means of X-band (9.4 GHz) time-resolved EPR (TREPR) spectroscopy. Theg values of Q1 in the ZnPc-3-NOPy and ZnPc-4-NOPy complexes were found to beg=2.0025 andg=2.0036, respectively. The obtainedg value (2.0036) for ZnPc-4-NOPy is in good agreement with the value (g=2.0037) of the Q1 state calculated under the strong-exchange limit. Theg value (2.0025) is just an average of the Q1 and D1 (g=2.0013) states for ZnPc-3-NOPy. Theg value of Q1 for zinc(II) meso-tetraphenylporphine (ZnTPP) ligated by 3-NOPy showed a slight shift (g=2.0027) at X-band and no shift (g=2.0031) at W-band from the calculatedg value (g=2.0031) (J. Fujisawa, Y. Iwasaki, Y. Ohba, S. Yamauchi, K. Koga, S. Karasawa, M. Fuhs, K. Mobius, S. Weber, Appl. Magn. Reson. 21, 483–493, 2001). These changes in theg value were found to originate from an averaging of the TREPR spectra over the Q1 and photoexcited doublet (D1) states via a fast intersystem crossing (ISC) process. The ISC rates between these two states were estimated by means of numerical calculations with the modified Bloch equations as 1.2·108 and 6·107 s−1 for the ZnTPP-3-NOPy complex at the X- and W-bands, respectively. The lower limit of the ISC rate was obtained as 109s−1 for the ZnPc-3-NOPy complex and the higher limit was found to be 3.1·108 s−1 for the ZnPc-4-NOPy complex.

Time-resolved EPR spectra of the lowest excited triplet state of boron subphthalocyanine chloride have been measured in toluene at various temperatures (5−360 K). On the basis of the observed and simulated spectra, electronic structure and molecular motion of the triplet state (1T) were analyzed, both in solid and fluid solution. The simulations were carried out using a model, which considers a temperature-dependent exchange between sites having different zero-field splitting (ZFS) parameters and molecular orientations. The ZFS parameter, D, was nearly the same at all temperatures examined. At very low temperatures, below 20 K, the spectrum was analyzed by a static model. At 40−120 K, two conformers with different ZFS parameter, E, have been found. The population ratio between the two conformers showed strong temperature dependence. These conformers were attributed to Jahn−Teller states and were identified by their different ZFS parameters. The exchange rate and activation energy of the conformers were co...

Recent advances in computational chemistry have significantly enhanced the capability of vibrational spectroscopy such as a Raman technique. It is now possible to simulate the Raman spectrum for large molecules such as proteins on the basis of ab initio quantum chemistry. Using both resonance Raman spectroscopy and quantum chemical calculations, we have determined the chromophore structures in the intermediate states of photoactive yellow protein(PYP). We have successfully extracted key information on the chromophore-protein interactions. The results permit us to propose structural models of the intermediates, which reveal the photocycle mechanism of PYP under physiological conditions.

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. We report the resonance Raman spectra for the long-lived blue-shifted intermediate of PYP whose chromophore is isotopically labeled with 13C at the carbonyl carbon atom or at the ring carbon atoms. Spectra have been also measured with PYP in D2O where the phenolic hydroxyl group of the chromophore is deuterated. All of the observed Raman bands are assigned on the basis of the observed isotope shifts and normal mode calculations using a density functional theory. The complete assignment provides a satisfactory framework for future investigations of the photocycle mechanism in PYP by vibrational spectroscopy.

Time-resolved EPR spectra were observed by means of the transient EPR technique for two kinds of fullerene dimers C120 and C120O in the lowest excited triplet (T1) states and compared with those of mono-adducts of fullerene (C60). It was found that zero-field splitting (zfs) parameters of both dimers are nearly the same as those of the monomers, indicating that the excitation is almost completely localized at one of the two fullerene units in the T1 state. © 2002 Elsevier Science B.V. All rights reserved.

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.

The photoexcited quartet (Q1) and doublet (D1) states of the complex of tetra-phenyl-porphine zinc (II) and a nitroxide radical have been studied in toluene solution by X-(9.4 GHz) and W-band (94 GHz) time-resolved electron paramagnetic resonance spectroscopy. The spin-polarized signals of the Q1 and D1 states are observed and assigned by analysis of theg-values. Line broadening and spin polarization mechanisms in this system are discussed.

Sublevel properties of the lowest excited triplet (T1) npstates of ¬-silyl and ¬-germyl ketones were examined by means of ODMR, time-resolved EPR and optical spectroscopy. The EPR parameters, D, E and g, population ratios, and triplet lifetimes were obtained. The D value and the triplet lifetime varied among the molecules. In contrast the E value and population ratio remains nearly the same. These properties together with their solvent dependence and emission properties are interpreted in terms of spin± orbit couplings between T1 (np ¤ ) and higher S1 (np ¤ ), T2 (pp ¤ ) and S2 (pp ¤ ) states. An origin of the remarkable red-shifts of 1;3 (np ¤ ) are discussed based on a model of delocalized n, p and pelectrons over the Si and Ge atoms. This model is also consistent with all the triplet properties obtained.

EPR signals of the triphenylsilyl and triphenylgermyl radicals were observed for the first time in fluid solution by direct photolysis of hexaphenyldisilane and hexaphenyldigermane. Observed electron spin polarization was interpreted in terms of a triplet mechanism (TM). Along with the main signal, the 29Si isotope signal was also observed and the polarization was tentatively explained by combination of a TM and a radical pair mechanism. An electron density on the silicon atom of the silyl radical was determined as 0.52 from the isotropic hyperfine coupling constant of 29Si. The triphenylstannyl radical was not observed with the time resolution (ca. 80 ns) of our system.

The lowest excited triplet (T1) state of 3,3′-biisoquinoline(dicyano)platinum(II), [Pt(CN)2(i-biq)] was examined by time-resolved electron paramagnetic resonance (TREPR) and optical techniques in comparison with that of a ligand 3,3′-biisoquinoline (i-biq). Trans and cis i-biqs were assigned from obtained and calculated zero-field splittings (zfs') in the T1 state. The zfs parameter D increased with complex formation from 2.66 to 4.66 GHz in a rigid dimethylformamide solution; the polarization pattern of the spectrum was very different between the ligand and the complex. The triplet lifetime (ca. 1 s) of i-biq is also remarkably shortened in the complex (ca. 2 ms). These facts were analyzed in terms of the spin-orbit coupling (soc) between T1 (ππ*) and 1,3dπ*. From the analyses, the soc matrix element and the magnitude of delocalization of π* electrons over the Pt atom are obtained as 65 cm-1 and 0.02, respectively. The solvent dependences of the D value and the triplet lifetime also support the importanc...

Characteristic luminescence from the assembled species of [Pt(CN) 2 (L)] (L = 3.3'-biisoquinoline (i-biq) and 2,2'-bipyridine (bpy)) has been found in a dilute EtOH-MeOH (4:1 v/v) glassy solution at 77 K. For the i-biq complex, a broad emission hand (λ max = 680 nm) appears with increasing concentration in addition to a 3 ππ * emission hand form the monomer complex (λ max = 528 nm). The broad band is attributable to the emission from the 3 MLCT state of the assembled species with the Pt-Pt intermolecular interaction. In the case of the bpy complex, similar broad emission is also observed as well as the sharp 3 ππ * emission. However, the broad emission has an excimeric character. The π-π interaction hetween the π-conjugated ligands would he an important factor in the assemblage of the platinum complexes in the dilute system.

The porphyrinato copper cation radical complexes were examined by EPR spectroscopy. The cation radical complexes having bulky peripheral substituents are present as monomers in fluid solution and show EPR spectra due to intramolecular spin coupling between the spins on copper and on the porphyrin ring. The cation radical complexes, having no such substituents, form a dimer in solution. On the other hand, when they are oxidized in frozen solution by γ-ray irradiation, the cation radical complexes are always formed as a monomer, showing a triplet state EPR due to the intramolecular spin coupling. The zero field splitting and spin exchange parameters, |D| and 2J, obtained from EPR measurements were classified into two groups by their magnitudes. The tetraphenylporphyrinato copper(II) cation radical complex, [CuII(tpp)]+, has a small |D| and a small negative 2J value, while the octaethylporphyrinato copper(II) cation radical complex, [CuII(oep)]+, has a large |D| and a large negative 2J value. The other porph...

The bisadduct of fullerene (1) having two nitroxide radicals at the trans-3 position was newly synthesized. The ground and photoexcited states of 1 were studied by continuous-wave and pulsed two-dimensional electron paramagnetic resonance spectroscopy. An exchange interaction between the radicals was evaluated to be much smaller than an isotropic hyperfine splitting, showing a doublet (S = 1/2) character in the ground state. It was found from a 2D nutation spectrum that a photoexcited state has a quintet character (S = 2) which is generated via interactions between photoexcited triplet fullerene (S = 1) and the two nitroxide radicals (S = 1/2).

An electron paramagnetic resonance (EPR) study of 3,4-fulleropyrrolidine-2-spiro-4′[2′,2′,6′,6′-tetramethyl]piperidine-1′-oxyl (1) was performed on the photoexcited quartet state in toluene glass. The spectrum of the |S,Ms〉=|3/2,±3/2〉⇔|3/2,±1/2〉 transitions was observed selectively by using a two-dimensional (2D) nutation method and analyzed with a spectral simulation in a randomly oriented system. A position of the nitroxide moiety was determined with respect to the zero-field splitting (zfs) axes of excited triplet fullerene (3C60) by taking into account of the dipolar–dipolar interaction between the radical and 3C60, the hyperfine coupling, the anisotropic g-value of the nitroxide radical, and the zfs of the 3C60 moiety. It was found that none of the zfs axes of the 3C60 moiety coincide with the local C2 axis of the molecule which is defined by the position of addition. A symmetry of the electronic structure in 3C60 is discussed on the basis of the result.

Electron spin polarizations were studied for a system of excited triplet (T1) phthalocyanine (MTNPc; M = H2, Zn) and a nitroxide radical (TEMPO) in toluene solution by means of time-resolved EPR (T...